The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics.

In this study, the complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) has been determined. The genome sequence was 22003 base pairs (bp) and it included thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and three putative control regions (CRs). The nucleotide composition of AT was 71.50%, with a slightly negative AT skewness (-0.04). Usually the standard start codon of the PCGs was ATN, while cox1, nad4L and cox3 began with TTG, TTG and GTG. The canonical termination codon was TAA, while nad5 and nad4 ended with incomplete stop codon T, and cox1 ended with TAG. The mitochondrial gene arrangement of eight species of the Hippolytidae were compared with the order of genes of Decapoda ancestors, finding that the gene arrangement order of the Lebbeus groenlandicus had not changed, but the gene arrangement order of other species changed to varying degrees. The positions of the two tRNAs genes (trnA and trnR) of the L. vittata had translocations, which also showed that the Hippolytidae species were relatively unconserved in evolution. Phylogenetic analysis of 50 shrimp showed that L. vittata formed a monophyletic clade with Lysmata/Exhippolysmata species. This study should be helpful to better understand the evolutionary status, and population genetic diversity of L. vittata and related species.

Introduction

The genus Lysmata is an important group in family Hippolytidae, contains more than 48 described species, most of which are small shrimp living in shallow waters [1,2]. For a long time, the classification of Hippolytidae was the most controversial family in Decapoda, especially the monophyly of Hippolytidae and the position of the genus Lysmata [3,4]. In the past few decades, the studies of Lysmata mainly focused on morphology, with relatively few studies on population genetic structure. Meanwhile, most of the selected marker genes are partial sequences of rrnL, rrnS and cox1, and these gene fragments often fail to provide enough information to make the study of population genetics and species evolution.

The mitogenome is a significant tool for studying identification and phylogenetic relationships in the different species [5]. In shrimps, the mitochondria is maternally inherited, usually is circular and approximately 15 to 20 kb in length, including thirteen PCGs, two rRNAs, twenty-two tRNAs and one CR. The mitogenome contains abundant gene information, and the phylogenetic tree based on the mitogenome sequences has the advantages of stable and reliable structure. Analyzing the genetic relationship of species through the establishment of the 13PCGs sequence of the mitogenome can better solve the problems encountered in species classification.

Lysmata vittata (Crustacea: Decapoda: Hippolytidae) belongs to a small marine ornamental shrimp, commonly known as peppermint shrimp, which is popular in the marine aquarium trade. The species has a special sexual system, ie, protandric simultaneous hermaphrodite (PSH) [3]. It is a member of the clean shrimp family, a common marine ornamental species that originated in the Indian Ocean-Pacific region, including coastal areas such as China, Japan, Philippines and Australia [6-8]. L. vittata prefers to move in the range of 2~50 m below the sea surface, usually hiding in the reef during the day and activating at night [9]. In recent years, with the continuous breakthroughs in genomics technology, the phylogenetic research of the Lysmata species has gradually moved from the morphological level to the genome level. As a relatively important marine ornamental species, the determination of L. vittata mitogenome is of great significance for the development of genetic diversity and evolutionary history of Lysmata.

In this study, the mitogenome of the L. vittata has been successfully determined, and its structure and phylogenetic status have been analyzed. This work should help to further understand the evolutionary relationship and population genetic diversity between the L. vittata and related species.

Materials and methods

Mitochondria DNA sequencing and genome assembly

Specimens of L. vittata were collected in Xiamen, Fujian province, China. The morphological characteristics of the species follow the previous description of Abdelsalam [1]. Approximately 5g of muscle tissue was harvested for mtDNA isolation using an improved extraction method [10]. After DNA isolation, the isolated DNA was purified according to manufacturer’s instructions (Illumina), and then 1 μg was taken to create short-insert libraries, whose insertion size was 430 bp, followed by sequencing on the Illumina Hiseq 4000 [11] (Shanghai BIOZERON Co., Ltd). The high molecular weight DNA was purified and used for PacBio library prep, BluePippin size selection, then sequenced on the Sequel Squencer.

The raw data obtained by sequencing was processed and then the duplicated sequences were assembled. The mitogenome was reconstructed using a combination of the PacBio Sequel and the Illumina Hiseq data. Assemble the genome framework by the both Illumina and PacBio using SOAPdenovo2.04 [12]. Verifying the assembly and completing the circle or linear characteristic of the mitogenome, filling gaps if there were. Finally, the clean data were mapped to the assembled draft mitogenome to correct the wrong bases, and the most of the gaps were filled through local assembly.

Validation of mitogenome data

In order to ensure the accuracy of the L. vittata mitogenome data, we resequenced the samples on the Illumina HiSeq X10 platform (Nanjing Genepioneer Biotechnologies Co. Ltd).

Genome annotation and sequence analysis

Mitogenome sequences were annotated using homology-based prediction and de novo prediction, and the EVidenceModeler v1.1 [13] was used to integrate the complete genetic structure. Twenty-two tRNAs and two rRNAs were predicted by tRNAscan-SE [14] and rRNAmmer 1.2 [15]. The circular of the complete L. vittata mitogenome graphical map was drawn using OrganellarGenomeDRAW v1.2 [16]. The RSCU of thirteen PCGs (remove incomplete codons) was calculated using MEGA 5.0 [17]. The composition skewness of each component of the genome was calculated according to the following formulas: AT-skew = (A-T)/(A+T); GC-skew = (G-C)/(G+C) [18]. The secondary cloverleaf structure of tRNAs was examined with MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py) [19].

Phylogenetic analysis

To reconstruct the phylogenetic relationship among shrimp, the PCGs sequences of the 49 Decapoda species were downloaded from GenBank database (S1 Table). The PCGs sequences of Harpiosquilla harpax (NC_006916) were used as outgroup. The nucleotide and amino acid sequences of 13 PCGs were aligned using MEGA 5.0 [17]. Gblocks was used to identify and selected the conserved regions [20]. Subsequently, Bayesian inference (BI) and Maximum likelihood (ML) analysis were utilized for reconstructing phylogenetic tree by MrBayes v3.2.6 [21] and PhyML 3.1 [22]. According to the Akaike Information Criterion (AIC) [23], TVM + I + G model was considered as the best-fit model for analysis with nucleotide alignments using jModeltest [24], and MtArt + I + G + F model was the optimal model for the amino acid sequence dataset using ProtTest 3.4.2 [25]. In BI analysis, two simultaneous runs of 10000000 generations were conducted for the matrix. Sampling trees every 1000 generations, and diagnostics were calculated every 5000 generations, with three heated and one cold chains to encourage swapping among the Markov-chain Monte Carlo (MCMC) chains. Additionally, the standard deviation of split frequencies was below 0.01 after 10000000 generations, and the potential scale reduction factor (PSRF) was close to 1.0 for all parameters. Posterior probabilities over 0.9 or bootstrap percentage over 75%, the results were regarded as credible [26,27]. The resulting phylogenetic trees were visualized in Fig Tree v1.4.0.

Results and discussion

Genome structure, organization and composition

The mitogenome of L. vittata was a typical circular molecule of 22003 bp in size. It contained 37 mitochondrial genes (thirteen PCGs, twenty-two tRNAs, two rRNAs and three CRs) (Fig 1 and S2 Table). Among the 37 genes, the coding direction of the twenty-three genes was clockwise (F-strand), and the coding direction of the remaining fourteen genes was counterclockwise (R-strand) (Fig 1 and S2 Table).

Fig 1

Mitogenome map of Lysmata vittata.

The genes outside the map were coded on the F strand, whereas the genes on the inside of the map are coded on the R strand. The middle black circle displays the GC content and the inside purple and green circle displays the GC skew.

The nucleotide composition of the mitogenome was biased toward A and T (T = 37.15%, A = 34.35%, C = 16.69%, G = 11.80%) (Table 1). The relatively AT contents of the complete mitogenome were calculated [mitogenome (71.50%), PCGs (69.79%), tRNAs (69.58%) and rRNAs (69.29%)] (Tables 1 and 2). However, with the exception of Thor amboinensis (73.10%), the AT content of L. vittata mitogenome was higher than other species in the Hippolytidae (Table 1). Among the nine species of Hippolytidae, the AT-skew values of L. vittata (-0.039) was similar with L. boggessi (-0.040), and the AT-skew values of Lebbeus groenlandicus (0.062), Exhippolysmata ensirostris (0.009) and Saron marmoratus (0.110) was positive. In addition, with the exception of Thor amboinensis (-0.081), the GC-skew value for L. vittata (Guangdong) was the biggest negative comparing to that of other mitogenomes (Table 1). By comparing the mitogenome sequence of L. vittata (Fujian) with that of L. vittata (Guangdong), it was found that the whole mitogenome sequence of L. vittata (Fujian) could completely overlap with L. vittata (Guangdong) except that it was 1146 bp bases longer than that of L. vittata (Guangdong). The base distribution of L. vittata (Guangdong) deletion was shown in S1 Fig. The reason for sequence deletion may be related to sequencing method and sequence splicing. All original sequence data in this study were submitted to the NCBI database under accession number MT478132.

Table 1

Composition and skewness of mitogenome in 9 Hippolytidae species.

Species	Size (bp)	T%	C%	A%	G%	A+T %	ATskewness	GCskewness
Whole genome
L. vittata	22003	37.15	16.69	34.35	11.80	71.50	-0.039	-0.172
L. vittata (Guangdong)	20857	36.87	17.03	34.36	11.74	71.23	-0.035	-0.184
L. amboinensis	16735	32.36	21.65	31.68	14.31	64.05	-0.011	-0.204
L. debelius	16757	34.11	19.70	33.04	13.15	67.15	-0.016	-0.199
L. boggessi	16979	35.01	19.55	32.33	13.10	67.34	-0.040	-0.197
L. groenlandicus	17398	30.37	21.37	34.41	13.85	64.78	0.062	-0.213
E. ensirostris	16350	31.93	21.31	32.52	14.24	64.45	0.009	-0.199
S. marmoratus	16330	30.21	21.70	37.68	10.42	67.89	0.110	-0.351
T. amboinensis	15553	37.01	14.54	36.09	12.36	73.10	-0.013	-0.081
PCGs
L. vittata	11144	41.09	15.25	28.70	14.96	69.79	-0.178	-0.010
L. vittata (Guangdong)	11285	41.17	15.18	28.75	14.90	69.92	-0.178	-0.009
L. amboinensis	11192	36.66	19.12	25.94	18.28	62.60	-0.171	-0.022
L. debelius	11162	38.66	17.17	27.40	16.78	66.05	-0.170	-0.011
L. boggessi	11165	38.83	17.65	27.02	16.50	65.85	-0.179	-0.034
L. groenlandicus	11175	37.19	18.93	25.66	18.23	62.85	-0.184	-0.019
E. ensirostris	11062	36.77	19.30	26.05	17.87	62.83	-0.171	-0.038
S. marmoratus	11135	37.53	17.45	28.50	16.52	66.03	-0.137	-0.027
T. amboinensis	11178	41.39	13.36	30.26	14.99	71.65	-0.155	0.058

Table 2

Composition and skewness of Lysmata vittata mitogenome.

Lysmata vittata	Size(bp)	T (%)	C (%)	A (%)	G (%)	A+T (%)	AT-skew	GC-skew
atp6	675	40.15	19.41	28.30	12.15	68.44	-0.17	-0.23
atp8	165	43.64	15.76	35.15	5.45	78.79	-0.11	-0.49
cob	1137	39.40	20.14	27.88	12.58	67.28	-0.17	-0.23
cox1	1614	37.73	17.91	27.76	16.60	65.49	-0.15	-0.04
cox2	693	37.95	19.77	28.43	13.85	66.38	-0.14	-0.18
cox3	756	39.29	18.25	27.91	14.55	67.20	-0.17	-0.11
nad1	927	44.01	10.79	27.29	17.91	71.31	-0.23	0.25
nad2	1005	43.28	18.01	29.05	9.65	72.34	-0.20	-0.30
nad3	354	42.66	18.93	26.27	12.15	68.93	-0.24	-0.22
nad4	1336	43.11	9.51	28.59	18.79	71.70	-0.20	0.33
nad4l	246	45.12	7.72	26.02	21.14	71.14	-0.27	0.46
nad5	1732	41.17	9.82	31.64	17.38	72.81	-0.13	0.26
nad6	504	44.64	17.06	28.57	9.72	73.21	-0.22	-0.27
tRNAs	1512	33.27	14.02	36.31	16.40	69.58	0.04	0.08
rRNAs	2315	32.40	11.88	36.89	18.83	69.29	0.06	0.23
CR1	650	42.15	9.85	38.31	9.69	80.46	-0.05	-0.01
CR2	3821	38.50	14.39	33.73	13.37	72.23	-0.07	-0.04
CR3	888	42.34	13.51	34.91	9.23	77.25	-0.10	-0.19

PCGs and codon usage

The PCGs region was 11144 bp long, and accounted 50.6% of the L. vittata mitogenome. Furthermore, a contrast of nucleotide composition, AT-skew value, and GC-skew value of PCGs from other species in the Hippolytidae were also exhibited in Table 1. Nine of thirteen PCGs (atp6, atp8, cob, cox1-3, nad2-3 and nad6) were encoded on the light (F) strand, while the other four genes (nad1, nad4L and nad4-5) were encoded on the heavy (R) strand (S2 Table). Each PCG was initiated by a canonical ATN codon (ATG for atp6, atp8, nad2-5 and cob; ATT for cox2 and nad1; ATC for nad6), except for cox1 (TTG), nad4L (TTG) and cox3 (GTG) (S2 Table). Two of the thirteen PCGs (nad5 and nad4) terminated with incomplete stop codon T, one PCG (cox1) terminated with stop codon TAG, and the other ten PCGs terminated with the canonical termination codon TAA (S2 Table).

The RSCU values of L. vittata mitogenome were analyzed and the results were shown in Table 3. The total number of codons in thirteen PCGs was 3714 except eleven canonical stop codons and two incomplete stop codons and the most common amino acids were Ile (AUR) (499), Phe (UUR) (357) and Leu2 (UUR) (315), whereas codons encoding Cys (UGR) (41) and Met (AUR) (24) were rare (Fig 2). The overall A + T content of thirteen PCGs was 69.79%, the AT-skews and GC-skews were negative which implied a higher occurrence of Ts and Cs than As and Gs (Table 1).

Table 3

The codon number and relative synonymous codon usage (RSCU) in L. vittata mitochondrial protein coding genes.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	300	1.68	UCU(S)	129	2.46	UAU(Y)	101	1.57	UGU(C)	32	1.56
UUC(F)	57	0.32	UCC(S)	29	0.55	UAC(Y)	28	0.43	UGC(C)	9	0.44
UUA(L)	283	3.13	UCA(S)	92	1.76	UAA(*)	10	0.29	UGA(W)	92	2.68
UUG(L)	32	0.35	UCG(S)	12	0.23	UAG(*)	1	0.03	UGG(W)	15	1
CUU(L)	131	1.45	CCU(P)	101	2.71	CAU(H)	53	1.47	CGU(R)	12	0.4
CUC(L)	33	0.36	CCC(P)	14	0.38	CAC(H)	19	0.53	CGC(R)	2	0.07
CUA(L)	59	0.65	CCA(P)	28	0.75	CAA(Q)	55	1.62	CGA(R)	38	1.26
CUG(L)	5	0.06	CCG(P)	6	0.16	CAG(Q)	13	0.38	CGG(R)	11	0.36
AUU(I)	266	1.6	ACU(T)	85	1.95	AAU(N)	108	1.65	AGU(S)	45	0.86
AUC(I)	42	0.25	ACC(T)	23	0.53	AAC(N)	23	0.35	AGC(S)	7	0.13
AUA(I)	191	1.15	ACA(T)	61	1.40	AAA(K)	83	1.77	AGA(S)	93	3.08
AUG(M)	24	1	ACG(T)	5	0.11	AAG(K)	11	0.23	AGG(S)	25	0.83
GUU(V)	95	1.82	GCU(A)	93	2.14	GAU(D)	56	1.51	GGU(G)	61	1.06
GUC(V)	8	0.15	GCC(A)	25	0.57	GAC(D)	18	0.49	GGC(G)	20	0.35
GUA(V)	87	1.67	GCA(A)	50	1.15	GAA(E)	64	1.35	GGA(G)	106	1.84
GUG(V)	19	0.36	GCG(A)	6	0.14	GAG(E)	31	0.65	GGG(G)	43	0.75

Fig 2

RSCU and Codon distribution in the mitogenome of L. vittata.

The left ordinate represents RSCU, and the right ordinate represents the number of the Codon distribution.

Transfer RNAs and ribosomal RNAs

The mitogenome of L. vittata contained twenty-two tRNAs and these genes ranged from 60 (trnA) to 77 bp (trnN) (S2 Table). The tRNAs showed a strong A +T bias (69.58%), while they also exhibited positive AT-skew (0.04) and GC-skew (0.08) (Table 1). Eight tRNAs [trnQ (CAA), trnC (UGC), trnY (UAC), trnF (UUC), trnH (CAC), trnP (CCA), trnL1 (CUA) and trnV (GUA)] were present on the R strand and the remaining fourteen were present on the F strand (S2 Table). The examined secondary structure of twenty-two tRNAs was shown in S2 Fig. The other twenty-one tRNAs had typical cloverleaf secondary structure except that trnS1 (AGA) lacked the dihydropyridine (DHU) arm [18,19,27,28] (S1 Fig). In the secondary structure of the tRNAs, the most common non-Watson–Crick base pair was G–U (e.g. trnC, trnE), followed by U–U (e.g. trnA, trnC) [19]. In addition, several mismatches were common in tRNAs, such as A–C (e.g. trnA), C–U (e.g. trnA, trnG) and A–A (e.g. trnM, trnS1) (S1 Fig).

Two rRNA genes were found on the R strand. The rrnL was 1494 bp and rrnS was 821 bp, one located between trnL1 and trnV and another located between trnV and CR1 (S2 Table and Fig 1). The total A+T content of the two rRNAs was 69.29%, with a positive AT-skew (0.06) (Table 2).

Overlapping and intergenic regions

The mitogenome of L. vittata contained four overlapping regions, these four pairs of genes were presented: atp8/atp6, trnE/trnF, nad4/nad4L and trnL1/rrnL, with the longest 23 bp overlap located between trnL1 and rrnL (S2 Table). The 27 intergenic regions were found with a length varying from 2 ~ 3821 bp (S2 Table). Three putative CRs had been identified in L. vittata mitogenome. The CR1 was located between rrnS and trnI, with a length of 650 bp, and the A+T content was 80.46%. The CR2 was located between cox1 and trnL2, with a length of 3821 bp, and the A+T content was 72.23%. The CR3 was located between trnL2 and cox2, with a length of 888 bp, and the A+T content was 77.25% (Tables 2 and S2).

To our knowledge, the complete mitogenome sequence of L. vittata is the longest in the existing research on shrimp. How multiple CRs were generated and evolved in the mitogenome of Lysmata is a novel problem that has not yet been solved, and more mitogenomes of Lysmata are still needed to clarify the mechanism forming this phenomenon.

Gene rearrangement

In terms of gene rearrangement, compared with the genes order of the ancestor of Decapoda [20,29], the order of the genes of L. groenlandicus remains unchanged, and all species of the Lysmata had multiple CR regions. Among them, L. amboinensis, L. debelius and L. boggessi had 2 CR regions, L. vittata has three CR regions and the positions of the two tRNA genes (trnA and trnR) had been translocated. In addition, the mitochondrial genes order of E. ensirostris, S. marmoratus and T. amboinensis all had varying degrees of translocation compared with the gene order of Decapoda (Fig 3). The position of cox2 and trnL2 of E. ensirostris was translocated, and the trnC and gene block (trnM-nad2-trnW) of S. marmoratus were translocated. T. amboinensis produced more translocations, including two gene block (nad6-cob-trnS2 and nad5-trnH-nad4-nad4l) translocations and single tRNA (trnQ, trnT, trnE, trnH, trnY, trnP, trnC and trnM) translocations (Fig 3). In fact, gene rearrangement was a very common phenomenon in the mitogenome and the rearrangement mainly occurred in tRNA genes. Gene arrangement was stable, and it could be used as an important phylogenetic marker in the analysis of evolutionary perspective on shrimp. Comparing the order of the mitochondrial genes of various species of Hippolytidae, it indicates that the species of Hippolytidae are not conserved in evolution.

Fig 3

Linear representation of gene rearrangements of Hippolytidae species.

The taxonomic status of genus Lysmata within Hippolytidae has been a highly contentious issue for a long time. In this study, using ML and BI analysis methods, phylogenetic analysis was performed based on the nucleotide and amino acid sequences of thirteen PCGs of the species in S1 Table, and the analysis results were presented (Figs 4 and 5). The phylogenetic tree based on the nucleotide sequence of thirteen PCGs showed that Lysmata and Exhippolysmata formed a monophyletic group, while S. marmoratus, L. groenlandicus and T. amboinensis was clustered into a monophyletic group with species of Alpheidae and Palaemonidae (Fig 4). This analysis supported Christoffersen’s [30,31] proposal to classify the Lysmata into the same classification level as the Lysmatidae. The phylogenetic tree based on the amino acid sequence of 13 PCGs revealed that the species of Hippolytidae clustered into a large branch, among which Lysmata-Exhippolysmata formed a monophyletic branch, which was in sister relationship with S. marmoratus-L. groenlandicus/T. amboinensis (Fig 5). The topological structures of the phylogenetic trees constructed based on the nucleotide sequence and amino acid sequence were slightly different within the Hippolytidae, but the monophyleticity of Lysmata-Exhippolysmata had been fully verified in previous studies [32-35]. Furthermore, the phylogenetic analyses confirmed that L. vittata (Fujian) and L. vittata (Guangdong) were closely related. The two shrimps were clustered together and the branch length was zero. Especially their branch nodes were strongly supported (ML BP = 100%; BI PP = 1), indicating that there was almost no difference between L. vittata (Fujian) and L. vittata (Guangdong). The phylogenetic relationship among other suborder/superfamily of Decapoda was similar to Ma et al. [36] research.

Fig 4

Phylogenetic tree inferred from nucleotide sequences of 13 PCGs of the mitogenome using ML and BI methods (BP/PP).

Fig 5

Phylogenetic tree inferred from amino acid sequences of 13 PCGs of the mitogenome using ML and BI methods (BP/PP).

Conclusion

In this study, we successfully obtained the mitogenome sequence of the L. vittata, which was also the first species of the Hippolytidae to publish the mitogenome sequence in the GenBank database. The genome sequence was 22003 base pairs (bp) and it included 37 genes and three CRs. Each PCGs was initiated by a canonical ATN codon, except for cox1, nad4L and cox3, which were initiated by a TTG, TTG and GTG. Two of the thirteen PCGs (nad5 and nad4) terminated with incomplete stop codon T, and one (cox1) terminated with stop codon TAG. The AT-skew (-0.04) and the GC-skew (-0.17) were both negative in the mitogenomes of L. vittata. Compared with the gene order of a Decapoda ancestor, the gene arrangement order of the L. vittata has changed. Futhermore, phylogenetic analyses showed that L. vittata formed a monophyletic branch with other species of the genus Lysmata/Exhippolysmata.

Comparison of the difference interval between L. vittata (Fujian) and L. vittata (Guangdong) mitogenome sequence.

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Predicted secondary structure for the tRNAs of Lysmata vittata mitogenome.

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List of species used to construct the phylogenetic tree.

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Summary of Lysmata vittata mitogenome.

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29 Jul 2021

PONE-D-21-22008

The complete mitogenome of  Lysmata vittata (Crustacea: Decapoda: Hippolytidae) and its phylogenetic position in Decapoda

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Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presented the first record of mitochondrial genome of the shrimp Lysmata vittata (Crustacea: Decapoda: Hippolytidae) and used it together with mitogenomes of the close families from the GenBank for reconstruction of phylogenetic tree. The data is valuable but only one mitogenome sequence was included in this manuscript. Nowadays, sequencing and describing a mitogenome is routine work and I can not find the novelty of the manuscript. In the manuscript, authors only described the mitogenome sequence and did not compared its structure with close species. Also, authors stated that phylogeny of Hippolytidae is problematic but the study is unable to solve the existing problem. In addition, even though authors stated that “we are the first to publish the mitochondrial genome sequence of the Hippolytidae species in the GenBank database”, a number of mitogenome sequence of Hippolytidae and Lysmatidae are available in the GenBank, such as Lysmata boggessi (GenBank no. MZ144584), Lysmata debelius (GenBank no. MW691200), Saron marmoratus (GenBank no. MT795210) and Exhippolysmata ensirostris (GenBank no. MK681888). It is surprise that authors did not make comparison between L. vittata mitogenome with these species. Moreover, these species were not included in the phylogenetic tree reconstruction. Because of that, the finding and discussion of the manuscript are poor.

Reviewer #2: This manuscript is written with minor grammatical errors. Please improve the manuscript again. The experimental design is properly set up and the problem statement is well defined. I would like to suggest the authors discuss and make a comparison with the available complete mitogenome of the same species, L. vittata from Guangdong province China. It seems that the size of the mitogenomes of both individuals is different even they were collected not far away from each other as Guangdong and Fujian are next to each other. The author should also include both individuals in the phylogenetic tree and the gene arrangement comparison. Apart from this, I also suggest the authors compare all the genes from the mitogenome of each individual and discuss. From this, authors can determine the individual variation of L. vittata from China. Please also improve the phylogenetic tree figures as the figures are quite blurry.

Reviewer #3: The manuscript is well designed and written, the results are sound and helpful for better understanding the mitogenome of Decapoda and for the phylogenetics. Here I have several concerns as below,

The authors should tell the readers why you carried out this study, is there any problem need to be solved in the Background section.

The authors analyzed the phylogenetic relationship under the order Decapoda, however, they introduce limited information in the Introduction section, I suggest the authors to add some information about crabs.

At line 65, what do “fresh leaves” mean?

I wonder for the phylogenetic analysis, did the authors used crabs? if not, they could not use Decapoda.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Hongyu Ma

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Sep 2021

Reviewer #1: The manuscript presented the first record of mitochondrial genome of the shrimp Lysmata vittata (Crustacea: Decapoda: Hippolytidae) and used it together with mitogenomes of the close families from the GenBank for reconstruction of phylogenetic tree. The data is valuable but only one mitogenome sequence was included in this manuscript. Nowadays, sequencing and describing a mitogenome is routine work and I can not find the novelty of the manuscript. In the manuscript, authors only described the mitogenome sequence and did not compared its structure with close species. Also, authors stated that phylogeny of Hippolytidae is problematic but the study is unable to solve the existing problem. In addition, even though authors stated that “we are the first to publish the mitochondrial genome sequence of the Hippolytidae species in the GenBank database”, a number of mitogenome sequence of Hippolytidae and Lysmatidae are available in the GenBank, such as Lysmata boggessi (GenBank no. MZ144584), Lysmata debelius (GenBank no. MW691200), Saron marmoratus (GenBank no. MT795210) and Exhippolysmata ensirostris (GenBank no. MK681888). It is surprise that authors did not make comparison between L. vittata mitogenome with these species. Moreover, these species were not included in the phylogenetic tree reconstruction. Because of that, the finding and discussion of the manuscript are poor.

Response: Many thanks for your professional suggestions. According to your suggestion, we have added the structural comparison and phylogenetic analysis of L. vittata mitogenome and its relative species in this article. Please see lines 130-143, 214-225 and 240-257. Your suggestions are of great help to us in improving the quality of article.

Reviewer #2: This manuscript is written with minor grammatical errors. Please improve the manuscript again. The experimental design is properly set up and the problem statement is well defined. I would like to suggest the authors discuss and make a comparison with the available complete mitogenome of the same species, L. vittata from Guangdong province China. It seems that the size of the mitogenomes of both individuals is different even they were collected not far away from each other as Guangdong and Fujian are next to each other. The author should also include both individuals in the phylogenetic tree and the gene arrangement comparison. Apart from this, I also suggest the authors compare all the genes from the mitogenome of each individual and discuss. From this, authors can determine the individual variation of L. vittata from China. Please also improve the phylogenetic tree figures as the figures are quite blurry.

Response: Thanks for your suggestions. This manuscript has been revised by an English speaking person. In addition, we have compared and discussed L. vittata (Fujian) and L. vittata (Guangdong) mitogenome sequences and hope that it is now clearer. Please see lines 135-143. Apart from this, we have also improved the phylogenetic tree figures.

Reviewer #3: The manuscript is well designed and written, the results are sound and helpful for better understanding the mitogenome of Decapoda and for the phylogenetics. Here I have several concerns as below,

The authors should tell the readers why you carried out this study, is there any problem need to be solved in the Background section.

Response: Thank you for the suggestion. The purpose and meaning of this research have been added in the introduction section. Please see lines 32-40 and 56-65.

The authors analyzed the phylogenetic relationship under the order Decapoda, however, they introduce limited information in the Introduction section, I suggest the authors to add some information about crabs.

Response: We are grateful for the suggestion. The content of the introduction section has been revised. At present, there are relatively few studies on the complete mitogenomes of Hippolytidae species, so we have selected some species under the order Decapoda to participate in the phylogenetic analysis. The main purpose of our phylogenetic analysis is to understand the classification status of L. vittata in the Hippolytidae, and our analysis results also illustrate the monophyleticity of the genus Lysmata and controversy exists in the classification of some species of Hippolytidae. Therefore, there is no information about crabs in the introduction section, but we added crab information when we performed phylogenetic analysis.

At line 65, what do “fresh leaves” mean?

Response: We apologize for the writing error in the original manuscript. The "fresh leaves" has been revised in the text. Please see line 70.

I wonder for the phylogenetic analysis, did the authors used crabs? if not, they could not use Decapoda.

Response: Thank you for your precious advice. Those advice are all valuable and very helpful for revising and improving our paper. We have added crab data to the phylogenetic analysis content. Please see Supplementary Table 1.

We would like to thank the referee again for taking the time to review our manuscript.

Submitted filename: Response to Reviewers-20210904.doc

Click here for additional data file.

22 Sep 2021

PONE-D-21-22008R1The complete mitogenome of  Lysmata vittata (Crustacea: Decapoda: Hippolytidae) and its phylogenetic position in DecapodaPLOS ONE

Dear Dr. Zhu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Author’s efforts to improve the submitted manuscript following reviewer’s comments have been done. Therefore, the current version of manuscript is much better than previous one. However, similar to the first comment, the only addition of a single new mitogenome, which does not show special meaning of mtgenome structure, to a crab molecular phylogeny is still problem. In addition, one more crucial problem is that the authors should justify why authors construct the phylogenetic trees for the decapods and what phylogenetic problem authors want to solve in the article. If the authors would like to present a novel decapod tree more comprehensive families and taxonomic representatives should be included. If the authors want to know the phylogenetic position of the Lysmata vittata, the tree will be enough with related species and taxa of L. vittate. In spite of these comments the final decision from the editor charging the article will be respected.

Reviewer #3: The authors have revised the manuscript according my comments, so now I have no more comments. The authors have revised the manuscript according my comments, so now I have no more comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Hongyu Ma

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Oct 2021

Reviewer #1: Author’s efforts to improve the submitted manuscript following reviewer’s comments have been done. Therefore, the current version of manuscript is much better than previous one. However, similar to the first comment, the only addition of a single new mitogenome, which does not show special meaning of mtgenome structure, to a crab molecular phylogeny is still problem. In addition, one more crucial problem is that the authors should justify why authors construct the phylogenetic trees for the decapods and what phylogenetic problem authors want to solve in the article. If the authors would like to present a novel decapod tree more comprehensive families and taxonomic representatives should be included. If the authors want to know the phylogenetic position of the Lysmata vittata, the tree will be enough with related species and taxa of L. vittate. In spite of these comments the final decision from the editor charging the article will be respected.

Response: Many thanks for your professional suggestions. We found that the mitogenome structure of L. vittata is different from other species in the same genus. Part of the mitogenome of L. vittata has translocations. There are 3 CR regions, and the length of the genome sequence is about 7 kb longer than that of normal shrimp species. Therefore, this manuscript analyzes the special mitogenome structure of L. vittata. In addition, in the phylogenetic analysis, there are currently limited mitogenome data of Hippolytidae species (all available data was collected), we borrowed the mitogenome data of some species in the Decapoda to conduct phylogenomics and population genetics analysis. Although the dispute over the taxonomic status of Hippolytidae still cannot be resolved, we are making more efforts to resolve this dispute. The purpose of establishing phylogenetic tree is to determine the phylogenetic position of the Lysmata vittata. In order to make this manuscript more express the author's thoughts, we have revised the title of the article, called "The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics." Thanks again for your valuable comments on our manuscript.

Reviewer #3: The authors have revised the manuscript according my comments, so now I have no more comments. The authors have revised the manuscript according my comments, so now I have no more comments.

Response: Thank you for taking the time to review our manuscript.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

22 Oct 2021

The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics

PONE-D-21-22008R2

Dear Dr. Zhu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: the authors have revised the manuscript according my comments, I think it is suitable to publish it.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Hongyu Ma

27 Oct 2021

PONE-D-21-22008R2

The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics

Dear Dr. Zhu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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