Relaciones filogenéticas de algunos géneros de la familia Lutjanidae inferidas a partir de regiones mitocondriales, con una nota sobre la taxonomía de Pinjalo pinjalo

Cecilia Chu¹, Mohammed Rizman-Idid^1,2*, Chong Ving Ching^1,2

¹ Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Lembah Pantai, Kuala Lumpur, Malaysia.

² Institute of Ocean and Earth Sciences, University of Malaya, 50603 Lembah Pantai, Kuala Lumpur, Malaysia. * Corresponding author. Email: rizman@um.edu.my.

Received March 2013, ]]> received in revised form July 2013,
accepted October 2013.

ABSTRACT

Phylogenetic relationships of 43 species in 11 genera, representing four subfamilies of the family Lutjanidae and two genera of the family Caesionidae, were inferred using mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI). Further assessment using the mtDNA control region (CR) was carried out to infer the relationship between the Indian and western Pacific types of Lutjanus russellii collected from the coast of Peninsular Malaysia. A total of 11 and 12 species were sequenced for COI and CR genes, respectively. Clade formation reflects, to some extent, the species groupings based on morphological characteristics and their biogeography. The close phylogenetic relationship between Pinjalo pinjalo and the Lutjanus red snappers (Lutjanus malabaricus and Lutjanus sebae) warrants a taxonomic revision of the former as the two genera are currently separated based on non-exclusive morphological characters. A sequence divergence of 4.2% between the Indian and western Pacific types of L. russellii as well as the morphological and biogeographical differences may suggest two separate species.

Key words: Lutjanus, Pinjalo, COI, barcoding, taxonomic revision.

RESUMEN

Las relaciones filogenéticas de 43 especies pertenecientes a 11 géneros, representando cuatro subfamilias de la familia Lutjanidae y dos géneros de la familia Caesionidae, fueron inferidas a partir del gen mitocondrial citocromo c oxidasa subunidad I (COI). Además, se usó la región control (RC) del ADN mitocondrial para inferir la filogenia entre los tipos de Lutjanus russellii del océano Índico y el Pacífico occidental recolectados en las costas de la península de Malasia. Se generaron secuencias de COI (11 especies) y RC (12 especies). Los clados generados concuerdan con las agrupaciones propuestas anteriormente basadas en la morfología y biogeografía de las especies. La relación filogenética cercana entre Pinjalo pinjalo y los pargos rojos Lutjanus malabaricus y Lutjanus sebae justifica la revisión taxonómica de los mismos ya que los caracteres morfológicos empleados para diferenciar ambos géneros son ambiguos. La divergencia de las secuencias (4.2%) entre los dos tipos de L. russellii (Índico y Pacifico occidental), así como la caracterización morfológica y la información biogeográfica permiten sugerir que son dos especies distintas.

Palabras clave: Lutjanus, Pinjalo, COI, código de barras genético, revisión taxonómica.

INTRODUCTION

About 112 species of snappers (family Lutjanidae) are reported to exist in the warm seas of the Indian Ocean and the tropical and subtropical parts of the western Pacific Ocean (Masuda 1984). The largest species diversity occurs in the genus Lutjanus, with 64 out of a total of 72 species found in the subfamily Lutjaninae (Anderson and Allen 2001). Lutjaninae is composed of the genera Lutjanus, Pinjalo, Macolor, Ocyurus, and Rhomboplites, the last two being monotypic genera found only in western Atlantic waters (Allen 1985). Malaysian snappers are represented by 38 species in 10 genera, which include Lutjanus, Pinjalo, and Macolor (Chong et al. 2010).

Previously, Johnson (1980) suggested monophyly of Lutjanus species based on their primitive and derivative morphological characters, but recent DNA sequence based phylogenies have rendered the genus Lutjanus as paraphyletic due to the phylogenetic associations of some Lutjanus species with other genera of the families Lutjanidae and Caesionidae (Chow and Walsh 1992, Miller and Cribb 2007, Gold et al. 2011).

In the present study, we hypothesize that the genus Pinjalo is related to Lutjanus, specifically Pinjalo pinjalo to Lutjanus malabaricus and Lutjanus sebae. Morphologically, the red Pinjalo snapper resembles the two Lutjanus red snappers and has been often misidentified as the latter (Zhang et al. 2006). Phylogenetic affinities between the Indian Ocean (with body stripes) and the western Pacific (without body stripes) types of Lutjanus russellii were also investigated as the two types have never been stated in previous phylogenetic studies.

The mitochondrial DNA cytochrome c oxidase subunit I (COI) was used to barcode and infer phylogenetic relationships of Lutjanidae, since many sequences are available for comparison from previous studies (Ward and Holmes 2007, Steinke et al. 2009, Victor et al. 2009, Asgharian et al. 2011, Gold et al. 2011, Lakra et al. 2011, Zhang and Hanner 2011). The relationship between the two conspecifics of L. russellii was further investigated using the mitochondrial DNA control region (CR) sequence since it better resolves relationships among populations, subspecies, or recently diverged species (Parker et al. 1998, Shaw et al. 2000).

MATERIALS AND METHODS

Sampling

A total of 441 snapper specimens were collected between August 2009 and December 2010 from the coastal waters of Peninsular Malaysia and neighboring islands using trawl nets, hook and lines, and traps. Fish from known locations were also purchased from commercial and artisanal landing sites. Muscle tissues or fin clips of each collected specimen were preserved immediately in 95% ethanol in the field, before keeping whole specimens in 10% formalin, which were subsequently measured, photographed, and identified following the identification keys of Anderson and Allen (2001). Two to five specimens of each of the 10 Lutjanus species (L. argentimaculatus, L. carponotatus, L. fulviflamma, L. johnii, L. lutjanus, L. madras, L. malabaricus, L. quinquelineatus, L. sebae, and L. vitta) and one Pinjalo species (P. pinjalo) were used for the phylogenetic study. For L. russellii, a total of 15 specimens (12 Indian and 3 western Pacific) were used for the phylogenetic study.

Ethanol-preserved tissue samples were digested using 10 mg L^-1 proteinase K, in 300 extraction buffer with 2% sodium dodecyl sulfate (SDS). DNA was extracted using the modified phenol-chloroform method (Taggart et al. 1992).

PCR amplification and sequencing

The partial fragment of the COI gene was amplified using the barcoding primers FishF1, FishR1, FishF2, and FishR2 (Ward et al. 2005). We prepared 50 of polymerase chain reaction (PCR) mixtures containing 5 |j.L of 10x PCR buffer, 5 mM MgCl₂, 0.05 mM dNTP mix, 0.1 |oM of each primer, and 1.2 U Taq polymerase (Fermentas), with 5-10 ng of each template DNA. PCR was performed using a MultiGene TC9600-G thermal cycler (Labnet International Inc.). Initial denaturation step was at 95 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 0.5 min, annealing at 54 °C for 0.5 min, and elongation at 72 °C for 1 min. The cycles ended with a final elongation step at 72 °C for 10 min and held at 4 °C.

The left domain of CR was amplified using primers Pro889U20 and TDKD1291L21 (Salini et al. 2006). The 50 µL PCR mixtures included 5 µL of 10× PCR buffer, 5 mM MgCl₂, 0.4 mM dNTP mix, 0.8 µM of each primer, and 4 U Taq polymerase (Fermentas), with 5-10 ng of each template DNA. PCR was performed using an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). The initial denaturation for PCR was at 94 °C for 1.5 min, followed by 35 cycles of denaturation at 94 °C for 5 s, annealing at 50 °C for 0.5 min, and elongation steps at 72 °C for 0.5 min. Final elongation was at 72 °C for 5 min and held at 4 °C.

The PCR products were purified using the GeneJET PCR Purification Kit (Fermentas), following the manufacturer's protocol. Forward and reverse sequencing of the purified PCR products was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem).

Sequence analysis

The DNA sequences were checked and edited using Sequence Scanner (Applied Biosystems) to create a consensus sequence for each specimen and they were deposited in GenBank. With the inclusion of reference sequences from GenBank, multiple sequence alignment and analysis of nucleotide variation of COI and CR sequences was done in MEGA4.0 (Tamura et al. 2007). All DNA sequences analyzed in this study are listed in table 1.

Phylogenetic analyses

The evolutionary model for COI sequences was chosen based on the Akaike Information Criterion (AIC) using Modeltest3.7 (Posada and Crandall 1998), whereas the corrected AIC was adopted for CR sequences in jModeltest (Posada 2008). By incorporating the respective evolutionary models, phylogenetic trees were constructed separately for both sets of sequences by Bayesian inference analysis. The Bayesian analysis was implemented in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) over 8,000,000 generations via four Markov Chain Monte Carlo chains, with tree sampling every 100 generations. Only branches with more than 0.70 posterior probabilities were considered. The COI-based phylogenetic tree was rooted with Aphareus rutilans (subfamily Etelinae) with the addition of the genera Pristipomoides and Etelis (subfamily Etelinae), Apsilus (subfamily Apsilinae), and Symphorichthys (subfamily Paradicichthyinae) to achieve better tree resolution among the Lutjaninae species. For the CR-based phylogenetic tree, Etelis carbunculus was used to root the tree.

RESULTS

DNA sequence variation

A total of 48 COI sequences were directly obtained from this study, disregarding two specimens of L. argentimaculatus that failed to amplify for COI. The 606 bp COI sequence alignment contained 219 variable sites, of which 212 were parsimony informative. The transition/transversion ratio was 2.9. G-bias was observed at both the second and third codon positions of COI with nucleotide compositions A = 24.9%, C = 28.8%, G = 18.4%, and T = 27.9%.

The CR sequence alignment was 461 bp in length, which includes 14 to 76 bp sized indels. There were 276 variable and 269 parsimony informative sites with transition/transversion ratio of 0.78. The bases were fractioned as A = 35.7%, C = 19.6%, G = 13.8%, and T = 30.9%.

Evolutionary model

The Modeltest result suggested the Tamura-Nei evolutionary model with invariable and gamma distribution (TrN + I + G; I = 0.6289; G = 1.3905) for COI sequences, whereas for the CR sequences, the program jModeltest suggested the three-parameter model with invariable sites and gamma distribution (TPM3uf +1 + G; I = 0.2940; G = 1.2110). Base frequencies for COI were 0.2899, 0.3106, 0.1216 and 0.2779 for A, C, G, and T, respectively, with incorporated rate matrix for the substitution model in the Bayesian inference analysis: [A-C] = 1.0000; [A-G] = 19.1232; [A-T] = 1.0000; [C-G] = 1.0000; [C-T] = 11.1862; [G-T] = 1.0000. Unequal base frequencies were also observed in CR, with 0.3801, 0.2024, 0.1120, and 0.3056 for A, C, G, and T, respectively. The incorporated rate matrix for CR was [A-C] = 0.6564; [A-G] = 3.9776; [A-T] = 1.0000; [C-G] = 0.6564; [C-T] = 3.9776; [G-T] = 1.0000.

COI-based phylogenetic tree

The COI phylogram shows that the subfamilies Etelinae, Apsilinae, and Paradicichthyinae are basal to Lutjaninae (fig. 1). All species in Lutjaninae are monophyletic except for L. bohar because L. bohar (EF609394) did not cluster with its conspecific L. bohar (JF952787).

The Lutjaninae species were defined into six major clades, based on a combination of well-supported branches and previous clade definitions (Miller and Cribb 2007, Victor et al. 2009, Wang et al. 2010, Gold et al. 2011). Clades A-E are supported with over 0.90 posterior probabilities, whereas clade F with only 0.73. Branching order is resolved for clades A-C; however, clades D-F form a polytomy. Clade A consists of P. pinjalo, L. malabaricus, L. adetii, and L. sebae, with an average sequence divergence of 12.9% (±1.2); the lowest sequence divergence is 11.8% between L. adetii and L. malabaricus, while the highest is 14.0% between P. pinjalo and L. sebae. Clade B consists of Macolor niger and two species of the family Caesionidae (Pterocaesio diagramma and Caesio caerulaurea), with an average sequence divergence of 10.9% (±0.4). Clades C and D comprise Lutjanus species from the western Atlantic and eastern Pacific, whereas the Lutjanus species in clades E and F are mostly from the western Pacific and Indian Ocean. Ocyurus chrysurus is found in clade C with L. analis, L. buccanella, L. synagris, L. mahogoni, L. vivanus, L. purpureus, and L. peru, with an average sequence divergence of 6.3% (±0.6), ranging from 1.9% to 8.9%. Clade D only contains L. griseus, L. jocu, and L. apodus. The interspecies divergences in clade D are the lowest observed amongst the Lutjaninae species, with an average sequence divergence of 2.5% (±1.4), ranging from 1.0% to 3.1%. Clade E contains L. lemniscatus, L. lutjanus, L. vitta, L. madras, L. fulviflamma, L. ehrenbergii, L. carponotatus, and both types of L. russellii, with an average sequence divergence of 9.0% (±0.6), ranging from 6.1% to 11. 9%. The maximum divergence in clade E is shared between both types of L. russellii with L. madras. A separate branching between the Indian and western Pacific types of L. russellii is supported by 4.2% pairwise sequence divergence. The western Atlantic L. cyanopterus and eastern Pacific L. novemfasciatus cluster unexpectedly in clade F together with the following western Pacific Lutjanus: L. rivulatus, L. stellatus, L. johnii, L. bohar (JF952787), L. quinquelineatus, L. notatus, L. kasmira, L. bengalensis, and L. argentimaculatus; the average sequence divergence is 12.4% (±0.8). In clade F, the lowest sequence divergence is 2.9% between L. novemfasciatus and L. cyanopterus, whereas sequence divergences between the western Pacific Lutjanus range from 5.2% to 16.4%.

CR-based phylogenetic tree

The CR phylogram (fig. 2) indicates the monophyly and affinities of 12 Lutjaninae species, with three well supported clades corresponding to clades A, E, and F in the COI phylogeny (fig. 1). The tree affirms the clustering of P. pinjalo, L. malabaricus, and L. sebae as a clade (clade A). In clade E, the two types of L. russellii cluster with L. carponotatus, L. fulviflamma, L. lutjanus, L. madras, and L. vitta, whereas clade F contains L. argentimaculatus, L. quinquelineatus, and L. johnii.

The CR tree also shows the separation of the two types of L. russellii, with pairwise sequence divergence of 10.8%. The intraspecies divergences of other species range from 0.5% to 6.7%, with an average of 1.8% (±0.8).

DISCUSSION

The present COI phylogeny is congruent with phyloge-nies of Lutjanidae from other studies that used different mitochondrial and nuclear genes (Miller and Cribb 2007, Guo et al. 2007, Wang et al. 2010, Gold et al. 2011). Our clades A, C, D, and F of Lutjaninae correspond to clades A, C, D, and F of Gold et al. (2011), whereas our clades B and E correspond to species clusters in Miller and Cribb (2007). Additionally we propose the basal position of Paradicichthyinae to Lutjaninae, in addition to Apsilinae and Etelinae (Johnson 1980, Gold et al. 2011).

Despite having fewer taxa in the CR phylogeny, we found that affinities between species are congruent with those inferred by COI. Such congruence between CR and other mitochondrial genes may indicate a lower effect of homoplasy in CR, as previously reported (Bernatchez and Danzmann 1993, Zhu et al. 1994), and thus the usefulness of CR for testing phylogenetic relationships between closely related species of Lutjaninae.

Our phylogram also supports the close relationship between L. sebae, L. malabaricus, and L. adetii as inferred in previous studies (Guo et al. 2007, Miller and Cribb 2007, Wang et al. 2010, Gold et al. 2011), with the addition of P. pinjalo into this basal clade. In fact, P. pinjalo shares similar morphological characteristics with L. malabaricus and L. sebae: dorsal fin with XI spines and 13 to 14 soft rays, body depth 2.7 times in standard length, vomerine tooth patch crescentic without posterior extensions, smooth tongue, and total gill rakers ranging from 22 to 23. Based on the taxonomic keys of Lutjanidae (Anderson and Allen 2001), both Lutjanus and Pinjalo have 20 or less gill rakers on the lower limb of the first gill arch, but are separate genera based on the head profile, longitudinal scale row pattern, and the absence or presence of anterior fang-like canines. However, these distinguishing characters are not exclusive as some are shared by a few Lutjanus species. Head and trophic features are also usually subjected to convergent evolution (Gold et al. 2011) that confounds reliable taxonomic classification. Alternatively, colorations and body patterns show better correlation to phylogenetic relationships (Shaw et al. 2000, Wang et al. 2010) , as also observed between P. pinjalo, L. malabaricus, and L. sebae.

While the affinities among L. russellii, L. fulviflamma, L. carponotatus and L. vitta have been reported in Miller and Cribb (2007), we have further separated the two types of L. russellii. The 4.2% divergence in COI sequences between the two conspecifics of L. russellii supports the recognition for separate species (Avise 2000, Zhang and Hanner 2011). Our CR phylogeny also confirms such separation with expectedly higher divergences. Besides being geographically separated, the adults of the Indian type of L. russellii have body stripes, whereas the adults of the western Pacific type of L. russellii lack body stripes (Anderson and Allen 2001). Therefore, the Indian and western Pacific types of L. russellii should be recognized as two separate species based on the high level of divergences, geographical separation, and morphological dissimilarities.

In summary, the phylogenetic tree inferred using COI and CR supports the monophyly of all the Lutjanus species analyzed except for the two "types" of L. bohar. We propose that P. pinjalo be subsumed into Lutjanus as molecular and morphological evidences have revealed the close affinity of P. pinjalo with the Lutjanus red snappers. Further research using both mitochondrial and nuclear genes to clarify whether the two types of L. russellii are different species is also warranted.

ACKNOWLEDGMENTS

This research was fully funded by the University of Malaya through UMRG and PPP research grants (RG032/ 09SUS and PS225/2009C, respectively). Our appreciation goes to all the fishermen and lab-mates that assisted in the sampling of specimens.

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