Molecular Phylogeny of Parapenaeopsis Alcock,1901(Decapoda: Penaeidae) Based on Chinese Materials and 16S rDNA and COI Sequence

LI Xinzheng,XU Yanand KOU Qi

1) Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P.R.China

2) University of Chinese Academy of Sciences, Beijing 100039, P.R.China

1 Introduction

The genusParapenaeopsisAlcock,1901 (Decapoda,Penaeidae) is a common penaeid genus in coastal water of tropical and subtropical seas with moderate body size.There have been 21 described species (Sakai and Shinomiya,2011),and all of them are commercially important.Some of them,such asP.hardwickii(Miers,1878),P.cultrirostrisAlcock,1906 andP.tenella(Bate,1888),are high-yield economic fishery species in China.

In accordance with Liu and Wang (1987),there have been sevenParapenaeopsisspecies reported from the Chinese coastal shallow water.They areP.cornuta(Kishinouye,1900),P.cultrirostrisAlcock,1906,P.hardwickii(Miers,1878),P.hungerfordiAlcock,1905,P.incisaLiu and Wang,1987,P.sinicaLiu and Wang,1987,andP.tenella(Bate,1888).Because of the similarity among these species and the infraspecific variation in morphology,it is difficult to distinguish these species exactly.For example,the male ofP.hardwickiiandP.cultrirostrisis easily distinguishable by their length and form of their rostra,but the female of these two species is very hard to be identified.Usually the female is classified according to the accompanying male.The complex ofP.cornuta,P.sinicaandP.incisais another difficult problem in identification.They are very similar in morphology,which makes it very difficult to correctly distinguish from each other by the traditional morphological way.Therefore,the systematic position of the above five species is questionable and a reliable identification method is necessary.

The phylogenetic research of decapod crustaceans by comparing various gene sequences has been widely used for species identification (Martinet al.,2009; Chuet al.,2009; Toonet al.,2009; Kouet al.,2013).The mitochondrial large subunit ribosomal RNA (16S rDNA) and cytochrome C oxidase subunit I (COI) are often employed in such studies.Chanet al.(2008) studied the phylogenetic relationship among the genera of the Penaeidae based on 16S rDNA sequence.Wetzeret al.(2009) studied the evolutionary origin of the gall crabs(Family Cryptochiridae) based on 16S rDNA sequence.Maet al.(2009) studied the phylogenetics of bacteria from diseasedApostichopus japonicas(Holothuroidea,Echinodermata) based on 16S rDNA sequence.Zhanget al.(2012) studied the diversity and bioactivity of actinomycetes from the marine sediments of the Yellow Sea based on 16S rDNA sequence.In order to exactly identify theParapenaeopsisspecies in the Chinese coastal shallow waters,we analyzed the molecular phylogenetic relationship among the sevenParapenaeopsisspecies from Chinese waters by comparing their 16S rDNA and COI sequences.

2 Materials and Methods

2.1 Samples

The materials used in the present study are listed in Table 1,including seven species,24 individuals from different areas in Chinese waters,which have been deposited in the Marine Biological Museum,Chinese Academy of Sciences (MBMCAS) in the Institute of Oceanology,Chinese Academy of Sciences (IOCAS),Qingdao,preserved in 75% ethanol.Penaeus monodonFabricius 1798 with the register number NC-oo2184 in GenBank was selected as outgroup.

Table 1 Data of specimens used in the present study

2.2 DNA Extraction,PCR Amplification and Sequencing

Muscle of 30–100 mg of each specimen was taken from the pleopods,or tail fan,or posterior pleomere,followed by rinsing in 80%,90% and 100% ethanol sequentially for two hours.After completely dehydrated,the total DNA of each specimen was isolated by phenol-chloroform extraction (Kocheret al.,1989).Then the DNA extract was dissolved in 40–100 μL of ddH2O.

Partial segments of 16S rDNA and COI gene were amplified by polymerase chain reaction (PCR).All 16S rDNA fragments were amplified with primer 16SAR(5’–GCC TGT TTA TCA AAA ACA T–3’) and primer 16SBR (5’–CCG GTC TGA ACT CAG ATC ACG T–3’)(Palumbi,1996).All COI fragments were amplified with primer HCO2198 (5’–GGT CAA CAA ATC ATA AAG ATA TTG G–3’) and primer LCO1490(5’–TAA ACT TCA GGG TGA CCA AAA AAT CA–3’ ) (Folmeret al.,1994).The reaction was carried aout in a 25 μL volume contained 100 ng DNA,2.5 μL 10× buffer,1.5 μL 25 mmol L?1MgCl2,0.5 μL each 10 μmol L?1primer,2.5 μL 10 mmol L?1dNTP,1 unitTaqDNA polymerase (the TaKaRa Biotechnology Co.,Ltd.,Dalian,China) and ddH2O.The reaction was thermocycled by denaturing at 94℃ for 90 s;followed by 33–35 cycles of denaturingat 94 ℃ for 30s,annealing at 46℃ for 30s (16S rDNA) or 52 ℃ for 30s(COI) and extending at 72 ℃ for 60s; and an extra extension at 72 ℃ for 10min.The PCR product was detected by 1.2% agarose gel electrophoresis,and then was purified and sequenced by Invitrogen Co.,Ltd.,Shanghai,China.

2.3 Data Processing

The sequences were aligned by the CLUSTAL X 1.81(Thompsonet al.,1997).Then the number of variable sites,the parsimony information,the nucleotide frequencies,Tamura-Nei distance (Tamura and Nei,1993) and R value (Transition/Transversion ratio) were calculated with MEGA 3.0 (Kumaret al.,2004).The phylogenetic trees were constructed using Maximum likelihood (ML) analysis with PhyML 3.0 (Guindonet al.,2010) and Bayesian inference (BI) analysis with Beast v1.6.1 (Drummondet al.,2012).The best-fit models of DNA substitution for ML and BI analysis were selected by ModelTest version 3.7(Posada and Crandall,1998).

For ML analysis,the confidence level at each branch was estimated by 1000 bootstrap replicates (BP).For BI analysis,the Markov chains were run for 10000000 generations,with sampling every 1000 generations.After the first 25% trees were discarded as burn-in,the remaining trees were used to construct the 50% majority rule consensus tree and estimate the posterior probabilities (PP).

3 Results

3.1 Phylogenetic Relationship Between Parapenaeopsis cultrirostris and P.hardwickii

The lengths of rostrum and carapace of three male individuals ofParapenaeopsis cultrirostris(the rostrum is short,coutel-like,Fig.1A),three male individuals ofP.hardwickii(the rostrum is long,saber-like,Fig.1B) and six female individuals (their rostra are saber-like,Fig.1C,D) of these two species were measured.The results (Table 2) showed that the carapace length (CL) of the three males ofP.hardwickiiwas shorter than their rostral length (RL),and the mean CL/RL (ratio of CL to RL) was 0.75 ± 0.02; the CL of the three males ofP.cultrirostriswas longer than their RL,and the mean CL/RL was 1.79 ±0.10; the CL of the six female individuals of the two species was longer than their RL,and the mean of CL/RL was 0.85 ± 0.12.The rostral form of the female specimens was like that of maleP.hardwickii,but the CL/RL was like that of maleP.cultrirostris.The CL/RL ratios of the males and the females were not correlative with their body sizes.

Fig.1 Anterior part of Parapenaeopsis cultrirostris and P.hardwickii showing different rostral types.A,male of Parapenaeopsis cultrirostris (CULT-M-3); B,male of Parapenaeopsis hardwickii (HARD-M-2); C,female (H-C-F-5,same collection data with CULT-M-3); D,female (H-C-F-2,same collection data with HARD-M-2).

Table 2 The carapace length (CL),rostral length (RL) and CL/RL of the individuals of Parapenaeopsis hardwickii and P.cultrirostris

By aligning 16S rDNA and COI sequence of three male individuals ofParapenaeopsis cultrirostris,two male individuals ofP.hardwickii,six female individuals of the two forms,460 bp for 16S rDNA (Fig.2) and 625 bp for COI (Fig.3) alignments were obtained,respectively.Among the 11 specimens,no variable nucleotide site was found in 16S rDNA,and only two variable nucleotide sites were found in COI gene (variation rate is 0.32%),Both variable nucleotide sites in the COI gene fragment were in the second code,causing non-synonymous mutation: Y→F (The site 370 of the individuals CULT-M-1 and CULT-M-2,A→T) and E→G (the site 592 of the individuals CULT-M-1 and HARD-M-1,A→G).

The results of sequence analysis of 16S rDNA and COI indicated thatParapenaeopsis cultrirostrisandP.hardwickiishould belong to the same species.Because the CL/RL ratios are not correlative with the body size of male specimens,the short and long rostra are not correlative with growth phase of the shrimps.In fact,there was not distinct difference in body size between the two forms when they were collected in the same trawling.So,we cognized that the species has dimorphic rostra in males:long and saber-like,or short and coutel-like.

Fig.2 Alignment of partial sequences of 16S rDNA of 11 specimens of Parapenaeopsis cultrirostris and P.hardwickii.‘·’means identical sites to those of CULT-M-1,‘-’ means gaps introduced for optimizing the alignments.

Fig.3 Alignment of partial sequences of COI of 11 specimens of Parapenaeopsis cultrirostris and P.hardwickii.‘·’means identical sites to those of CULT-M-1,‘-’ means gaps introduced for optimizing the alignments.

3.2 Phylogenetic Relationship Among Parapenaeopsis sinica,P.cornuta and P.incisa

Fig.4 Alignment of partial sequences of 16S rDNA of nine specimens of Parapenaeopsis sinica (‘sin03233’,‘sin03235’,‘sin03236’),P.cornuta (‘cor12303’,‘cor12304’,‘cor12305’) and P.incisa (‘inc03295’,‘inc12306’,‘inc12307’).‘·’means identical sites to those of ‘sin03233’,‘-’ means gaps introduced for optimizing the alignments.

By aligning 16S rDNA and COI sequence of nine individuals ofP.sinica,P.cornutaandP.incisa(three individuals per species) with CLUSTALX 1.8,472 bp for 16S rDNA and 602 bp for COI alignments were obtained,respectively (Figs.4,5).Only one intra-species variable nucleotide site in 16S rDNA fragments was found in specimens ofP.incisa,i.e.,A→G (‘inc03295’→ ‘inc12307’) at site 285.No variable nucleotide site in COI alignment fragments was found in specimens of the same species.

Fig.5 Alignment of partial sequences of COI gene of nine specimens of Parapenaeopsis sinica (‘sin03233’,‘sin03235’,‘sin03236’),P.cornuta (‘cor12303’,‘cor12304’,‘cor12305’) and P.incisa (‘inc03295’,‘inc12306’,‘inc12307’).‘·’means identical sites to those of ‘sin03233’.

Many variable nucleotide sites were found among the specimens of different species in the three species.All the nucleotide variations happened at the first and the third code positions and caused synonymous mutation.The nucleic acid sequence divergence rates of the alignment fragments of 16S rDNA and COI amongP.sinica,P.cornutaandP.incisaare shown in Table 2.In 16S rDNA alignments,28,23,and 28 variable nucleotide sites were observed between specimens ofP.sinicaandP.cornuta,P.cornutaandP.incise,andP.sinicaandP.incisa,respecitively.The nucleotide site variation rates were 5.82%,4.78% and 5.82%,respectively.In COI alignments,there were 71 (9 in first code,62 in third code),75(3 in first code,72 in third code),and 91 (8 in first code,83 in third code) variable nucleotide sites between specimens ofP.sinicaandP.cornuta,P.cornutaandP.incise,andP.sinicaandP.incisa,respecitively.The nucleotide site variation rates were 11.79%,12.46% and 15.12%,respectively.The distinct nucleotide site variation rates were 4.78%–5.82% in 16S rDNA,11.79%–15.12% in COI gene sequences among the three species.Therefore,the molecular analysis results indicateP.sinica,P.cornutaandP.incisaare different species.

3.3 Phylogenetic Relationship Among Chinese Parapenaeopsis Species

There have been sevenParapenaeopsisspecies reported from Chinese waters.Because the nucleotide site variations of alignments of 16S rDNA and COI gene were approximately 0% among conspecific specimens (Table 1),we chose the partial sequences of 16S rDNA and COI gene of one specimen from one species.BecauseParapenaeopsis cultrirostrisandP.hardwickiiwere supposed to be the same species (see above 3.1 and 3.2),we chose CULT-M-1 as the representative of these two forms.The specimens of ChineseParapenaeopsisspecies used in the phylogenetic relationship study were:P.cornuta–cor12303,P.cultrirostrisandP.hardwickii–CULT-M-1,P.hungerfordi–hun03298,P.incisa–inc03295,P.sinica–03233,P.tenella–ten03231.

Fig.616S rDNA partial sequences alignment of individuals of Chinese Parapenaeopsis species.‘·’ means identical sites to those of ‘sin03233’,‘-’ means gaps introduced for optimizing the alignments.

Fig.7 Alignment of partial sequences of COI of individuals of Chinese Parapanaeopsis species.‘·’ means identical sites to those of‘sin03233’.

By aligning 16S rDNA and COI gene sequence of the specimens of the above six ChineseParapenaeopsisspecies and the outgroupPenaeus monodon,481bp for 16S rDNA and 602 bp for COI alignment was obtained,respectively (Figs.6,7).The result of aligning partial protein sequences of COI is shown in Fig.8.

The variation rates of variable nucleotide sites of partial sequence of 16S rDNA among the studied individuals of ChineseParapenaeopsisspecies are shown in Table 3.The genetic distances and transition/transversion among the studied shrimp species based on partial sequence of 16S rDNA of the studied individuals are shown in Table 4.

Table 3 Variation rate (%) of partial sequence of 16S rDNA among the studied individuals of Parapenaeopsis species

Table 4 Genetic distance (below the diagonal) and transition/transversion (above the diagonal) among Chinese Parapenaeopsis species and Penaeus monodon (outgroup) based on partial sequences of 16S rDNA

Fig.8 Alignment of partial protein sequence of COI of individuals of Chinese Parapanaeopsis species.‘·’ means identical sites to those of ‘sin03233’.

In 481 bp of the aligned 16S rDNA sequence of the sixParapenaeopsisforms,there were 95 variable sites,of which 50 were parsimony-informative.The average base frequencies were 31.1% A,35.8% T,12.0% C,and 35.8%G (A%+T%=66.9%),a moderate AT bias which is consistent with the previous reports for AT rich 16S rDNA sequence in crustaceans (e.g.,Tamet al.,1996; Tam and Kornfield,1998).The lowest sequence divergence of 4.78% was found betweenP.incisaandP.cornuta.The 12.06% divergence betweenP.hungerfordiandP.cornutawas the highest among the studied species.The average nucleotide divergence rate of 16S rDNA among conspecific individuals was 0.02%,while the average rate among the studied species of the genus was 9.40%.The pattern of nucleotide substitutions evidently favored transition over transversion,and the average ratio of transition/transversion (Rvalue) was 2.8 in the genus.

The variation rates of variable nucleotide sites of partial sequence of COI gene and variable amino acid sites of partial protein sequence of COI among the studied individuals of ChineseParapenaeopsisspecies are shown in Table 5.The genetic distances (D) and transition/transversion (R) among the studied species based on partial sequence of COI gene of the studied individuals are shown in Table 6.The nucleotide frequencies of partial sequences of COI gene of the studied individuals of ChineseParapenaeopsisspecies are shown in Table 7.The frequences of nucleotide pairs of partial sequences of COI gene of the studied individuals of ChineseParapenaeop-sisspecies are shown in Table 8.

Table 5 Variation rates of partial sequence of COI gene (below the diagonal) and variation rates of partial protein sequence of COI (above the diagonal) among the studied individuals of Chinese Parapenaeopsis species (%)

Table 6 Genetic distances (below the diagonal) and transition/transversion (above the diagonal) among Chinese Parapenaeopsis species and Penaeus monodon (outgroup) based on partial sequences of COI gene

Table 7 Nucleotide frequencies of partial sequences of COI gene of the studied individuals of Chinese Parapenaeopsis species (%)

Table 8 Frequences of nucleotide pairs of partial sequences of COI gene of the studied individuals of Chinese Parapenaeopsis species

In 602 bp of partial sequence of COI gene,there were 181 variable nucleotide sites which evenly distributed over the sequence.Of these variable sites,109 were parsimony-informative.In the 181 variable nucleotide sites,23 distribute at the position of the first code,one at the second code,157 at the third code.The base composition was also AT rich (mean values 28.0% A,32.1% T,21.6%C,and 18.3% G; A+T=60.1%).The bias was the highest at position of the third code,with mean values of 43.6 %A,33.9% T,18.0% C,4.5% G.The result of an underrepresentation of G at the third code was similar to previous reports in insects such asDrosophila(Gleasonet al.,1997) and crustaceans (e.g.,Metapenaeopsis,Tonget al.,2000).

The pattern of nucleotide substitutions of COI gene also favored transitions over transversions with mean R value 1.4 and the R values of three codes were distinct.The R value of the first code was up to 17.0 (very strong bias),while that of the third code was just 1.2.Although the nucleotide divergences of COI gene varied from 11.79% to 18.60% among the studied species,there were only two variable sites (divergence rate=0.37%) in amino acid sequences among the six forms.So,only 1.10% nucleotide mutations were non-synonymous mutations,including CULT-M-1,No.260 of second code,nucleotide mutation: C→T (amino acid: S→L),and ‘hun03298’,‘ten03231’ and CULT-M-1,No.448 of first code,nucleotide mutation: T→A (amino acid: S→T).The other 98.90% mutations were synonymous mutations,while 22 synonymous mutations at the first code were caused by transversions between C and T and all corresponding amino acids were L.The average nucleotide divergence rate of COI gene among the studied conspecific individuals was 0%.The average rate among the studied species of the genus was 16.21%,including 5.54% at the first code; none at the second code,43.23% at the third code.

The best-fit model of the 16S rDNA and COI dataset suggested by ModelTest 3.7 was TVM+I and GTR+I+G,respectively.As the tree topologies derived from the ML and BI analyses are congruent,only the phylogenetic tree resolved by Bayesian Inference analysis are shown(Figs.9,10).

The 16S rDNA and COI phylogenetic trees show thatP.cornuta,P.incisaandP.sinicaform a branch,which is also supported by both maximum likelihood and Bayesian inference analyses (PP = 1.00,BP = 100%; PP = 1.00,BP =90%).Within this branch,P.cornutaandP.incisaalways cluster together with moderate to high supports (PP = 0.98,BP = 78%; PP = 0.74,BP = 88%).P.tenella,P.cultrirostrisandP.hungerfordiform another branch.However,this branch is not well supported (PP = 0.93,BP = 42%; PP= 0.74,BP = 68%) and the internal relationship is not stable.

Fig.9 Phylogenetic tree obtained by Bayesian inference analysis of Chinese Parapenaeopsis species based on partial sequences of 16S rDNA gene.

Fig.10 Phylogenetic tree obtained by Bayesian inference analysis of Chinese Parapenaeopsis species based on partial sequences of COI gene.

4 Discussion

4.1 Systematic Positions of Parapenaeopsis hardwickii Miers 1878 and P.cultrirostris Alcock 1906

The present study proved thatParapenaeopsis cultrirostrisandP.hardwickiifound in Chinese waters are the same species with dimorphic rostrum in males.If this situation also occurs in the area out of Chinese waters,the two forms can be cognized as the same species conclusively.In that case,according to the ‘International Code of Zoological Nomenclature’ (1999),the valid name of the species will beParapenaeopsis hardwickiiMiers 1878 (originallyPenaeus hardwickiiMiers 1878),and theParapenaeopsis cultrirostrisAlcock 1906 (originallyParapenaeopsis aculptilisvar.cultrirostrisAlcock 1906)will be a junior and invalid synonym of thePenaeus hardwickiiMiers 1878.More specimens,especially those out of Chinese waters need to be verified before this conclusion could be confirmed.

4.2 Systematic Positions of Parapenaeopsis sinica,P.cornuta and P.incisa

The nucleotide site variation rate of the aligned sequences of 16S rDNA and COI gene amongParapenaeopsis sinica,P.cornutaandP.incisavaried between 4.78% and 5.82% and between 11.79% and 15.12% respectively,much higher than the intraspecific mean variation rate ofParapenaeopsis(0.05% in partial sequence of 16S rDNA,0% in partial sequence of COI gene),but are at the same level of the interspecific variation rate of the genus (see Tables 3 and 5).Thus these three species are different species.The mean variation rate of 16S rDNA and COI gene among the six studied species ofParapenaeopsiswas 9.42% and 16.21%,respectively,which are a little higher than those in the three species.This indicated that the phylogenetic relationship among these three species is closer than that among other species of the genus.This is also identical to the morphological situation as they belong to the same species complex.Additionally,their close relationship is also supported by the present phylogenetic analyses.They group together and occupy the highest clade,implying they are advanced species in the genus.

Simonet al.(1994) proved that there were multiple substitutions and sequence saturation among the distantly relative species.According to their observation,the saturation degree of substitutions at every site since the divergence of the two sequences can be measured by transition/transversion ratio (Rvalue).When the ratio of the recent divergent sequences is higher than 2,the sequence substitutions are far from the saturation state.As the ratio descends and the saturation state elevates,the multiple substitutions happens more frequently.So the transition/transversion ratio among the closely related species is higher than that among the distantly relative species.The previous observations in arthropod mitochondrial DNA showed a strong bias for transitional substitutions between closely related species (Gleasonet al.,1997;Chuet al.,2003).TheRvalues of both 16S rDNA and COI gene amongP.sinica,P.cornutaandP.incisaare evidently higher than those among otherParapenaeopsisspecies.Additionally,theRvalues betweenP.cornutaandP.incisais the highest among the three species,im-plying that these two species are the closest in the genus.

4.3 Phylogenetic Relationship Among Chinese Parapenaeopsis species

Recently,Sakai and Shinomiya (2011) divided the original genusParapenaeopsisinto 8 genera,mainly basing on the various structures of the male petasma,viz.Alcockpenaeopsis,Arafurapenaeopsis,Batepenaeopsis,Ganjampenaeopsis,Holthuispenaeopsis,Kishinouyepenaeopsis,MierspenaeopsisandParapenaeopsis.According to the new classification,P.hardwickiiandP.cultrirostrisbelong to the genusMierspenaeopsis,P.sinica,P.cornutaandP.inciseare included in the genusKishinouyepenaeopsis,whileP.hungerfordiandP.tenellaare transformed toAlcockpenaeopsisandBatepenaeopsis,respectively.Although the classification ofParapenaeopsisis not definite,our analysis supported this radical separation to a certain extent.In the present phylogenetic trees of the six ChineseParapenaeopsisspecies,P.sinica,P.cornutaandP.inciseoccupy an independent and advanced branch,revealing a close and stable relationship among them.Hence it is reasonable to assign these three species to the same genus.In contrast,the other three species form another branch,the internal topologies are unstable and the supports are not high.This might suggest thatP.tenella,P.cultrirostrisandP.hungerfordihave relatively remote relationships between each other.So they may be classified into different genera.

Although 16S rDNA and COI gene are well-known genetic markers for analyzing the phylogenetic relationship of congeneric species,they still have obvious deficiency in the phylogenetic analysis of the genusParapenaeopsis.Sometimes the accurate systematic positions cannot be determined only by these two genes,which have been found in caridean molecular phylogenetic analysis (Xuet al.,2005).Therefore,more genetic markers such as 18S rRNA and 28S rRNA gene are supposed to be used for further study of the phylogenic relationship in genusParapenaeopsis.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos.31071889 and 30499340).We sincerely thank Prof.Linsheng Song (IOCAS) and his group for the guidance and support during the experimental study.We are grateful to the managers of the Marine Biological Museum in the IOCAS for helping us sort the samples.Thanks are also due to the anonymous reviewers for their invaluable comments to the manuscript.

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