Phylogenetic relationships among anchovies, sardines, herrings and their relatives (Clupeiformes), inferred from whole mitogenome sequences

Molecular Phylogenetics and Evolution 43 (2007) 1096–1105 www.elsevier.com/locate/ympev

Phylogenetic relationships among anchovies, sardines, herrings and their relatives (Clupeiformes), inferred from whole mitogenome sequences Sébastien Lavoué a,¤, Masaki Miya b, Kenji Saitoh c, Naoya B. Ishiguro a, Mutsumi Nishida a a

Department of Marine Bioscience, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan b Department of Zoology, Natural History Museum and Institute, Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan c Tohoku National Fisheries Research Institute, Shinhama, Shiogama, Miyagi 985-0001, Japan Received 11 June 2006; revised 20 September 2006; accepted 22 September 2006 Available online 7 October 2006

Abstract The relationships among and within the main lineages of the order Clupeiformes have been explored in few morphological studies and still remain poorly understood. Using whole mitogenome sequences, we inferred the relationships among 25 clupeiform species, sampled from each clupeiform family and subfamily, and a large selection of non-clupeiform teleosts. Our character sets, including unambiguously aligned, concatenated mitogenome sequences that we have divided into four (1st and 2nd codon positions, tRNA genes, and rRNA genes) or Wve partitions (same as before plus the transversions at 3rd codon positions, using ‘RY’ coding), were analyzed by the partitioned Bayesian method. The result strongly supported the monophyly of the Clupeiformes within the Otocephala, with Denticeps clupeoides as the sister group of a clade comprising all the remaining clupeiforms species ( D suborder Clupeoidei). Within the Clupeoidei, the family Engraulidae was the sister group of the remaining taxa, comprising members of Sundasalangidae, Pristigasteridae, Clupeidae and Chirocentridae. Relationships among the latter four families remained ambiguous. In particular, the position of the Chirocentridae was diYcult to estimate possibly owing to its higher molecular evolutionary rate. Of the Wve subfamilies in the family Clupeidae, monophylies of three (Alosinae, Clupeinae and Dorosomatinae) were statistically rejected. Instead, our mitogenomic data provide strong support for new clades within the Clupeidae, some of which are composed of members of more than one of the previously accepted subfamilies. © 2006 Elsevier Inc. All rights reserved. Keywords: Mitogenomics; Basal teleostei; Otocephala; Clupeomorpha; Long PCR

1. Introduction The Wshes of the order Clupeiformes include anchovies, herrings, sardines, menhadens, shads, gizzard shads, wolf herrings and their relatives. Of crucial importance in world Wsheries, global capture production of the Clupeiformes was 19,000,000 tons in 2003, representing about 25% of the total annual catch of all Wshes (marine and freshwater), which is far more than for any other single systematic group of Wshes (FAO, Fishery statistics 2003, http:// www.fao.org/W/statist/statist.asp). In some regions, local Wshery economies are highly dependent on the population

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Xuctuation of a single clupeiform species. One of the most famous examples is the heavily exploited Peruvian anchovy (Engraulis ringens). A cyclic climatic change modifying the Humboldt Current ecosystem along the coasts of Peru and Chile in 1972–73 provoked the collapse of anchovy’s populations and the bankruptcy of the local Wshery economy in Peru (Niquen and Bouchon, 2004; Whitehead, 1985). In regard to their economical importance, the clupeiforms have been the subject of many studies covering diverse biological areas related to Wsheries sciences, yet relatively few studies have addressed the relationships among these Wshes. A well-supported phylogenetic hypothesis among the major groups of clupeiforms is still lacking. The clupeiforms are distributed worldwide, with 401 valid species (FishBase, ver. April 2006: http://www.Wshbase.org/search.cfm). Most of them inhabit marine tropical

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and sub-tropical coastal areas, with several groups being euryhaline and anadromous (Whitehead, 1985). Several species, from various lineages of Clupeiformes, are also known to live exclusively in freshwater environments, such as the African pellonulins (Whitehead, 1985; Whitehead et al., 1988). Such particularities make this group of great interest for studies of the mechanisms of marine/freshwater transitions and their associated physiological adaptations. The Clupeiformes, as restrictively deWned by Greenwood et al. (1966), is a well-accepted group, which is supported by several complex anatomical characters (Di Dario, 2004; Grande, 1985; Lauder and Liem, 1983; Nelson, 1973). The current classiWcation of extant taxa, as reviewed by Nelson (2006), subdivides the Clupeiformes into two suborders, the monospeciWc Denticipitoidei (Denticeps clupeoides) and the Clupeoidei. The Clupeoidei comprise several subgroups to which various authors have attributed diVerent taxonomic ranks (from the subfamilial to superfamilial level). Nelson (2006), following Grande (1985), subdivides the Clupeoidei into four families: the Engraulidae (two subfamilies, 16 genera, 139 species), the Pristigasteridae (two subfamilies, 9 genera, 37 species), the Chirocentridae (one genus, 2 species) and the Clupeidae (Wve subfamilies, 66 genera, 216 species). Recently, Siebert (1997) and Ishiguro et al. (2005) proA

vided evidence for the inclusion of the paedomorphic Wshes of the family Sundasalangidae (one genus, seven species) within the Clupeiformes based on morphological and molecular evidence, respectively. Since the publication by Greenwood et al. (1966), a handful of phylogenetic hypotheses have been proposed for the Clupeiformes (Fig. 1A–E) (Di Dario, 2002; Grande, 1985; Lauder and Liem, 1983; Nelson, 1967, 1970; Sato, 1999; Whitehead, 1985). All of these hypotheses share four important similarities. (1) They are based solely on the comparison of morphological characters. No higher level hypothesis based on a molecular dataset has ever been published for this group. (2) Their authors divided the Clupeiformes in a similar way (with 9 to 11 subgroups at the familial/subfamilial level), although some authors questioned the validity of certain subgroups, such as the Alosinae, Clupeinae and Dorosomatinae (Grande, 1985; Nelson, 1970; Sato, 1994). However, because an alternative natural arrangement proved diYcult to justify, these subfamilies were conservatively maintained, pending additional data (Grande, 1985). (3) The relationships among the major lineages of Clupeoidei and, in particular, among the clupeid subfamilies were mostly unresolved (Fig. 1A–E). Lastly, (4) none of these previous hypotheses included the family Sundasalangidae. C

B

D

1097

E

Fig. 1. Five diVerent phylogenetic hypotheses for clupeiforms based on morphological characters (A–E). Asterisks following subfamily names (A and C) indicate that the authors of these hypotheses questioned the monophyly of these subfamilies, but they keep them as “groups of convenience.” Arrows indicate the main modiWcations introduced by Grande (1985) and Di Dario (2002).

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In the past Wve years, the technique to amplify the entire mitochondrial genome (i.e., the mitogenome) by long PCR has made possible a large-scale mitogenomic project that aims to provide new insights into the relationships among teleost Wshes (Inoue et al., 2001a,b, 2003; Inoue et al., 2004; Ishiguro et al., 2005; Ishiguro et al., 2003; Lavoué et al., 2005; Miya et al., 2001; Miya and Nishida, 2000; Miya et al., 2005; Miya et al., 2003; Saitoh et al., 2003; Yamanoue et al., 2004). As a part of this eVort, we address here the phylogenetic relationships among 25 clupeiform taxa representing all subfamilies currently recognized. The purpose of this study is to test the monophyly and interrelationships of higher-level groups of Clupeiformes. We discuss our phylogenetic results with reference to the previous morphologybased studies. 2. Material and methods 2.1. Taxonomic sampling Our taxonomic sampling included at least one representative for each family and subfamily of the Clupeiformes that have been commonly recognized. In total, we determined the nucleotide sequences of 19 new mitogenomes from clupeiform taxa. We combined these new mitogenomic data with the six previously published clupeiform taxa and a selection of 19 non-clupeiform taxa that we have selected from the Otocephala (10), Elopomorpha (5) and Euteleostei (4). We chose three Osteoglossomorpha taxa as operational outgroup to root our phylogenetic trees. The list of taxa examined in this study is provided in Table 1, along with DDBJ/EMBL/GenBank accession numbers of the corresponding mitogenomic sequences. Most of the specimens used in this study are deposited in the collection of the Muséum National d’Histoire Naturelle, Paris (MNHN), the National Science Museum, Tokyo (NSMT) and the Musée Royal de l’Afrique Centrale, Tervuren (MRAC). The collection accession numbers are available upon request to the Wrst author. 2.2. DNA extraction, long PCR, short PCR and sequencing A portion of the epaxial musculature or of the pelvic Wn was removed from fresh specimens from each specimen and immediately preserved in 99.5% ethanol. Total genomic DNA was extracted using the Qiagen DNeasy tissue kit following the manufacturer’s protocol. Then, the mitogenomes were puriWed in their entirety in two or three reactions using a long PCR technique (Cheng et al., 1994) and designed Wsh-versatile long PCR primers (locations and sequences of these primers are available upon request to the Wrst author). The long PCR products were diluted with distilled water (1:15) for subsequent use as templates for the short PCR. Eighty-Wve Wsh-versatile PCR primers were used in various combinations to amplify short (<1500 bp), contiguous, overlapping segments of the entire mitogenome for each of the 19 species

(a list of these primers available upon request to the Wrst author). Long PCR and subsequent short PCR were carried out as previously described (Miya and Nishida, 1999). Double-stranded DNA products were Wrst puriWed using an ExoSap enzyme reaction, before being used as a template for direct cycle sequencing with dye-labeled terminators (Applied Biosystems). We used the same primers for sequencing as those used for short PCR. All sequencing reactions were performed according to the manufacturer’s instructions. Labeled fragments were run on a Model 3130xl DNA automated sequencer (Applied Biosystems). 2.3. Sequences editing and alignment The sequence electropherograms were edited with EditView ver. 1.0.1 (Applied Biosystems). Sequencher software package ver. 4.1.2 (Gene Codes) and DNASIS ver. 3.2 (Hitachi Software Engineering) were used to concatenate and check the consensus mitogenomic sequences, before exporting them to phylogenetic software programs. For each individual protein-coding gene, we aligned by eye the sequences for the 47 species, with respect to the translated amino acid sequence. All stop codons were excluded from the subsequent phylogenetic analyses, as well as ambiguous alignment stretches at the 5⬘ and 3⬘ ends for some protein-coding genes and the heterogeneous base composition ND6 gene (Miya and Nishida, 2000). The 12S and 16S rRNA sequences, as well as the concatenated 22 tRNA genes were aligned using the software Proalign ver. 0.5 (Loytynoja and Milinkovitch, 2003), and default setting parameters. Regions with posterior probabilities of 650% were excluded from the subsequent phylogenetic analyses. Our complete data matrix includes 14,498 positions, of which 8622 were variable and 7491 were parsimoniously informative. 2.4. Phylogenetic analysis Simmons and Miya (2004) empirically demonstrated that Bayesian analysis is the most accurate method for inferring phylogeny from a mitogenomic dataset. Following the Simmons and Miya (2004) recommendations, we used this method for two diVerent character matrices. The Wrst matrix (dataset #1, 10,868 positions) includes concatenated nucleotide sequences from 12 protein-coding genes except the third codon positions (7260 positions), 22 transfer RNA genes (1600 positions) and the two ribosomal RNA genes (2008 positions). The second (dataset #2, 14,025 positions) includes the same set of characters plus the transversions at the third codon positions of proteincoding genes, Wrst replacing the purines (C/T) with‘Y’, and the pyrimidines (A/G) with ‘R’ (Phillips et al., 2004). Since MrBayes estimated unnecessarily transitional changes when ‘R/Y’ coding is used, we arbitrarily recoded ‘Y’ and ‘R’ with ‘A’ and ‘C’, respectively, and we set a single rate category for this partition (lset nst D 1) instead of 6.

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Table 1 List of species examined in this study ClassiWcation

Species

Accession Nos.

Reference

Osteoglossomorpha Family Hiodontidae Family Osteoglossidae Family Pantodontidae

Hiodon alosoides (RaWnesque) Osteoglossum bicirrhosum (Cuvier) Pantodon buchholzi Peters

AP004356 AB043025 AB043068

Inoue et al. (2003) Inoue et al. (2001b) Inoue et al. (2001b)

Elopomorpha Family Elopidae Family Megalopidae Family Notacanthidae Family Anguillidae Family Muraenidae

Elops hawaiensis Regan Megalops atlanticus Valenciennes Notacanthus chemnitzi Bloch Anguilla japonica Temminck & Schlegel Gymnothorax kidako (Temminck & Schlegel)

AB051070 AP004808 AP002975 AB038556 AP002976

Inoue et al. (2004) Inoue et al. (2004) Inoue et al. (2003) Inoue et al. (2001b) Inoue et al. (2003)

Denticeps clupeoides Clausen Sardinops melanostictus (Jenyns) Clupea pallasii Valenciennes Clupea harengus Linnaeus Sprattus sprattus (Linnaeus) Sardinella maderensis (Lowe) Sardina pilchardus (Walbaum) Ethmalosa Wmbriata (Bowdich) Alosa alosa (Linnaeus) Alosa pseudoharengus (Wilson) Spratelloides delicatulus (Bennett) Spratelloides gracilis (Temminck & Schlegel) Etrumeus teres (DeKay) Jenkinsia lamprotaenia (Gosse) Dorosoma petenense (Günther) Nematalosa japonica Regan Pellonula leonensis Boulenger Odaxothrissa vittata Regan Engraulis japonicus Temminck & Schlegel Engraulis encrasicolus (Linnaeus) Coilia nasus Temminck & Schlegel Chirocentrus dorab (Forsskål) Ilisha elongata (Bennett) “Ilisha” africanaa (Bloch) Sundasalanx mekongensis Britz & Kottelat

AP007276 AB032554 AP009134 AP009133 AP009234 AP009143 AP009233 AP009138 AP009131 AP009132 AP009144 AP009145 AP009139 AP006230 AP009136 AP009142 AP009232 AP009231 AB040676 AP009137 AP009135 AP006229 AP009141 AP009140 AP006232

Lavoué et al. (2005) Inoue et al. (2000) This study This study This study This study This study This study This study This study This study This study This study Ishiguro et al. (2005) This study This study This study This study Inoue et al. (2001a) This study This study Ishiguro et al. (2005) This study This study Ishiguro et al. (2005)

Order Gonorynchiformes Family Chanidae Family Gonorynchidae Family Phractolaemidae Family Kneriidae

Chanos chanos (Forsskål) Gonorynchus greyi (Richardson) Phractolaemus ansorgii Boulenger Grasseichthys gabonensis Géry

AB054133 AB054134 AP007280 AP007277

Saitoh et al. (2003) Ishiguro et al. (2003) Lavoué et al. (2005) Lavoué et al. (2005)

Order Cypriniformes Family Cyprinidae Family Cobitidae

Carassius auratus (Linnaeus) Cobitis striata Ikeda

AB006953 AB054125

Murakami et al. (1998) Saitoh et al. (2003)

Order Characiformes Family Characidae

Phenacogrammus interruptus (Boulenger)

AB054129

Saitoh et al. (2003)

Order Siluriformes Family Bagridae

Pseudobagrus tokiensis Döderlein

AB054127

Saitoh et al. (2003)

Order Alepocephaliformes Family Alepocephalidae Family Platytroctidae

Alepocephalus tenebrosus Gilbert Platytroctes apus Günther

AP004100 AP004107

Ishiguro et al. (2003) Ishiguro et al. (2003)

Protacanthopterygii Family Salmonidae Family Esocidae

Coregonus lavaretus (Linnaeus) Esox lucius Linnaeus

AB034824 AP004103

Miya and Nishida (2000) Ishiguro et al. (2003)

Otocephala Order Clupeiformes Family Denticipitidae Family Clupeidae

Clupeinae

Alosinae

Dussumeriinae

Dorosomatinae Pellonulinae Family Engraulidae

Family Chirocentridae Family Pristigasteridae Family Sundasalangidae

(continued on next page)

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Table 1 (continued) ClassiWcation

Species

Accession Nos.

Reference

Neoteleostei Family Gadidae

Gadus morhua Linnaeus

X99772

Paralichthys olivaceus (Temminck & Schlegel)

AB028664

Johansen and Bakke (1996) Saitoh et al. (2000)

Family Paralichthyidae

ClassiWcation follows Nelson (2006) with the exception of Otocephala, which groups Clupeiformes, Gonorynchiformes, Otophysi (i.e. Cypriniformes, Characiformes, Siluriformes and Gymnotiformes) and Alepocephaliformes (D Alepocephaloidei) (Ishiguro et al., 2003; Lavoué et al., 2005). Within the family Clupeidae, the subfamilial classiWcation is indicated. a According to Grande (1985), the genus Ilisha is not monophyletic: I. africana is distantly related to the other Ilisha species.

We set up four (dataset #1: Wrst and second codon positions of protein-coding genes, tRNA genes and rRNA genes) or Wve partitions (datasets #2: same four as for dataset #1 plus the transversions at the third codon positions). Partitioned Bayesian phylogenetic analyses were conducted with MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). We used the general time reversible model with some sites assumed to be invariable and with variable sites assumed to follow a discrete gamma distribution [GTR + I + G; (Yang, 1994)], as it was selected as the bestWtting model with Modeltest version 3.06 (Posada and Crandall, 1998). Parameter settings in MrBayes are as described in Lavoué et al. (2005). For each matrix, two independent Bayesian analyses were performed. The Markov chain Monte Carlo (MCMC) process was set so that four chains (three heated and one cold) ran simultaneously. On the basis of preliminary runs with varying cycles (0.5–3.0 £ 106), we estimated average log likelihood scores at stationarity. After reaching stationarity in the two independent runs, we continued the runs for 2.5 £ 106 cycles (25,000 trees) to conWrm lack of improvement in the likelihood scores. Parameter values and trees were sampled every 100 generations. For each dataset, 50% majority-rule consensus trees were calculated from the 50,000 trees pooled from the two runs. 2.5. Testing alternative topologies Alternative tree topologies, in which the monophyly of some previously recognized subgroups of Clupeiformes were individually constrained, were compared to our best hypothesis (from dataset #1) using the Bayes factor. We calculated Bayes factors using MrBayes 3.1.2. For this purpose, we conducted Partitioned Bayesian phylogenetic analyses using our dataset #1 and the same set of parameter values as previously described, but we constrained the topology of all the trees sampled from the chain to be congruent with the alternative hypothesis tested. Then, the harmonic means of likelihoods from the constrained analyses were calculated using the sump command in MrBayes 3.1.2, and they were compared to the harmonic means of likelihoods from the unconstrained analysis by calculating twice the diVerence (i.e., 2¤ln). Following Kass and Raftery (1995), a 2¤ln Bayes factor >10 is interpreted as a strong evidence against the alternative topology tested. Based on an empirical study,

however, Brandley et al. (2005) found that a 2¤ln Bayes factor of 10 could be too conservative of a threshold. 3. Results 3.1. Mitochondrial genome organisation Mitogenomes of the 19 newly determined species varied modestly in size from 16,617 bp (Spratelloides delicatulus) to 16,979 bp (Etrumeus teres). The genome content and organization of these 19 new mitogenomes follow the typical pattern for vertebrates so far examined. These mitogenomes include 12S and 16S rRNAs, 22 tRNAs, 13 protein-coding genes and the control region. The H-strand codes for most of these genes, except the ND6 and eight tRNA genes, which are coded on the L-strand. 3.2. Bayesian analysis Analysis of the complete data set, excluding all third codon positions (dataset #1), produced a well-resolved tree. Fig. 2 shows the 50% majority rule consensus of 50,000 combined samples from two independent Bayesian analyses of this data set, with branch lengths proportional to number of changes. In this tree, only four nodes did not receive a Bayesian posterior probability (BPP) 795%; all of them are within the suborder of Clupeoidei. In rooting this tree with the osteoglossomorph taxa, the Elopomorpha, the Euteleostei (sensu Johnson and Patterson (1996) and Lecointre and Nelson (1996), minus the Alepocephaloidei), and the Otocephala (including the Alepocephaloidei) are reciprocally monophyletic. Within the Otocephala, the Clupeiformes form a monophyletic group (BPP D 100%), which is the sister group of the clade (Alepocephaloidei, Ostariophysi)(BPP D 98%). Within the Clupeiformes, Denticeps clupeoides (suborder Denticipitoidei) is the sister group of the suborder Clupeoidei. The Clupeoidei appears as a well-deWned genetic group, as indicated by its long internal branch. Based on branch lengths and BPP, we identiWed four major lineages within this group: the Engraulidae (represented by Coilia and Engraulis), the Sundasalangidae (Sundasalanx), the Pristigasteridae (Ilisha), and the superfamily Clupeoidea (Chirocentridae and Clupeidae). The Engraulidae appear as the sistergroup of the remaining clupeoidei (BPP D 99%), whereas the

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1101

Fig. 2. Fifty percent majority rule consensus tree (with branch lengths proportional to number of changes) based on 50,000 pooled trees from two independent Bayesian analyses of the Wrst dataset (see “Section 2” for details). This tree was rooted with Osteoglossum bicirrhosum, Pantodon buchholzi and Hiodon alosoides. Numbers at internal branches indicate Bayesian posterior probabilities (shown as percentages). On the right side, the familial classiWcation within the Clupeiformes is indicated by black rectangles; and two well-supported lineages within the Clupeidae, named A1 and A2, are indicated by open rectangles. It should be noted that four of the Wve traditional subfamilies of the Clupeidae are not monophyletic. The arrowhead indicates the topological diVerence with the tree based on the analysis of our dataset #2.

relative relationships among the Sundasalangidae, Pristigasteridae and Clupeoidea are weakly supported (BPP D 66%). Within the Clupeoidea, we recognize Wve lineages: the genus Etrumeus, the genus Chirocentrus, the tribe Spratelloidini (Spratelloides and Jenkinsia), the clade (Clupea, Sprattus), and a clade grouping all other clupein taxa (i.e., Sardinella, Sardinops and Sardina) along with the alosin taxa (Alosa and Ethmalosa), dorosomatin taxa (Nematalosa and Dorosoma) and pellonulin taxa (Odaxothrissa and Pellonula). Chirocentrus (Chirocentridae) is nested within the subfamily Dussumieriinae, making the Clupeidae paraphyletic. However, when we test the alternative topology, in which the monophyly of the Dussumieriinae is constrained, the resulting Bayes factor is only of 11 (Table 2). Moreover, since Chirocentrus dorab exhibits the longest (by far) terminal branch of all clupeiform taxa sampled, its unorthodox position could be diYcult to estimate. The non-monophyly of the clupeid subfamilies Alosinae, Clupeinae and Dorosomatinae are strong results (1160 > Bayes factors > 38, Table 2). When the transversions at the 3rd codon positions are included in the analysis (dataset #2), the tree topology

within the Clupeiformes is congruent with the previous analysis from the dataset #1, with the notable exception of the position of the clade (Chirocentrus, (Spratelloides, Jenkinsia)) relative to the Pristigasteridae and Clupeidae. In this tree (not shown), the Clupeiformes were again robustly recovered as monophyletic, with Denticeps clupeoides as the sistergroup of the suborder Clupeoidei, and the Engraulidae as the sister group of the remaining clupeoids. Within the Clupeidae, the topology is the same as in the previous analysis (dataset #1) except for the position of the clade (Chirocentrus, (Spratelloides, Jenkinsia)). 4. Discussion Due to the high economic value of these Wshes and the importance of accurate identiWcations for stock management, the taxonomy of the clupeiforms has been thoroughly studied and is considered to be relatively well understood (reviewed in Whitehead (1985) and Whitehead et al. (1988)). However, the relationships among the main clupeiform lineages are still mostly unresolved based on

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Table 2 2ln Bayes factors result of comparison of alternative topologies, in which the monophylies of speciWc clades were constrained, using MrBayes 3.1.2 Alternative topologies tested Unconstrained topology (L0) Monophyly of Clupeidae constraineda Monophyly of Dussumieriinae constraineda Monophyly of Alosinae¤ constrained Monophyly of Clupeinae constrained Monophyly of Dorosomatinae constrained Monophyly of Pellonulinae + Dussumieriinae constrained Pristigasteridae sister group of the remaining Clupeoidei

ln ¡128,545.22 ¡128,554.45 ¡128,550.9 ¡129,005.86 ¡129,125.5 ¡128,564.64 ¡129,062.52 ¡128,585.5

2ln Bayes factors

Evidence against alternative topologies tested

— 18.46 11.36 921.28 1160.56 38.84 1034.14 80,56

— Very strong (>10) Very strong (>10) Very strong (>10) Very strong (>10) Very strong (>10) Very strong (>10) Very strong (>10)

To calculate these factors, we use our dataset #1 that excludes the third codon positions of the protein coding genes. A 2ln Bayes factor >10 is considered as a very strong evidence against the alternative hypothesis tested. However, as discussed by Brandley et al. (2005), such threshold could be considered as too conservative. a Excluding Chirocentrus dorab (Chirocentridae). ¤ Sensu Grande (1985), i.e. including Ethmalosa.

morphological characters (Fig. 1A–E). Our study is the Wrst one that investigates higher relationships of Clupeiformes based on molecular data. 4.1. The sister group of the Clupeiformes The order Clupeiformes is monophyletic (Di Dario, 2004; Grande, 1985; Greenwood et al., 1966; Nelson, 1973). Traditionally, three unambiguous morphological characters support its monophyly: (1) a distinctive type of ear-swimbladder connection, (2) the architecture of the neurocranium (presence of a recessus lateralis) and (3) the supporting skeleton of the caudal Wn, which is unique among all teleosts (Greenwood et al., 1966). We conWdently place the Clupeiformes close to the Ostariophysi; with the addition of the Alepocephaloidei, this group makes up the Otocephala sensu lato. The close relationship between Clupeiformes and Ostariophysi has been supported by several molecular (Ishiguro et al., 2003; Lavoué et al., 2005; Lê et al., 1993; Zaragüeta-Bagils et al., 2002) and morphological studies (Arratia, 1997; Johnson and Patterson, 1996) and is now well accepted (Lecointre and Nelson, 1996). In contrast, the addition of the Alepocephaloidei within the Otocephala is novel and deserves further investigation. Resolving the question of the position of the Alepocephaloidei relative to the Clupeiformes and the Ostariophysi is central to understanding the origin and the diversiWcation of the Otocephala. 4.2. Denticeps: the sister group of Clupeoidei Our results support a sister relationship between the primitive looking African Denticeps clupeoides (suborder Denticipitoidei) and the remaining extant Clupeiformes (suborder Clupeoidei) (Di Dario, 2004; Greenwood, 1968). The monophyletic Clupeoidei appears as a genetically welldeWned group, supported by a long branch. 4.3. Higher-level relationships within the Clupeoidei Four families are commonly recognized within the Clupeoidei: the Clupeidae, the Engraulidae ( D Engraulididae),

the Chirocentridae and the Pristigasteridae (Nelson, 1970; Nelson, 2006; Whitehead, 1985; Whitehead et al., 1988). The respective monophylies of the Engraulidae, the Chirocentridae and the Pristigasteridae are all well supported by morphological characters (Grande, 1985; Nelson, 1967, 1970), while the limits and content of the family Clupeidae have sometimes varied. Nevertheless, most of the recent studies agreed with the deWnition of Clupeidae given by G.J. Nelson (Nelson, 1970), which (1) excludes the Pristigasteridae; and (2) includes the Dussumieriinae (for alternative hypotheses consult Whitehead (1963a) and Lauder and Liem (1983)). The recognition of the family Congothrissidae (Poll, 1964), for the single African species Congothrissa polli, is still debated (Gourène and Teugels, 1994; Roberts, 1972; Taverne, 1977), although Grande (1985) supports the inclusion of Congothrissa within the Clupeidae (subfamily Pellonulinae). The relationships among the clupeoid families have been explored by several authors, but they remain mostly unresolved (Fig. 1A–E) with the exception of the hypothesis proposed by Di Dario (2002)(Fig. 1E). G.J. Nelson (1967, 1970) examined the morphology of the gill-arch structure of extant Clupeoidei (Fig. 1A). He found that the condition observed within the Pristigasterinae is suYciently distinct from the Clupeidae to exclude the Pristigasterinae from the Clupeidae. Other inter-familial relationships were left unresolved. Based on an extensive osteological comparison employing wide sampling of both extant and fossil taxa, Grande (1985) provided the most complete comparative work on this group to date. He diagnosed all of the four families previously recognized (Fig. 1C). However, with the exception of the hypothesized sister relationship between the Chirocentridae and the Clupeidae ( D the superfamily Clupeoidea), Grande left unresolved the other interfamilial relationships. Later, Patterson and Johnson (1995) proposed an additional synapomorphy—the “rib/epicentral fusion”—uniting the Chirocentridae to the Clupeidae. In a partially unpublished Ph.D. dissertation, Sato (1994, 1999) examined the osteology of this group (Fig. 1D). He recognized only three families within the Clupeoidei: the Engraulidae, the Chirocentridae and the Clupeidae (including the

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Pristigasteridae). The relative positions among these three families remain unresolved. Recently, Di Dario (2002) presented three osteological synapomorphies supporting a sister relationship between the superfamily Clupeoidea (Grande, 1985; Patterson and Johnson, 1995) and the Engraulidae; in consequence, the Pristigasteridae represents the sister group to this clade (Fig. 1E). The South-East Asian freshwater paedomorphic Wshes of the family Sundasalangidae (monogeneric, Sundasalanx; seven species) were originally thought to be related to the osmeroid family Salangidae because of their overall resemblance (Roberts, 1981). Recently, Siebert (1997) and Ishiguro et al. (2005) supported the inclusion of the family Sundasalangidae within the Clupeiformes based on morphological and molecular evidence, respectively. However, none of these authors proposed a clear placement of this group within the Clupeiformes. Siebert (1997) hypothesized a possible relationship between Sundasalanx and Jenkinsia (Clupeidae; Dussumieriinae), whereas the incomplete taxonomic sampling within the Clupeiformes (only six taxa) used by Ishiguro et al. (2005) did not allow these authors to propose a robust hypothesis. Our study supports the respective monophylies of the Engraulidae and Pristigasteridae, but not for the Clupeidae sensu Nelson (1970), because Chirocentrus (Chirocentridae) is nested within the Clupeidae. However, the higher rate of molecular evolution in Chirocentrus, the single genus of the family Chirocentridae, makes diYcult the estimation of its phylogenetic position. Our results support Nelson’s (1970) removal of the subfamily Pristigasterinae from the Clupeidae and elevation of it to the familial level. Expanding our taxonomic sampling within the Clupeiformes allows us to conWrm the insertion of Sundasalangidae within the Clupeoidei, although its relative position among the other clupeoid families is still unclear. In agreement with the conclusions of Britz and Kottelat (1999) but in contrast to those of Siebert (1997), we rejected a close relationship between Sundasalanx and Jenkinsia. Inferred relationships among the clupeoid families are not well supported by our mitogenomic dataset and are rather unstable from one analysis to the other. Our results only moderately support the sister relationship between the Engraulidae and the remaining clupeoids. On the other hand, we did not detect any evidence supporting any alternative hypothesis, in particular the Pristigasteridae as the sister group of the remaining clupeoids (Di Dario, 2002) (Table 2). 4.4. Towards a natural subfamilial arrangement within the Clupeidae The family Clupeidae (sensu Nelson, 1970) is the most speciose family within the Clupeiformes, with about 216 species and 66 genera (Fishbase, ver. April 2006: http:// www.Wshbase.org/search.cfm). The Clupeidae are commonly divided into Wve subfamilies, Dussumieriinae, Alosi-

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nae, Dorosomatinae, Clupeinae and Pellonulinae (Grande, 1985; Nelson, 1970; Whitehead, 1985; Whitehead et al., 1988). Several authors noted that at least three of these subfamilies – the Alosinae, Dorosomatinae and Clupeinae – are certainly not monophyletic (Grande, 1985; Nelson, 1970), but they left unchanged their classiWcation pending clariWcation from additional studies. Thus, Grande (1985), who failed to Wnd any derived character supporting the monophylies of these three groups, wrote, “the greatest remaining problem in clupeomorph systematics is to discover how the members of these three groups are interrelated within Clupeoidei.” In agreement with the remarks of Nelson (1970) and Grande (1985), the mitogenomic data do not support the monophylies of the subfamilies Alosinae, Dorosomatinae and Clupeinae (Fig. 2, Table 2). Only the subfamily Pellonulinae is recovered as monophyletic in our study, although we have examined only two closely related African species from the Pellonulinae (tribe Pelonulini), Pellonula and Odaxothrissa (Gourène and Teugels, 1994; Grande, 1985). Future studies should provide a better test of the monophyly of this subfamily by adding other representatives; in particular, from the East-African tribe of Ehiravini (Bertin, 1943; Gourène and Teugels, 1994; Stiassny, 2002; Whitehead, 1963b) or from some other possibly related Indo-PaciWc genera (Grande, 1985). We do not Wnd any evidence for a close relationship between the Dussumieriinae and the Pellonulinae as it has sometimes been suggested (Poll et al., 1965; Roberts, 1972) (Table 2). Instead of the conventional subfamilial arrangement, our data support the recognition of a distinct group within the Clupeidae (named A; Fig. 2) that excludes the representatives of the subfamily Dussumieriinae (Jenkinsia, Spratelloides and Etrumeus) and the clade (Clupea, Sprattus). Moreover, this clade A is divided into two well-supported clades, the Wrst one (named A1) includes the genera Sardina, Sardinops and Alosa; and the second one (named A2) includes all other clupeid genera examined in this study (i.e., Sardinella (Clupeinae), Ethmalosa (Alosinae), Dorosoma and Nematalosa (Dorosomatinae), and Pellonula and Odaxothrissa (Pellonulinae)). This phylogenetic arrangement matches rather well with some ideas expressed by Nelson (1967, 1970) even though our taxonomic sampling is incomplete. For example, Nelson (1967) already noted the distinctiveness of Clupea, Sprattus and some dussumieriins from the rest of the Clupeidae (our clade A) “ƒin having near the medial end of E4 a foramen for the fourth eVerent branchial artery.” A second character shared by both of these groups is the reduction of the abdominal scutes. Later, Nelson (1970) wrote, “It seems possible also that other “clupeine” genera (e.g., Sardina and Sardinops) really are close relatives of Alosa and should be classiWed with that genus.” The monophyly of our clade A could be supported by the non-overlap of the gillrakers, as again noted by Nelson (1970).

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Acknowledgments We warmly thank the following people who provide us some samples: Jean-Dominique Durand (IRD), Fumito Mutoh (Fisheries Research Agency), Marc Jerome (IFREMER), Robert Sabatier and Ilaria Guarniero (CdL Aquacoltura ed Ittiopatoloogia). For providing assistance during Weld trips in Gabon and for help in collecting sample of Odaxothrissa vittata, S.L. thanks Victor Mamonekene (Université de Brazzaville) and André Kamdem Toham (WWF-International). We also thank Mitsugu M. Yamauchi, Kohji Mabuchi, Jun G. Inoue and Yusuke Yamanoue for their assistance in the laboratory work, and John P. Sullivan and Matthew E. Arnegard for reading and commenting an earlier version of this manuscript. S.L. thanks Kyoko Koda, Yukiko Watanabe and Misako Seimiya for their greatly appreciated help in the administrative jungle. This study was supported by Research Grants No. 12NP0201, 15380131, 15570090, 15-3601, and 17207007 from the Japan Society for the Promotion of Science. S.L. was successively supported by a Postdoctoral Fellowship (No. 3601) of the Japan Society for the Promotion of Science (JSPS) and by a Postdoctoral Fellowship “Lavoisier-Japon” of the French Foreign Ministry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev. 2006.09.018. References Arratia, G., 1997. Basal teleosts and teleostean phylogeny. Palaeo Ichthyologica 7, 5–168. Bertin, L., 1943. Revue critique des Dussumiériidés actuels et fossiles. Description d’un genre nouveau. Bull. Inst. Océanograph. 40, 1–32. Brandley, M.C., Schmitz, A., Reeder, T.W., 2005. Partitioned Bayesian analyses, partition choice, and the phylogenetic relationships of scincid lizards. Syst. Biol. 54, 373–390. Britz, R., Kottelat, M., 1999. Sundasalanx mekongensis, a new species of clupeiform Wsh from the Mekong basin (Teleostei: Sundasalangidae). Ichthyol. Explor. Freshwaters 10, 337–344. Cheng, S., Higuchi, R., Stoneking, M., 1994. Complete mitochondrial genome ampliWcation. Nat. Genet. 7, 350–351. Di Dario, F., 2002. Evidence supporting a sister-group relationship between Clupeoidea and Engrauloidea (Clupeomorpha). Copeia, 496–503. Di Dario, F., 2004. Homology between the recessus lateralis and cephalic sensory canals, with the proposition of additional synapomorphies for the Clupeiformes and the Clupeoidei. Zool. J. Linn. Soc. 141, 257–270. Gourène, G., Teugels, G.G., 1994. Synopsis de la classiWcation et phylogénie des Pellonulinae de l’Afrique Occidentale et Centrale (Teleostei; Clupeidae). J. African Zool. 108, 77–91. Grande, L., 1985. Recent and fossil Clupeomorph Wshes with materials for revision of the subgroups of Clupeoids. Bull. Amer. Mus. Nat. Hist. 181, 231–372. Greenwood, P.H., 1968. The osteology and relationships of the Denticipitidae, a family of clupeomorph Wshes. Bull. Br. Mus. (Nat. Hist.) Zool. 16, 213–273. Greenwood, P.H., Rosen, D.E., Weitzman, S.H., Myers, G.S., 1966. Phyletic studies of teleostean Wshes, with a provisional classiWcation of living forms. Bull. Amer. Mus. Nat. Hist. 131, 339–456.

Huelsenbeck, J.P., Ronquist, F., 2001. Mrbayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2000. Complete mitochondrial DNA sequence of the Japanese sardine Sardinops melanostictus. Fish. Sci. 66, 924–932. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2001a. Complete mitochondrial DNA sequence of the Japanese anchovy Engraulis japonicus. Fish. Sci. 67, 828–835. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2001b. A mitogenomic perspective on the basal teleostean phylogeny: resolving higher-level relationships with longer DNA sequences. Mol. Phylogenet. Evol. 20, 275–285. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2003. Basal actinopterygian relationships: a mitogenomic perspective on the phylogeny of the “ancient Wsh”. Mol. Phylogenet. Evol., 26. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2004. Mitogenomic evidence for the monophyly of elopomorph Wshes (Teleostei) and the evolutionary origin of the leptocephalus larva. Mol. Phylogenet. Evol. 32, 274–286. Ishiguro, N.B., Miya, M., Inoue, J.G., Nishida, M., 2005. Sundasalanx (Sundasalangidae) is a progenetic clupeiform, not a closely-related group of salangids (Osmeriformes): mitogenomic evidence. J. Fish Biol. 67, 561–569. Ishiguro, N.B., Miya, M., Nishida, M., 2003. Basal euteleostean relationships: a mitogenomic perspective on the phylogenetic reality of the “Protacanthopterygii”. Mol. Phylogenet. Evol. 27, 476–488. Johansen, S., Bakke, I., 1996. The complete mitochondrial DNA sequence of Atlantic cod (Gadus morhua): relevance to taxonomic studies among codWshes. Mol. Mar. Biol. Biotechnol. 5, 203–214. Johnson, G.D., Patterson, C., 1996. Relationships of Lower Euteleostean Fishes. In: Stiassny, M.L.J., Parenti, L.R., Johnson, G.D. (Eds.), Interrelationships of Fishes. Academic Press, NEwYork, pp. 251–332. Kass, R.E., Raftery, A.E., 1995. Bayes Factors. J. Am. Stat. Assoc. 90, 773–795. Lauder, G.V., Liem, K.F., 1983. The evolution and interrelationships of the actinopterygian Wshes. Bull. Mus. Comp. Zool. 150, 95–197. Lavoué, S., Miya, M., Inoue, J.G., Saitoh, K., Ishiguro, N.B., Nishida, M., 2005. Molecular systematics of the gonorynchiform Wshes (Teleostei) based on whole mitogenome sequences: Implications for higher-level relationships within the Otocephala. Mol. Phylogenet. Evol. 37, 165–177. Lê, H.L.V., Lecointre, G., Perasso, R., 1993. A 28S rRNA-based phylogeny of the Gnathostomes: Wrst steps in the analysis of conXict and congruence with morphologically based Cladograms. Mol. Phylogenet. Evol. 2, 31–51. Lecointre, G., Nelson, G.J., 1996. Clupeomorpha, sister-group of Ostariophysi. In: Stiassny, M.L.J., Parenti, L.R., Johnson, G.D. (Eds.), Interrelationships of Wshes. Academic Press, New York, pp. 193–207. Loytynoja, A., Milinkovitch, M.C., 2003. A hidden Markov model for progressive multiple alignment. Bioinformatics 19, 1505–1513. Miya, M., Kawaguchi, A., Nishida, M., 2001. Mitogenomic exploration of higher teleostean phylogenies: a case study for moderate-scale evolutionary genomics with 38 newly determined complete mitochondrial DNA sequences. Mol. Biol. Evol. 18, 1993–2009. Miya, M., Nishida, M., 1999. Organization of the mitochondrial genome of a deep-sea Wsh, Gonostoma gracile (Teleostei : Stomiiformes): Wrst example of transfer RNA gene rearrangements in bony Wshes. Mar. Biotechnol. 1, 416–426. Miya, M., Nishida, M., 2000. Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximumparsimony optimality criterion. Mol. Phylogenet. Evol. 17, 437–455. Miya, M., Satoh, T.R., Nishida, M., 2005. The phylogenetic position of toadWshes (order Batrachoidiformes) in the higher ray-Wnned Wsh as inferred from partitioned Bayesian analysis of 102 whole mitochondrial genome sequences. Biol. J. Linn. Soc. 85, 289–306. Miya, M., Takeshima, H., Endo, H., et al., 2003. Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences. Mol. Phylogenet. Evol. 26, 121–138.

S. Lavoué et al. / Molecular Phylogenetics and Evolution 43 (2007) 1096–1105 Murakami, M., Yamashita, Y., Fujitani, H., 1998. The complete sequence of mitochondrial genome from a gynogenetic triploid “ginbuna” (Carassius auratus langsdorW). Zool. Sci. 15, 335–337. Nelson, G.J., 1967. Gill arches of teleostean Wshes of the family Clupeidae. Copeia 1967, 389–399. Nelson, G.J., 1970. The hyobranchial apparatus of teleostean Wshes of the families Engraulidae and Chirocentridae. Am. Mus. Novit. 2410, 1–30. Nelson, G.J., 1973. Relationships of clupeomorphs, with remarks on the structure of the lower jaw in Wshes. In: Greenwood, P.H., Miles, R.S., Patterson, C. (Eds.), Interrelationships of Wshes. Academic press, London, pp. 333–349. Nelson, J.S., 2006. Fishes of the World, fourth ed. John Wiley and Sons, NewYork. Niquen, M., Bouchon, M., 2004. Impact of El Nino events on pelagic Wsheries in Peruvian waters. Deep-Sea Res. Part II 51, 563–574. Patterson, C., Johnson, G.D., 1995. The intermuscular bones and ligaments of teleostean Wshes. Smithson. Contrib. Zool. 559, 1–85. Phillips, M.J., Delsuc, F., Penny, D., 2004. Genome-scale phylogeny and the detection of systematic biases. Mol. Biol. Evol. 21, 1455–1458. Poll, M., 1964. Une famille dulçicole nouvelle de poissons africains: les Congothrissidae. Acad. R. Sci. d’Outre-Mer 15, 1–40. Poll, M., Whitehead, P.J.P., Hopson, A.J., 1965. A new genus and species of Clupeoid Wsh from West Africa. Bull. Acad. R. Belg. Cl. Sci. 51, 277–292. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Roberts, T.R., 1972. Osteology and description of Thrattidion noctivagus, a minute, new freshwater clupeid Wsh from Cameroon, with a discussion of Pellonulin relationships. Breviora 382, 1–25. Roberts, T.R., 1981. Sundasalangidae, a new family of minute freshwater salmoniform Wshes from Southeast Asia. Proc. Calif. Acad. Sci. 42, 295–302. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Saitoh, K., Hayashizaki, K., Yokoyama, Y., Asahida, T., Toyohara, H., Yamashita, Y., 2000. Complete nucleotide sequence of Japanese Xounder (Paralichthys olivaceus) mitochondrial genome: structural properties and cue for resolving teleostean relationships. J. Hered. 91, 271–278. Saitoh, K., Miya, M., Inoue, J.G., Ishiguro, N.B., Nishida, M., 2003. Mitochondrial genomics of ostariophysan Wshes: perspectives on phylogeny and biogeography. J. Mol. Evol. 56, 464–472.

1105

Sato, Y., 1994. Cladistic-phenetic relationships and the evolutionary classiWcation of the suborder Clupeoidei (Pisces, Teleostei). Unpublished thesis (in Japanese), Tokyo Metropolitan University, Tokyo. Sato, Y., 1999. Methods for evolutionary classiWcation. In: Matsuka, K., Miya, M. (Eds.), Natural History of Fishes. Hokkaido University, Sapporo, pp. 24–41 (in Japanese). Siebert, D.J., 1997. Notes on the anatomy and relationships of Sundasalanx Roberts (Teleostei, Clupeidae), with descriptions of four new species from Borneo. Bull. Br. Mus. (Nat. Hist.) Zool. 63, 13–26. Simmons, M.P., Miya, M., 2004. EYciently resolving the basal clades of a phylogenetic tree using Bayesian and parsimony approaches: a case study using mitogenomic data from 100 higher teleost Wshes. Mol. Phylogenet. Evol. 31, 351–362. Stiassny, M.L.J., 2002. Revision of Sauvagella Bertin (Clupeidae; Pellonulinae; Ehiravini) with a description of a new species from the freshwaters of Madagascar and diagnosis of the Ehiravini. Copeia, 67–76. Taverne, L., 1977. Le complexe squelettique mésethmoidien de Congothrissa et la validité de la famille des Congothrissidae au sein de l’ordre des clupéiformes sensu stricto (Pisces, Teleostei). Rev. Zool. Afr. 91, 330–336. Whitehead, P.J.P., 1963a. A contribution to the classiWcation of clupeoid Wshes. Ann. Mag. nat. Hist. 5, 737–750. Whitehead, P.J.P., 1963b. A revision of the recent round herrings (Pisces: Dussumieriidae). Bull. Br. Mus. (Nat. Hist.) Zool. 10, 305–380. Whitehead, P.J.P., 1985. Clupeoid Wshes of the World (Suborder Clupeoidei): An annotated and illustrated catalogue of the herrings, sardines, pilchards, sprats, shads, anchovies and wolf herrings. Part 1. Chirocentridae, Clupeidae and Pristigasteridae. FAO Fish. Synop. 125, 1–303. Whitehead, P.J.P., Nelson, G.J., Wongratana, T., 1988. Clupeoid Wshes of the World (Suborder Clupeoidei): An annotated and illustrated catalogue of the herrings, sardines, pilchards, sprats, shads, anchovies and wolf herrings. Part 2. Engraulididae. FAO Fish. Synop. 125, 305–579. Yamanoue, Y., Miya, M., Matsuura, K., Katoh, M., Sakai, H., Nishida, M., 2004. Mitochondrial genomes and phylogeny of the ocean sunWshes (Tetraodontiformes : Molidae). Ichthyol. Res. 51, 269–273. Yang, Z., 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39, 306–314. Zaragüeta-Bagils, R., Lavoué, S., Tillier, A., Bonillo, C., Lecointre, G., 2002. Assessment of otocephalan and protacanthopterygian concepts in the light of multiple molecular phylogenies. C.R. Biol. 325, 1191–1207.