ABSTRACT. Penaeid shrimps are an important resource in crustacean fisheries, representing more than the half of the gross production of shrimp worldwide. In the present study, we used a sample of wide-ranging diversity (41 shrimp species) and two mitochondrial markers (758 bp) to clarify the evolutionary relationships among Penaeidae genera. Three different methodologies of tree reconstruction were employed in the study: maximum likelihood, neighbor joining and Bayesian analysis. Our results suggest that the old Penaeus genus is monophyletic and that the inclusion of the Solenocera genus within the Penaeidae family remains uncertain. With respect to Metapenaeopsis monophyly, species of this genus appeared clustered, but with a nonsignificant bootstrap value. These results elucidate some features of the unclear evolution of Penaeidae and may contribute to the taxonomic characterization of this family.

Key words: Molecular evolution, Monophyly, Maximum likelihood, Crustacea, Old genus Penaeus

INTRODUCTION

Penaeid shrimps are the most important economic resource in the world’s crustacean fishery industry (Holthuis, 1980; Dall et al., 1990; Pérez-Farfante and Kensley, 1997), being responsible for more than half of the gross production of shrimp (FAO, 2000). Recently, several molecular phylogenetic studies have contributed to the clarification of the evolutionary history of the group (Baldwin et al., 1998; Gusmão et al., 2000; Tong et al., 2000; Maggioni et al., 2001; Lavery et al., 2004; Vázquez-Bader et al., 2004).

Most of the studies, however, have focused on the phylogenetic issues of individual penaeid genera. Only recently, Quan et al. (2004) and Vázquez-Bader et al. (2004) examined penaeid phylogenetics, but they were severely restricted by taxon sampling (12 species; Quan et al., 2004) or sequence length (around 300 bp; Vázquez-Bader et al., 2004). Thus, it remains to be seen whether their results would change if more data were made available.

In the present study, we used a sample of wide-ranging diversity (41 shrimp species), two mitochondrial markers (758 bp) and robust phylogenetic methodologies to clarify the evolutionary relationships among Penaeidae genera.

MATERIAL AND METHODS

The taxonomic classification proposed by Pérez-Farfante and Kensley (1997) was adopted in this study. Sequences for two mitochondrial genes, namely 16s rRNA gene (16S) and cytochrome oxidase subunit 1 gene (CO1), were downloaded from GenBank for thirty-nine Penaeidae species. Additionally, two species of the closely related Solenoceridae family and the Brachyura species Portunus trituberculatus (as the outgroup, see Quan et al., 2004) were included (accession numbers are presented in Table 1).

The sequences were aligned using the ClustalW algorithm available at http://www.ebi.ac.uk/clustalw (Thompson et al., 1994) and subsequently checked by visual inspection. All gaps were excluded from the alignment, resulting in a concatenated sequence 758 bp long.

The phylogenetic tree and branch support values were estimated using three different methodologies of phylogenetic reconstruction: 1) maximum likelihood (ML), 2) distance and 3) Bayesian inference (BI). In the ML approach, a heuristic algorithm was necessary due to computer time limitations. Two initial trees, obtained by stepwise addition or by neighbor-joining (NJ), were swapped using the tree bisection and reconnection method. Because both stepwise addition and NJ approaches yielded exactly the same final topology, the NJ + tree bisection and reconnection method was used for the computation of the branch supporting values, since it is a much faster algorithm. In this case, a nonparametric bootstrap was performed with 100 pseudoreplicates. The best-fit model (GTR + G + I; a = 0.5995 and I = 0.529) was selected through a hierarchical likelihood ratio test on the Modeltest 3.06 software (Posada and Crandall, 1998). All ML analyses were performed with the PAUP* 4.0 software (Swofford, 2002).

For distance analysis, a nonparametric bootstrap was performed on 1000 pseudo-replicates, using the NJ algorithm and Jukes-Cantor as the model of substitution. The choice of this model was based on Nei and Kumar (2000) (overall media: p distance = 0.141 transitions/tranversions = 1.444). Distance analysis was carried out with MEGA 3 (Kumar et al., 2004).

Finally, Bayesian posterior probabilities were calculated from 9,000 sample trees, using the model selected through Modeltest 3.06. The first 100,000 generations of the Markov chain Monte Carlo were not used to ensure that the chain would be sampled on a stationary portion. The other parameters were set according to the MrBayes 3.0 program default (Ronquist and Huelsenbeck, 2003).

RESULTS AND DISCUSSION

The ML analysis of the concatenated sequence of the 42 species studied resulted in the phylogenetic tree shown in Figure 1. Controversial issues of Penaeidae phylogenetics and aspects that were never properly tested are discussed below.

The first important result of our tree is that the genus Solenocera clustered with Parapenaeus fissuroides (hereafter, support values for ML: 41; BI: 63; NJ: 44), and this clade appeared to be associated with Metapenaeopsis spp (ML: 61; BI: 94; NJ: 39). This is an important result because it argues against the monophyletic status of the Penaeidae family, since Solenocera belongs to a different family.

Nonetheless, support values of this association are too low to firmly contest the monophyletic condition of Penaeidae. In fact, even in a more restricted analysis including 12 Penaeoidea species (Quan et al., 2004), the bootstrap value for the group formed by the Solenocera and P. fissuroides + M. barbata clade was still not significant (75%). Another study (Vázquez-Bader et al., 2004) showed that the Solenocera genus is included within Penaeidae, but again a non-significant bootstrap value was assigned to the branch (less than 74%). Naturally, if this outcome proves consistent, the Solenocera genus will not be deemed suitable for an outgroup in penaeid phylogenetics, as has been the case in some studies (Tong et al., 2000; Lavery et al., 2004). This clearly indicates an unstable condition of the group, and thus, a more detailed study is apparently needed to put these matters to rest.

The second major result considers the monophyletic condition of the Metapenaeopsis genus. Tong et al. (2000) have already analyzed the phylogenetic relations within species of this genus. Unfortunately, however, they used an improper outgroup (S. koelbeli), and in addition, a single species outside the Metapenaeopsis genus was included in the analysis (P. monodon). More recently, Quan et al. (2004) and Vázquez-Bader et al. (2004) studied the phylogenetic position of the genus within Penaeioidea, but again, a single species of Metapenaeopsis was included in the analyses. Consequently, the monophyletic status of the genus was never properly tested previously with molecular data. In our tree, all seven Metapenaeopsis species were grouped with relatively high support values (ML: 78; BI: 100; NJ: 73). With regard to morphology, the diagnostic characteristics for the genus are the asymmetrical petasma and the unpaired and uninvaginated seminal receptacles (Vázquez-Bader et al., 2004).

The group formed by Farfantepenaeus, Fenneropenaeus, Marsupenaeus, Melicertus, Litopenaeus, and Penaeus, also known as the “old Penaeus genus,” clustered with high support values (ML: 91; BI: 98; NJ: 89). This result is very interesting and may eventually be used to justify a separate intermediate taxonomic level for the classification of this group. Our results also suggest the monophyletic condition of Fenneropenaeus with a high ML bootstrap value and significant supports in BI and NJ (ML: 90; BI: 100; NJ: 95). As expected, the monophyly of Farfantepenaeus (ML: 86; BI: 100; NJ: 99) and Litopenaeus (ML: 98; BI: 100; NJ: 97) was confirmed (Maggioni et al., 2001; Lavery et al., 2004) with high support values, which corroborates the classification based on thelycum morphology (Pérez-Farfante and Kensley, 1997). The group formed by Melicertus + Marsupenaeus genera (ML: 81; BI: 98; NJ: 82) is also supported by morphological data (Pérez-Farfante and Kensley, 1997), since both subgenera usually have telson with tree pairs of movable lateral spines. In our tree, Marsupenaeus is not clearly included in the Melicertus group, but the BI support value strongly suggests that they are closely related genera.

The genus Penaeus appears to be paraphyletic with Fenneropenaeus spp, clustering between the two Penaeus lineages: P. monodon and P. esculentus + P. semisulcatus. Nonetheless, this result needs verification because the support values are particularly low (ML: 44; BI: NA; NJ: NA).

The most basal lineages that appear in the tree are the Metapenaeus genus, followed by a clade formed by T. curvirostris + P. hardwickii + X. kroyeri. The support values for the deeper branches, however, are too low to firmly establish the relation among these penaeid lineages (ML: 16; BI: 62; NJ: 46). Still, the monophyly of Metapenaeus is attested by significant supporting values (100%) in the Bayesian and NJ analyses (ML: 93).

This inclusive analysis of Penaeidae phylogenetics helped to examine some aspects of the unclear evolutionary relation among Penaeidae genera, which were never properly addressed until now. In the future, the phylogeny obtained here can be used for taxonomic purposes and possibly in a divergence time analysis which may also clarify the origin and diversification of the family. It is important to note that even when using the combined sequences of two markers (16S and COI) and a comprehensive sample (including 42 Penaeid species), the phylogenetic relation within the family remains unstable. Obviously, more genes must be sequenced to clarify the taxonomic issues of the family.

ACKNOWLEDGMENTS

The authors wish to thank CNPq (Brazilian research council) and FAPERJ (Rio de Janeiro’s research foundation) for funding which enabled us to conclude this work. C.M. Voloch was funded by CNPq and CAPES fellowships whereas CNPq supported P.R. Freire. This study is part of the Ph.D. thesis of C.M. Voloch at the Genetics Program at Universidade Federal do Rio de Janeiro.

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