Complete Mitochondrial Genomes of Two Oriental Sweetlips, Plectorhinchus orientalis and Plectorhinchus vittatus (Perciformes: Haemulidae) with the Molecular Analysis on their Synonym Controversies

Rishen Liang*, Meng Zhou, Zhenxiang Lin, Guozhang Li, Yuan Chen, Xuan Lin and Zaohe Wu

College of Animal Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, China

ABSTRACT

Plectorhinchus orientalis and Plectorhinchus vittatus were two species of colorful coral reef sweetlips which distribute in the areas of Indo-Western Pacific. The two species have long been considered as a synonym (oriental sweetlips) in morphology. In order to investigate the validity of two species at the molecular level, complete mitochondrial genomes of the two species were first determined. The genomes were 16,546 bp (P. orientalis) and 16,545 bp (P. vittatus) in size, respectively, which both consisted of a typical structure of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and one noncoding control region. Genomic composition, organization and gene order were similar to that obtained in most vertebrates. By comparative analysis of the two genomes, 941 variable sites (5.69%) were found. Sequence divergences of 13 protein-coding genes, 2 rRNA genes and one control region which are commonly used as molecular markers between the two species ranged from 2.4% (12S rRNA) to 11.2% (ND6), all divergence values were larger than 2%. Great variations of Cyt b and COI between P. orientalis and P. vittatus were revealed to be 6.0% and 7.4%, respectively, which were largely greater than the threshold of species diagnosis divergence value of 2%. Inner individual divergence values of each species were less than 0.5%. In the molecular phylogenetic trees, the longer branch length also clearly distinguished the independent placement of the two species. These results revealed great genetic differences between P. orientalis and P. vittatus, and strongly suggested that they might be two distinct species and should not be placed as synonym.

Article Information

Received 11 August 2017

Revised 10 October 2017

Accepted 13 November 2017

Available online 19 March 2019

Authors’ Contribution

RL and ZW designed the research and wrote the manuscript. RL and MZ collected the samples. RL, ZL and GL carried out experiments. YC and LX analyzed sequencing data and experimental results.

Key words

Complete mitochondrial genome, Plectorhinchus orientalis, Plectorhinchus vittatus, Molecular phylogeny, Mitogenome.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.3.871.885

* Corresponding author: cheetahliang@126.com

0030-9923/2019/0003-0871 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

The vertebrate mitochondrial genome (mitogenome) is a small, double-stranded and circular DNA ranging in size from 15-20 kb. The structural gene organization is quite conserved within vertebrate mitogenome which contains 13 protein-coding genes, 2 ribosomal RNA genes (12S and 16S), 22 transfer RNA genes and one noncoding control region. In addition, mtDNA possesses two main non-coding regions involved in replication and transcription processes: the control region or D-loop and the light-strand replication origin or OL (Clayton, 1992; Shadel and Clayton, 1997; Boore, 1999). The mitochondrial DNA has been extensively used as a marker for molecular evolution, genetic diversity studies and phylogenetic analyses due to its compact size, maternal inheritance, rapid evolutionary rate and lack of recombination (Inoue et al., 2001; Miya et al., 2003; Saitoh et al., 2006; Kawahara et al., 2008). In fish, the mitogenome was first reported in freshwater loach (Crossostoma lacustre) by Tzeng et al. (1992). At the time of writing, complete mitogenomes from more than 472 species in Perciformes are available in NCBI database.

Family Haemulidae belongs to the suborder Percodei of order Perciformes, which comprised two subfamilies: Plectorhynchinae (sweetlips) and Haemulinae (grunts). Sweetlips are globally marine fish and mainly found in the oceanic waters of tropical, subtropical water along the Indo-Western Pacific. Most colorful sweetlips represent a drastic change in their external coloration appearance from juvenile to adult, which makes difficulties in species identification. Two oriental sweetlips Plectorhinchus orientalis (Bloch, 1793) and Plectorhinchus vittatus (Linnaeus, 1758) are common colorful sweetlips and mainly distributed in the areas of Indo-Western Pacific. P. vittatus has been widely known as P. orientalis (Satapoomin and Randall 2000), and most studies suggested P. orientalis and P. vittatus refer to the same species: Oriental sweetlips (McKay, 1984, 2001; Randall and Lim, 2000; Gillibrand et al., 2007; Unsworth, 2010) and considered P. orientalis as synonym of P. vittatus (Randall and Johnson, 2000; McKay, 2001; Randall, 2005; Froese and Pauly, 2014). Randall and Johnson (2000) has resurrected P. vittatus (Linnaeus) as an older name. Recently, some studies have separated oriental sweetlips from the Indian Ocean and Pacific Ocean populations and considered P. vittatus as the Indian Ocean populations (Indian Ocean oriental sweetlips) and P. orientalis (oriental sweetlips) as the Pacific Ocean populations. Despite a large number of morphology-based hypotheses, no molecular studies have been examined on the two debated species. As mention above, mitochondrial DNA has become a very useful marker for species identification and phylogenetic studies. Thus, further studies of genetic characteristics of P. orientalis and P. vittatus based on multiple mitochondrial gene markers appear necessary to recover conclusive phylogenetic relationships of the two species and resolve the classification and identification problem.

In the present study, we first reported the complete mitogenome sequences of P. orientalis and P. vittatus and further compared their genome structure and organization, nucleotide variation and codon usage. We also reported the phylogenetic analysis using protein sequences from P. orientalis, P. vittatus and other Haemulidae species to clarify the interrelationship of the two species and investigate their evolutionary position within Haemulidae.

 

Materials and Methods

Sweetlips samples collection and genomic DNA extraction

Sample of P. orientalis was collected in its natural habitats, the coral reef areas of Jiyin Island in Paracel Islands. Sample of P. vittatus was a vouchered specimens obtained from SAIAB (South African Institute for Aquatic Biodiversity). A small piece of fresh muscle was sampled and preserved in 95% ethanol and total genomic DNA was extracted using a Tissue Genomic DNA Extraction kit (Omega, USA) following the manufacturer’s protocol and stored at -20°C. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals, and protocols were approved by the Institutional Animal Care and Use Committee in Zhongkai University of Agriculture and Engineering, Guangdong, China.

Primer design and long-PCR amplification

The complete mitochondrial genomes of P. orientalis and P. vittatus were both amplified using long PCR technique (Miya and Nishida, 1999). Three sets of primers were designed from conservative regions of the genes for 16SrRNA , CO II and tRNA(Leu) to efficiently amplify the complete mitochondrial genome in three long-PCRs using EX Taq DNA polymerase (TaKaRa, Japan). All PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, German).

The purified PCR products were then cloned into the pUCm-T cloning vectors (Invitrogen, USA) and sequenced according to the manufacturer’s protocol by using T7 and M13R inner primers of pUCm-T vector. Primer walking method was applied for sequencing the DNA fragment. Each segment overlapped the next contig by 80–120 bp to ensure the accuracy of the sequence.

Sequence analysis

The primary DNA sequence data was characterized by BLAST tools at NCBI database (www.ncbi.nlm.nih.gov). Then the DNA sequences were edited and analyzed by the computer programs Vector NTI Suite 8 (Invitrogen, USA). The locations of the 13 protein-coding genes were determined by comparisons with nucleotide or amino acid sequences of other bony fish mitochondrial genomes. The 22 tRNA genes and their anticodon sequences were identified by their proposed cloverleaf secondary structures drawn by tRNAscan-SE software (Lowe and Eddy, 1997). The two ribosomal RNA (rRNA) genes and the control region were identified by sequence homology and proposed secondary structure (Gutell et al., 1993).

Phylogenetic analysis

To determine the phylogenetic position of P. orientalis and P. vittatus in the subfamily Plectorhynchinae, sequences of Cytb and COI genes of 21 ingroup species in Plectorhynchinae and 4 outgroup species in Haemulinae from GenBank were selected (Table I). individuals of certain species Besides, in order to examine the evolutionary status of Haemulidae of the phylogenetic relationships in Percoidei species, 12 protein-coding sequences of 42 species belonged to 25 families in the suborder Percoidei and one outgroup species Salarias fasciatus in suborder Blennioidei were also selected (Table II). Both datasets were aligned with Clustal X 1.85 (Thompson et al., 1994) with default gap penalties. The molecular phylogeny was analyzed by Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The Modeltest 3.7 software (Posada and Crandall, 1998) was used as a guide to determine the best-fit evolutionary model under the Akaike information criterion (AIC) (Posada and Buckley, 2004) and the GTR (general time reversible) model was chosen in the data computing.

 

Table I.- Species used for Percoidei phylogenetic analysis, along with GenBank accession numbers and reference.

Families	Species	GenBank No.	References
Percoidei
Serranidae	Epinephelus coioides	EU043376	Zhuang et al., 2009
	Anyperodon leucogrammicus	GQ131336	Unpublished
Carangidae	Carangoides armatus	AP004444	Miya et al., 2003
	Caranx melampygus	AP004445	Miya et al., 2003
	Seriola lalandi	AB517557	Iguchi et al., 2012
Latidae	Lates calcarifer	DQ010541	Lin et al., 2006
Toxotidae	Toxotes chatareus	AP006806	Yagishita et al., 2009
Haemulidae	Diagramma pictum	AP009167	Yamanoue et al., 2007
	Parapristipoma trilineatum	AP009168	Yamanoue et al., 2007
	Hapalogenys nigripinnis	HM754620	Liang et al., 2012
	Plectorhinchus orientalis		This study
	Plectorhinchus vittatus		This study
Centrarchidae	Micropterus salmoides	DQ536425	Broughton and Reneau, 2006
	Micropterus dolomieu	AB378750	Mukai and Sato, 2009
Emmelichthyidae	Emmelichthys struhsakeri	AP004446	Miya et al., 2003
Monodactylidae	Monodactylus argenteus	AP009169	Yamanoue et al., 2007
Oplegnathidae	Oplegnathus fasciatus	DQ872160	Oh et al., 2007
	Oplegnathus punctatus	AP011066	Yagishita et al., 2009
Kyphosidae	Labracoglossa argentiventris	AP011062	Yagishita et al., 2009
	Scorpis lineolata	AP011063	Yagishita et al., 2009
	Kyphosus cinerascens	AP011061	Yagishita et al., 2009
Acropomatidae	Doederleinia berycoides	AP009181	Yamanoue et al., 2007
Caesionidae	Pterocaesio tile	AP004447	Miya et al., 2003
Lutjanidae	Lutjanus malabaricus	FJ824741	Unpublished
	Lutjanus sebae	FJ824742	Unpublished
Terapontidae	Rhynchopelates oxyrhynchus	AP011064	Yagishita et al., 2009
Chaetodontidae	Chaetodon auripes	AP006004	Yamanoue et al., 2007
	Heniochus diphreutes	AP006005	Yamanoue et al., 2007
Sciaenidae	Larimichthys polyactis	GU586227	Cheng et al., 2012
	Sciaenops ocellatus	JQ286004	Cheng et al., 2012
Lethrinidae	Lethrinus obsoletus	AP009165	Yamanoue et al., 2007
	Monotaxis grandoculis	AP009166	Yamanoue et al., 2007
Kuhliidae	Kuhlia mugil	AP011065	Yagishita et al., 2009
Pomacanthidae	Chaetodontoplus septentrionalis	AP006007	Yamanoue et al., 2007
	Centropyge loricula	AP006006	Yamanoue et al., 2007
Sparidae	Pagrus major	AP002949	Miya et al., 2001
	Pagellus bogaraveo	AB305023	Ponce et al., 2008
	Acanthopagrus latus	EF506764	Xia et al., 2008
Centracanthidae	Spicara maena	AP009164	Yamanoue et al., 2007
Pseudochromidae	Labracinus cyclophthalmus	AP009125	Mabuchi et al., 2007
Moronidae	Morone saxatilis	HM447585	Unpublished
Lateolabracidae	Lateolabrax japonicus	JQ860109	Unpublished
Sillaginidae	Sillago sihama	JQ048935	Unpublished
Blennioidei
Blenniidae	Salarias fasciatus	AP004451	Miya et al., 2003

 

Table II.- Species used for Haemulidae phylogenetic analysis, along with GenBank accession numbers of COI and Cyt b gene sequences.

Species	GenBank Accession No.
	Cyt b	COI
Ingroup species
Plectorhinchus albovittatus	JX042230	JX042260
Plectorhinchus chaetodonoides	JX042231-JX042234	JX042261- JX042264
Plectorhinchus chubbi	JX042235	JX042293
Plectorhinchus cinctus	JX042236 -JX042238	JX042265- JX042267
Plectorhinchus diagrammus	JX042239- JX042241	JX042268- JX042270
Plectorhinchus flavomaculatus	JQ741559- JQ741561	JF494169- JF494171
Plectorhinchus gaterinus	JX042289- JX042290	JX042291- JX042292
Plectorhinchus gibbosus	JX042242- JX042243	JX042271- JX042272
Plectorhinchus lineatus	JX042244- JX042246	JX042273 -JX042275
Plectorhinchus macrolepis	HQ676733	HQ676788
Plectorhinchus orientalis	JX042247- JX042249	JX042276- JX042278
	JX042256	JX042285
Plectorhinchus picus	JX042250- JX042251	JX042279- JX042280
Plectorhinchus plagiodesmus	JX042252	JX042281
Plectorhinchus playfairi	JX042253	JX042282
Plectorhinchus polytaenia	JQ741565	JQ741334
Plectorhinchus schotaf	JX042254	JX042283
Plectorhinchus sordidus	JX042255	JX042284
Plectorhinchus vittatus	JX042257- JX042259	JX042286 -JX042288
Diagramma pictum	JX042214 -JX042217	JX042226- JX042229
Diagramma centurio	JX042212- JX042213	JX042224-JX042225
Parapristipoma trilineatum	JX042210 -JX042211	JX042222- JX042223
Outgroup species
Pomadasys maculatus	JX042209	JX042221
Haemulon aurolineatum	JX042208	JX042220
Anisotremus virginicus	JX042206	JX042218
Conodon nobilis	JX042207	JX042219

 

BI analysis was implemented in Mrbayes version 3.1.2 (Ronquist and Huelsenbeck, 2003), The MCMC (Markov Chain Monte Carlo) simulation was run for 10 million generations, with trees sampled and saved every 1000 generations (10,000 trees saved per run), and the bootstrap value of internal branch of the phylogenetic tree was supported by the posterior probabilities.

 

Results

Genome information

The complete mitochondrial genomes of P. orientalis and P. vittatus were sequenced as 16,546 bp and 16,545 bp in length, which have been submitted to GenBank database (Accession no. KP966562, KP976103). The structural organization and arrangement of both genomes consisted of 13 typical vertebrate protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and one putative control region (Fig. 1A, B; Table III). Overall base compositions of L-strand nucleotide sequences in the two mitochondrial genome were as follows, P. orientalis: A, 27.9%; G, 16.4%; T, 24.7%; and C, 31.0%, A+T=52.6%C+G=47.4%; P. vittatus: A, 28.0%; G, 16.4%; T, 24.7%; and C, 30.9%, A+T=52.7%C+G=47.3% (Table IV).

 

Table III.- Main charateristic of mitochondrial genome of P. orientalis and P. vittatus.

Gene	Stra-nd	P. orientalis			P. vittatus			Codon
		Position From -to	Nucle-otide Size /bp	Interg-enic nucle-otide	Position From-to	Nucle-otide Size/ bp	Interg-enic nucle-otide	Start	Stop
tRNAPhe	H	1-69	69	0	1-69	69	0
12S rRNA	H	70-1020	951	0	70-1021	952	0
tRNAval	H	1021-1091	71	0	1022-1092	71	0
16S rRNA	H	1092-2786	1695	0	1093-2783	1691	0
tRNALeu(UUR)	H	2787-2860	74	0	2784-2857	74	0
ND1	H	2861-3835	975	5	2858-3832	975	5	ATG/ ATG	TAA/ TAG
tRNAIle	H	3841-3910	70	-1	3838-3907	70	-1
tRNAGln	L	3910-3980	71	-1	3907-3977	71	-1
tRNAMet	H	3980-4049	70	0	3977-4046	70	0
ND2	H	4050-5096	1047	0	4047-5093	1047	0	ATG/ ATG	TAA/ TAA
tRNATrp	H	5097-5168	72	0	5094-5165	72	0
tRNAAla	L	5169-5237	69	1	5166-5234	69	1
tRNAAsn	L	5239-5311	73	40	5236-5308	73	39
tRNACys	L	5352-5418	67	-1	5348-5415	68	-1
tRNATyr	L	5418-5488	71	1	5415-5486	72	1
COI	H	5490-7049	1557	-13	5488-7050	1563	-13	GTG/ GTG	AGG/ AGG
tRNASer(UCN)	L	7037-7107	71	3	7038-7108	71	3
tRNAAsp	H	7111-7182	72	3	7112-7183	72	4
COII	H	7186-7876	691	0	7188-7878	691	0	ATG/ ATG	T--/ T--
tRNALys	H	7877-7951	75	1	7879-7953	75	1
ATPase8	H	7953-8120	168	-10	7955-8122	168	-10	ATG/ ATG	TAA/ TAA
ATPase6	H	8111-8793	683	0	8113-8795	683	0	ATG/ ATG	TA-/ TA-
COIII	H	8794-9578	785	0	8796-9580	785	0	ATG/ ATG	TA- /TA-
tRNAGly	H	9579-9650	72	0	9581-9652	72	0
ND3	H	9651-9999	349	0	9653-10001	349	0	ATG/ ATG	T--/ T--
tRNAArg	H	10000-10068	69	0	10002-10070	69	0
ND4L	H	10069-10365	297	-7	10071-10367	297	-7	ATG/ ATG	TAA/ TAA
ND4	H	10359-11739	1381	0	10361-11741	1381	0	ATG/ ATG	T--/ T--
tRNAHis	H	11740-11808	69	0	11742-11810	69	0
tRNASer(AGY)	H	11809-11875	67	4	11811-11877	67	4
tRNALeu(CUN)	H	11880-11952	73	0	11882-11954	73	0
ND5	H	11953-13791	1839	-4	11955-13793	1839	-4	ATG/ ATG	TAG/ TAA
ND6	L	13788-14309	522	0	13790-14311	522	0	ATG/ ATG	TAA/ TAA
tRNAGlu	L	14310-14378	69	4	14312-14380	69	4
Cytb	H	14383-15523	1141	0	14385-15525	1141	0	ATG/ ATG	T--/ T--
tRNAThr	H	15524-15595	72	33	15526-15597	72	29
tRNAPro	L	15629-15698	70	0	15627-15696	70	0
D-loop	H	15699-16546	848	0	15697-16545	849	0

 

Table IV.- Base composition of mtDNA in P. orientalis and P. vittatus.

	P. orientalis						P. vittatus
	T%	C%	A%	G%	A+T%	C+G%	T%	C%	A%	G%	A+T%	C+G%
Mitochondrial genome	24.7	31.0	27.9	16.4	52.6	47.4	24.7	30.9	28.0	16.4	52.7	47.3
13 protein-coding genes	26.5	32.4	25.2	15.9	51.7	48.3	26.5	32.3	25.4	15.8	51.9	48.1
1st	20.0	28.9	25.7	25.5	45.7	54.4	20.0	29.7	26.2	24.6	46.2	54.3
2nd	40.0	27.8	18.3	13.5	58.3	41.3	39.0	27.9	18.4	14.5	57.4	42.4
3rd	19.0	40.5	31.5	8.7	50.5	49.2	21.0	39.3	31.7	8.3	52.7	47.6
22 tRNAs	23.8	25.9	29.9	20.4	53.7	46.3	24.0	25.7	29.8	20.4	53.8	46.1
2 rRNAs	20.6	26.2	32.5	20.8	53.1	47.0	20.4	26.3	32.5	20.8	52.9	47.1
Control region	29.8	25.8	29.5	14.9	59.3	40.7	29.1	25.9	29.6	15.4	58.7	41.3

 

Protein-coding genes

The 13 protein-coding genes in the two sweetlips mtDNA were similar in length to their corresponding vertebrate counterparts. The total length of the protein-coding genes was 11,435 bp in P. orientalis and 11,441 bp in P. vittatus without introns, accounting for 69.12% and 69.15% of the complete genome. A6 bp differences of the protein-coding genes was found between the two species in gene COI, which was 1557 in P. orientalis and 1563 in P. vittatus (Table III). All protein-coding genes use ATG as a start codon except for COI, which started with GTG. A diverse pattern of codon usage was found within stop codons: six genes ended with TAA: ND1 (P. orientalis), ND2, ATPase8, ND4L, ND5 (P. vittatus) and ND6, two ended with TAG: ND1 (P. vittatus) and ND5 (P. orientalis), one ended with AGG: COI, the remaining genes had incomplete stop codons, TA (ATPase6, COIII) or T: COII, ND3, ND4 and Cyt b. Among the 13 protein-coding genes, three reading-frame overlaps were found: the pair of gene ATPase 8 and ATPase 6 overlapped by 10 nucleotides, ND4L and ND4 overlapped by 7 nucleotides, ND5 and ND6 overlapped by 4 nucleotides, no overlaps were between ATPase and COIII.

The base composition of the two sweetlips mitochondrial protein-coding genes is summarized in Table IV. The most frequent nucleotides at the first, second and third codon positions were C (28.9%, 29.7%), T (40.0%, 39.0%), C (40.5%, 39.3%) in P. orientalis and P. vittatus, respectively. The frequency of nucleotide G at the third codon position was relatively low (only 8.7%, 8.3%), showing a strong bias against G at this position. In addition, pyrimidines at the second codon position were over-represented (T+C=67.8%, 66.9%).

Codon usage in 13 protein-coding genes of the two sweetlips were analyzed and shown in Table V. The total number of codons for 20 amino acids were 3789 (P. orientalis) and 3791 (P. vittatus) excluding start and stop codons. The most frequent used amino acids are Leu (17.5%, 17.2%), followed by Ile (7.3%, 7.2%), Ser (6.4%, 6.5%), Gly (6.2%, 6.2%), and Val (5.7%, 5.5%). For amino acids with four-fold degenerate third positions, codon families ending in C were the most frequent, followed by codons ending in A. Among two-fold degenerate codons, C appeared to be used more than T in pyrimidine codon families, whereas purine codon families ended mostly with A.

Transfer RNA genes and ribosomal RNA genes

The two sweetlips mitochondrial genomes contain 22 tRNA genes, which are interspersed between rRNA and protein-coding genes, ranging from 67 (tRNACys, tRNASer(AGY)) to 76 (tRNALys(UUR)) nucleotides in length (Table III). Two forms of tRNA-Leu (UUR and CUN) and tRNA-Ser (UCN-AGY) in the two sweetlips were identified. The anticodons of 22 tRNA genes were shown in Table III. Two ribosomal RNA genes, the small subunit (12S) and the large subunit (16S) of rRNAs were 951bp and 1,695 bp in P. orientalis and 952bp and 1,691 bp in P. vittatus, respectively. The two ribosomal RNA genes were located between the genes of tRNA-Phe and tRNA-Leu, separated by the gene tRNA-Val.

Noncoding sequence

The light strand replication origin (OL) was comprised of 40 nucleotides in P. orientalis, and 39 nucleotides in P. vittatus, which could be folded into a stable stem-loop secondary structure containing 22 bp in the stem, 15 or 16 bp in the loop. The conserved motif of the two sweetlips were also 5’-GGCGG-3’ found at the base of the stem within the tRNA-Cys gene. The control region was located between the tRNAPro and tRNAPhe genes and determined to be 848 bp in P. orientalis and 849 bp in P. vittatus. The control regions of the two species were both heavily biased towards A+T nucleotides with A+T content 59.3% in P. orientalis and 58.7% in P. vittatus (Table III).

 

Table V.- Codon usage in P. orientalis and P. vittatus mitochondrial protein-coding genes.

Amino acid number	Codon	P. orientalis		P. vittatus		Amino acid number	Codon	P. orientalis		P. vittatus
		n	(%)	n	(%)			n	(%)	n	(%)
Phe	TTT	68	1.8	68	1.8	Tyr	TAT	33	0.9	41	1.1
	TTC	164	4.3	145	3.8		TAC	85	2.2	80	2.1
Leu	TTA	52	1.4	47	1.2	Stop	TAA	0	0	7	0.2
	TTG	21	0.6	26	0.7		TAG	1	0	0	0
	CTT	133	3.5	136	3.6	His	CAT	27	0.7	25	0.7
	CTC	194	5.1	194	5.1		CAC	79	2.1	84	2.2
	CTA	194	5.1	190	5	Gln	CAA	87	2.3	86	2.3
	CTG	69	1.8	59	1.6		CAG	13	0.3	14	0.4
Ile	ATT	123	3.2	120	3.2	Asn	AAT	24	0.6	40	1.1
	ATC	157	4.1	153	4		AAC	97	2.6	80	2.1
	ATA	91	2.4	95	2.5	Lys	AAA	67	1.8	69	1.8
Met	ATG	59	1.6	64	1.7		AAG	7	0.2	3	0.1
Val	GTT	46	1.2	51	1.3	Asp	GAT	20	0.5	19	0.5
	GTC	79	2.1	67	1.8		GAC	56	1.5	55	1.5
	GTA	64	1.7	63	1.7	Glu	GAA	79	2.1	78	2.1
	GTG	27	0.7	25	0.7		CAG	19	0.5	16	0.4
Ser	TCT	46	1.2	46	1.2	Cys	TGT	4	0.1	7	0.2
	TCC	72	1.9	70	1.8		TGC	19	0.5	22	0.6
	TCA	62	1.6	61	1.6	Stop	TGA	97	2.6	96	2.5
	TCG	6	0.2	6	0.2	Trp	TGG	23	0.6	18	0.5
Pro	CCT	58	1.5	74	2	Arg	CGT	8	0.2	15	0.4
	CCC	110	2.9	112	3		CGC	19	0.5	23	0.6
	CCA	49	1.3	52	1.4		CGA	43	1.1	42	1.1
	CCG	8	0.2	10	0.3		CGG	5	0.1	11	0.3
Thr	ACT	44	1.2	46	1.2	Ser	AGT	12	0.3	10	0.3
	ACC	123	3.2	124	3.3		AGC	45	1.2	53	1.4
	ACA	125	3.3	118	3.1	Stop	AGA	0	0	5	0.1
	ACG	13	0.3	14	0.4		AGG	0	0	11	0.3
Ala	GCT	65	1.7	57	1.5	Gly	GGT	22	0.6	30	0.8
	GCC	147	3.9	148	3.9		GGC	89	2.3	80	2.1
	GCA	102	2.7	103	2.7		GGA	80	2.1	88	2.3
	GCG	25	0.7	15	0.4		GGG	46	1.2	37	1

The incomplete T/TA of the stop codon is not included.

 

Sequence comparison between P. orientalis and P. vittatus

The comparison analysis of 16 genes between P. orientalis and P. vittatus were showed in Table VI. The complete mitogenome of the two sweetlips were 16,546 bp (P. orientalis) and 16,545 bp (P. vittatus) in length, but there were 941 variable sites (5.69%) in the two mitogenomes, the sequence divergence value was revealed to be 6.5% based on the Kimura-2 Parameter model. The differences of 13 protein-coding genes, 2 ribosomal RNA genes and one control region which are commonly used as molecular markers between the two species were also analyzed. That showed great genetic differences between the two sweetlips existed in all 16 genes. The divergence values based on K-2-P model between P. orientalis and P. vittatus ranged from 2.4% (12S rRNA) to 11.2% (ND6) with nucleotide mutation ranged from 6 bp to 132 bp, all divergence values were larger than 2%, even in the two ribosomal RNA genes (12 and 16 rRNAs) which retained very low evolutionary rate, revealing a significant genetic differences between the two sweetlips at molecular level.

 

Table VI.- Comparison analysis of 16 gene sequences between P. orientalis and P. vittatus.

	Length (P. orientalis)	Length(P. vittatus)	Number of variable sites (bp)	Bases variation (%)	Sequence divergence (%)
D-loop	848	849	83	9.78	10.6
12S rRNA	951	952	22	2.31	2.4
16S rRNA	1695	1691	45	2.66	2.7
COI	1557	1563	94	6.03	6.4
COII	691	691	23	3.33	3.4
COIII	785	785	41	5.22	5.5
ND1	975	975	76	7.79	8.4
ND2	1047	1047	91	8.69	9.4
ND3	349	349	26	7.49	8
ND4L	297	297	12	4.04	4.2
ND4	1381	1381	109	7.89	8.5
ND5	1839	1839	132	7.18	7.8
ND6	522	522	53	10.15	11.2
Cytb	1141	1141	82	5.69	7.7
ATPase6	683	683	40	5.86	6.2
ATPase8	168	168	6	3.57	3.7
Whole sequences	16546	16545	941	5.69	6.5

 

Phylogenetic analysis

Molecular phylogenetic trees of 21 sweetlips (Plectorhynchinae) were constructed using ML and BI methods based on combined Cyt b and COI sequences, the trees were well divided the sweetlilps into three groups with high bootstrap values and posterior probabilities (Fig. 2). Group one composed sweetlips with beautiful coloration pattern, group two and three composed sweetlips with simple patterns and dark appearances. Two sweetlips P. orientalis and P. vittatus were positioned in group one, clustering together as sister species and closely related to the cluster P. lineatus and P. diagrammus which also possessed black stripes along the body, indicating their closed relationships with each other. Another phylogenetic trees constructed from 42 Percoidei species of 25 families formed 5 large groups, Diagramma pictum, Parapristipoma trilineatum, P. orientalis and P. vittatus formed a monophyletic Haemulidae in group I, sister to the cluster Lethrinus obsoletus + Monotaxis grandoculis. The species Hapalogenys nigripinnis, which was a Haemulidae member morphologically, was separated from Haemulidae group. It was placed in group IV and clustered with Sillago sihama, revealing its distant relationship with Haemulidae species.

 

Discussion

Similar organization of two mitogenomes with other vertebrates

The two mitogenomes both consisted of 13 typical vertebrate protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and one putative control region, which were similar to other vertebrate mitogenomes. As in other vertebrate species (Miya et al., 2003; Oh et al., 2007; Ponce et al., 2008; Catanese et al., 2010), most genes were encoded on the H-strand except for one protein-coding gene ND6 and 8 tRNA genes (Gln, Ala, Asn, Cys, Tyr, SerUCN, Glu and Pro) which were encoded on the L-strand. As shown in Table IV, the contents of A+T of the two sweetlips were both higher than that of C+G, indicating a strong compositional base bias in the mitochondrial genome which was in accordance with the variation tendency of the A+T content in other vertebrates.

For 13 protein-coding genes (Table V), variances were observed in stop codons, which seemed to be a tendency in fish mitochondrial genomes (Miya and Nishida, 1999; Ponce et al., 2008; Catanese et al., 2010). The three reading-frame overlaps observed between protein coding genes showed different from the previously reported species that 1 nucleotide was overlapped between the two genes (Oh et al., 2007; Ponce et al., 2008; Catanese et al., 2010). As shown in Table IV, the base composition of the two sweetlips mitochondrial protein-coding genes showed a strong bias against G, which seemed due to the hydrophobic characteristic of the proteins (Naylor et al., 1995). These typical features of protein-coding genes were also found in most vertebrates (Oh et al., 2007; Catanese, 2010; Cheng et al., 2012a, b). These codon usage patterns in protein coding genes were in accordance with the overall bias against G, because G was the least common third position nucleotide in all codons except for glycine, in which G was more frequent than T. All these features were very similar to other bony fish (Miya et al., 2003; Ponce et al., 2008; Cheng et al., 2012a, b).

 

For tRNAs, They showed the typical arrangement in vertebrates. Most of them could be folded into the typical secondary structure-canonical cloverleaf, which were determined by the tRNAscan-SE software (Lowe and Eddy, 1997). The two ribosomal RNA genes were located between the genes of tRNA-Phe and tRNA-Leu, separated by the gene tRNA-Val, as in other vertebrates (Miya et al., 2001; Oh et al., 2007; Catanese et al., 2010). For noncoding sequences, the light strand replication origin (OL) in P.orientlais and P. vittatus was positioned in a cluster of five tRNA genes known as the WANCY region between the tRNAAsn and tRNACys genes as most of the vertebrates.

The differences of mitogenomes between P. orientalis and P. vittatus

Avise and Walker (1999) revealed divergence values between species of vertebrates in Cyt b were ordinarily greater than 2%. Hebert (2003a) and (2003b) studied the COI sequences of 13320 species from 11 phyla and suggested that the threshold of divergence values in COI for species diagnosis was 2%. Intraspecific divergences were rarely greater than 2% and most were less than 1% (Hebert, 2003b; Avise, 2000). In our study, sequence divergences were revealed in COI and Cyt b between the two sweetlips were 6.4% and 7.7%, respectively, largely greater than the 2% value suggested by Hebert. The other 14 genes in Table VI were also larger than the threshold 2% value, even in the two highly conserved ribosomal RNA genes. In our previous studies, we had investigated the phylogenetic relationship of 17 sweetlips involved 3 individuals of P. orientalis and 3 individuals of P. vittatus based on Cyt b and COI genes (Liang et al., 2016). The result revealed that sequence divergences in each individual sample of P. orientalis and P. vittatus were all less than 0.5% in the two genes. The study also discovered that the divergence value between P. orientalis and P. vittatus was larger than some inter-sweetlips species values like Plectorhinchus chubbi vs Plectorhinchus sordidus (COI: 3.6%; Cytb: 3.4%), Plectorhinchus chubby vs Plectorhinchus plafairi (COI: 4.8%; Cytb: 5.0%) and Plectorhinchus sordidus vs Plectorhinchus plafairi (COI: 5.7%; Cytb: 4.5%). All these pieces of evidences indicated that great sequence variation and molecular differentiation were existed in the two sweetlips, suggested that they might be considered as two separate species.

Phylogenetic analysis of P. orientalis and P. vittatus

Phylogenetic trees constructed from 21 Haemulidae species based on the the combined sequences of Cyt b, COI were well divided the sweetlilps into three groups (Fig. 2). Group one composed sweetlips with beautiful coloration pattern, group two and three composed sweetlips with simple patterns and dark appearances, which was in accordance with traditional morphological classifications (McKay, 1984, 2001). The two sweetlips P. orientalis and P. vittatus were positioned in group one, clustering together as sister species and closely related to the cluster P. lineatus and P. diagrammus, which was consistent with the molecular phylogeny of Haemulidae species based on 16S rRNA and TMO-4C4. By morphological diagnosis, they all had black stripes and bands on their bodies. In the phylogenetic trees, the long branch clearly distinguished the separated evolutionary positions of the two orientalis sweetlips. Along with the great genetic differences result of sequence comparison analysis on 16 molecular marker genes above, we suggested that P. orientalis and P. vittatus might be placed as two distinct species and were not synonym.

The phylogenetic position of Haemulidae among the Percoidei was also examined in this study (Fig. 3). The tree revealed 4 Haemulidae species Diagramma pictum, Parapristipoma trilineatum, P. orientalis and P. vittatus formed a monophyletic group except for Hapalogenys nigripinnis, which was placed in an independent position out of the Haemulidae clusters. Such finding was not congruent with the traditional morphological studies (Shen, 1993; McKay, 2001; Nelson, 2006) but agreed with some other morphology and molecule-based studies which suggested that the genus Hapalogenys were removed from Haemulidae (Springer and Raasch, 1995; Leis and Carson-Ewart, 2000; Froese and Pauly, 2014). Recent phylogenetic analysis on the relationships of Hapalogenys and Haemulidae also revealed a distant relationship between them (Ren and Zhang, 2007; Sanciangco et al., 2011). Our result was in agreement with those molecular reports, indicating the fundamental status of Hapalogenys in the Percoidei may need to be redefined. The family Haemulidae was grouped with family Lethrinidae as sister lineage and was closely related to families Lutjanidae, Caesionidae, Emmelichthyidae and Monodactylidae. Traditionally, potential sister groups to Haemulidae as well as its placement among Percoidei were uncertain either in morphological or in molecular studies (Johnson, 1981; Sanciangco et al., 2011; Tavera et al., 2012). Recent studies on higher-level relationships of percomorphs and acanthomorphs have shown potential outgroups for Haemulidae on the basis of molecular characters but the relationships and placement of Haemulidae varied through time according to different authors (Dettai and Lecointre, 2005; Chen et al., 2007; Craig and Hastings, 2007; Smith and Craig, 2007; Li et al., 2009). The current studies on Haemulidae relationships by Tavera et al. (2012) have revealed Sillaginidae sister clade to Haemulidae, but the support values were relatively low. Our molecular phylogenetic study on the placement of Haemulidae among Percoidei included the previously suggested potential Haemulidae-close families. The result recovered Haemulidae sister to Lethrinidae and was placed in a cluster comprised of families Lutjanidae, Caesionidae, Emmelichthyidae and Monodactylidae, which were consistent with the studies of Tavera et al. (2012). However, the Percoidei is the largest suborder of the Perciformes and the sequences of most Percoidei species are not available. A complete mitochondrial genomes are needed to be sequenced for further detailed analysis of the evolutionary relationships of Haemulidae within Percoidei species.

 

Conclusions

The mitochondrial genome of two synonyms debated species P. orientalis and P. vittatus were first determined in this study. The lengths of the two mitogenome are 16,546 bp and 16,545 bp, which both consist of 13 typical vertebrate protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and one putative control region. Genomic composition, organization, and gene order of the two genomes are similar to that obtained in most vertebrates. All sequence divergences of 13 protein-coding genes, 2 rRNA genes and one control region which are commonly used as molecular markers between the two species are larger than the species diagnosis divergence value 2%. And divergence values of Cyt b (6.0%) and COI (7.4%) are also largely greater than 2%. Furthermore, molecular phylogenetic trees also clearly distinguish the independent position of the two species. These results above reveal great genetic differences between P. orientalis and P. vittauts, and strongly suggest that they are two distinct species and should not be placed as synonyms.

 

Acknowledgments

The study was supported by the Natural Science Foundation of Guangdong Province, China (2016A030310236) and Foundation for Young Talents in Higher Education of Guangdong, China (2014KQNCX164). We wish to thank Bernard Mackenzie (SAIAB South African Institute for Aquatic Biodiversity, Grahamstown, South Africa) for his kind assistance in providing samples of P. vittatus. We would also like to thank the College of Animal Science in South China Agricultural University for their laboratory facilities and experimental support to this study.

 

Statement of conflict of interest

Authors declare that they have no competing interests.

 

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