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A phylogeny of the fish family Sparidae (porgies) inferred from mitochondrial sequence data
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 425–434 www.elsevier.com/locate/ympev
A phylogeny of the fish family Sparidae (porgies) inferred from mitochondrial sequence dataq Thomas M. Orrella,* and Kent E. Carpenterb a
NOAA/NMFS Systematics Laboratory, Smithsonian Institution, NHB MRC-153, P.O. Box 37012, Washington, DC 20013-7012, USA b Department of Biology, Old Dominion University, Norfolk, VA 23529, USA Received 25 September 2001; revised 15 January 2004 Available online 5 March 2004
Abstract The porgies (Sparidae) comprise a diverse group of neritic fishes with a broad geographic distribution. We used mitochondrial DNA sequences from partial 16S ribosomal RNA and cytochrome b genes to reconstruct the phylogenetic history of these fishes. Sequences from 38 sparid species, 10 species in outgroups closely related to sparids, seven basal percoid species, and a non-perciform outgroup species were analyzed with parsimony and maximum likelihood. The Sparidae were monophyletic with the inclusion of Spicara, which is currently placed in the Centracanthidae. The genera Spicara, Pagrus, and Pagellus, were not monophyletic indicating a need for revision. Two main sparid lineages were recovered in all analyses, but the previously proposed six sparid subfamilies (Boopsinae, Denticinae, Diplodinae, Pagellinae, Pagrinae, and Sparinae) were not monophyletic. This suggests that dentition and feeding modes, upon which these subfamilies are based, were independently derived multiple times within sparid fishes. There was no evidence from the 16S or combined analyses for a monophyletic Sparoidea. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Perciformes; Percoidei; Sparoidea; Sparidae; phylogeny; mtDNA; 16S; rRNA; cyt b
1. Introduction The porgies (Percoidei: Sparidae) are primarily coastal fishes with about 110 species and 33 genera. Smith (1938) and Smith and Smith (1986) placed the genera of Sparidae in four subfamilies (Boopsinae, Denticinae, Pagellinae, and Sparinae) based primarily on dentition. Members of Boopsinae are herbivores and have compressed outer incisiform teeth and Denticinae are piscivores with enlarged canines in front and smaller conical teeth behind. Pagellinae lack canines, have small conical outer teeth, small inner molars, and are carnivorous on small invertebrates. Sparinae have jaws with bluntly rounded posterior molars and enlarged front teeth, and are carnivorous on crustaceans, mollusks, and small fishes. Akazaki (1962) further subdivided Sparinae q The authors would be happy to provided their 16S aligned data on request. * Corresponding author. Fax: 1-202-357-2986. E-mail address: [email protected] (T.M. Orrell).
1055-7903/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.01.012
by erecting two new subfamilies, Diplodinae and Pagrinae. Diplodinae have six to eight anterior teeth in the jaws and obliquely projecting incisors. Pagrinae have four canines on the upper jaw, four to six canines on the lower jaw, scales on the head extending to the interorbital region, and molar teeth in two series. Hanel and Sturmbauer (2000) used 16S sequences to estimate a phylogeny of northeastern Atlantic and Mediterranean sparids on which they mapped sparid trophic types. They examined a 486 bp fragment for 24 sparid fishes covering 10 sparid genera, and used the centracanthid Spicara as the outgroup. Their findings showed no support for three of the currently defined subfamilies (Boopsinae, Denticinae, and Sparinae) and that members of the genus Dentex are polyphyletic. Because Hanel and Sturmbauer (2000) used only the centracanthid Spicara as an outgroup, their study could not examine the placement of the sparids to closely related families. Orrell et al. (2002) inferred a phylogeny of representatives of all 33 sparid genera and a number of percoid outgroups using complete mitochondrial cytochrome b (cyt b) sequences.
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They also found the currently defined subfamilies to be non-monophyletic and found Spicara to be a member of the sparid ingroup. Day (2002) in a morphological analysis of most Sparidae genera found no support for the previously defined subfamilies and found Spicara to be nested deeply within the Sparidae. The Sparidae have variously been placed together with Caesionidae, Haemulidae, Lethrinidae, Lutjanidae, and Nemipteridae (Jordan and Fesler, 1893; Schultz, 1953). Akazaki (1962) used comparative morphology to define ‘‘spariform’’ fishes as including Lethrinidae, Nemipteridae, and Sparidae. Akazaki suggested that spariform fishes had three ‘‘stems’’: the primitive Nemipteridaestem; the intermediate Sparidae-stem; and the highly specialized Lethrinidae-stem. Johnson (1980) included AkazakiÕs three spariform families in his superfamily Sparoidea and he added Centracanthidae based on similarity with Sparidae of the maxillary–premaxillary distal articulation and other osteological characters. Centracanthidae were considered members of Sparidae by Jordan and Fesler (1893) and very closely related to Sparidae based on jaw morphology by Regan (1913) and Smith (1938). Johnson (1980) noted the close relationship of Centracanthidae to Sparidae, but he retained the family status of Centracanthidae, Apending a more complete understanding of sparoid interrelationships. Carpenter and Johnson (2002) concluded from a cladistic analysis of 54 morphological characters that the centracanthids were unresolved with respect to the Sparidae. They proposed the phylogenetic order to be Nemipteridae, Lethrinidae, Sparidae plus Centracanthidae. Here we report the results of a phylogenetic analysis of the sequences of partial mitochondrial 16S rRNA for 38 sparid taxa, 17 closely related percoid taxa, and one non-perciform outgroup. In addition, we report phylogenetic relationships estimated from combining the 16S sequences with cyt b sequences reported in Orrell et al. (2002). Parsimony and maximum likelihood analyses were used to test the monophyly of Sparidae, validity of the six subfamilies of Sparidae, evolutionary relationships of the 33 genera of the Sparidae, monophyly of Sparoidea, and relationship of sparoid fishes to other classically related percoids.
2. Materials and methods Materials examined, tissue preservation, DNA isolation, amplification, cloning, and sequencing for cyt b sequences are given in Orrell et al. (2002). Where possible the same voucher was used as a source of DNA for sequences of both cyt b and 16S. Sampling included all 33 recognized genera of the family Sparidae, other members of the superfamily Sparoidea (Centracanthidae, Lethrinidae, and Nemipteridae), and possible close outgroups in the Percoidei
(Caesionidae, Haemulidae, and Lutjanidae). The percoids, Centropomus and Moronidae + Lateolabrax, were used to root the resulting trees. The ostariophysin, Cyprinus carpio, was used as a distant outgroup. GenBank 16S sequences were used for C. carpio and Morone chrysops. Voucher designations and collection data are provided in Table 1. When possible, vouchers specimens were saved and deposited in regional or national museums. In some cases photographic vouchers were taken. In many cases, vouchers were too large to be preserved or preservation was not possible. All specimens were identified by co-authors or by regional taxonomic experts. Gill tissue or white muscle tissue was dissected from fresh or frozen samples and placed into a buffer solution of 0.25 M disodium ethylenediaminetetra-acetate (EDTA), 20% dimethyl sulfoxide (DMSO), saturated sodium chloride (NaCl), at pH 8.0 (Seutin et al., 1990), and stored at room temperature. The 16S primer sequences used for PCR amplification in this study were the 16Sar-50 (Palumbi et al., 1991) and 16Sbr-30 (Kocher et al., 1989). Primers were ordered from Genosys (Genosys Biotechnologies, The Woodlands, TX). A 50 ll PCR amplification of 16S was performed with 5–10 ng of each template DNA. The following reagents from the PCR Reagent System (Gibco-BRL Life Technologies) were used in each reaction: 5 ll 10 PCR Buffer plus Mg (200 mM Tris–HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2 ); 1 ll 10 mM dNTP Mix (10 mM each dATP, dCTP, dGTP, and dTTP); 50 pmols of each primer, 0.25 ll Platinum Taq DNA polymerase (5 U/ll). Platinum Taq was used to reduce the possibility of PCR miss-incorporations. All amplifications were performed on a MJ Research PTC200 thermocycler (Watertown, MA) with the following cycle parameters: initial denaturation of 95 °C for 4.0 min; 35 cycles of [denaturation 94 °C for 1.0 min, annealing 45–47 °C (depending on sample) for 1.0 min; extension 65 °C for 3.0 min]; final extension of 65 °C for 10 min; hold at 4 °C indefinitely. Amplified target sequences were cloned using the Invitrogen TA Cloning Kit (Invitrogen) and plasmid DNA was obtained by either standard plasmid preparation protocols Sambrook et al. (1989) or by using a PERFECTprep kit (50 – 30 ). Pure plasmid DNA was suspended in 65 ll diH2 0. At least two plasmids preparations were sequenced for each exemplar (both light and heavy strands) using dideoxynucleotide chain termination Sanger et al. (1977). Plasmid DNA was quantified using a DyNAQuant 200 flourometer (Amersham–Pharmacia Biotech) and approximately 300 fmol of plasmid DNA was used in each cycle sequencing reaction. Forward and reverse IRD800 flourescently labeled M13 primers (Li-Cor) were used for sequencing. A heat-stable DNA polymerase, Thermo Sequenase TM (Amersham–Pharmacia Biotech) was used to incorporate the IRD800 flourescently labeled primer during cycle sequencing. To relax structural
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Table 1 Collection data for samples used in this study Species
GenBank Accession No.
Voucher Number
Collection locality
Outgroup Taxa Centropomidae Centropomus undecimalis
AF247437
No voucher
Gulf of Mexico, Florida
Cyprinidae Cyprinus carpio
X61010
Sequences From GenBank (Chang et al., 1994)
Haemulidae Haemulon sciurus Pomadasys maculatus
AF247442 AF247443
No voucher No voucher
Florida, Big Pine Key, W. of Bridge Manila Fish Market, Luzon, Manila, Philippines
Lateolabracidae Lateolabrax japonicus Lateolabrax latus
AF247438 AF247439
VIMS 10381 MTUF 27451
Picture voucher, Market Sample, Japan Sasebo, Nagasaki Prefecture, Japan
Lethrinidae Lethrinus ornatus Lethrinus rubrioperculatus
AF247446 AF247447
USNM 345259 No voucher
Bolinao, Luzon, Philippines W. Australia CSIRO SS 8/95/45, Australia
Lutjanidae Caesio cuning Lutjanus decussatus
AF247444 AF247445
USNM 345193 USNM 346695
Fish market, Iloilo Panay, Philippines Fish market, northern Negros, Philippines
Moronidae Dicentrarchus punctatus Morone americana
AF247437 AF247440
No voucher No voucher
Fish market, Spain VIMS Trawl Survey, Chesapeake Bay, Virginia
Morone chrysops
AF055610
Morone saxatilis
AF247441
Sequences From GenBank (Tang et al., 1999) VIMS Uncat
AF247448
USNM 345202
AF247449
USNM 346853
Manila Fish Market, Luzon, Manila, Philippines Guimaras Island, Philippines
Sparidae Boopsinae Boops boops Crenidens crenidens Gymnocrotaphus curvidens Oblada melanura Pachymetopon aeneum Polyamblyodon germanum Sarpa salpa Spondyliosoma cantharus
AF247396 AF247397 AF247398 AF247399 AF247400 AF247401 AF247402 AF247403
No voucher No voucher RUSI 49447 No voucher RUSI 49672 RUSI 49690 RUSI 49456 ODU 2782
Fiumicino Fish Market, Italy Qatif Market, eastern Saudi Arabia Kenton-on-Sea, South Africa Spain, Azohia, Bay of Cartagena Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Fiumicino Fish Market, Italy
Denticinae Argyrozona argyrozona Cheimerius nufar Dentex tumifrons Petrus rupestris Polysteganus praeorbitalis Sparidentex hasta
AF247404 AF247405 AF247406 AF247407 AF247408 AF247431
RUSI 58449 RUSI 49443 AMS I.36450-002 RUSI 49684 RUSI 49686 ODU 2783
Durban Fish Market, South Africa Kenton-on-Sea, South Africa Nelson Bay, Australia Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Shuwaik Market, Kuwait City, Kuwait
Diplodinae Archosargus probatocephalus Diplodus bermudensis Diplodus cervinus Diplodus holbrooki Lagodon rhomboides
AF247414 AF247419 AF247420 AF247421 AF247423
VIMS 010192 No voucher RUSI 49680 ODU 2789 VIMS Uncat
Chesapeake Bay, Virginia Bermuda Kenton-on-Sea, South Africa Atlantic, South Carolina Florida Keys, Bahia Honda Ocean Side, Florida
Nemipteridae Nemipterus marginatus Scolopsis ciliatus
VIMS Trawl Survey, Chesapeake Bay, Virginia
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Table 1 (continued) Species
GenBank Accession No.
Voucher Number
Collection locality
Outgroup Taxa Pagellinae Boopsoidea inornata Lithognathus mormyrus Pagellus bogaraveo Pagellus bellottii
AF247409 AF247410 AF247411 AF247412
ODU ODU ODU ODU
St. Sebastian Bay, South Africa Fiumicino Fish Market, Italy Fiumicino Fish Market, Italy R/V African, Station 17491, South Africa
Pagrinae Argyrops spinifer Evynnis japonica Pagrus auratus Pagrus auriga Pagrus pagrus
AF247415 AF247422 AF247424 AF247425 AF247426
AMS I.36447-001 NSMT-P 47497 No voucher ODU 2786 ODU 2790
N. Territory, Australia Miyazaki, Kyushu Prefecture, Japan Sydney Fish Market, New Zealand V. Emmanul Fish Market, Rome, Italy Atlantic, South Carolina
Sparinae Acanthopagrus berda Calamus nodosus Chrysoblephus cristiceps Cymatoceps nasutus Porcostoma dentata Pterogymnus laniarius Rhabdosargus thorpei Sparodon durbanensis Sparus auratus Stenotomus chrysops
AF247413 AF247416 AF247417 AF247418 AF247427 AF247428 AF247429 AF247430 AF247432 AF247433
USNM 345989 No voucher RUSI 49441 RUSI 49445 RUSI 58450 No voucher RUSI 49683 RUSI 49673 ODU 2787 No voucher
Philippines, Manila, Market Atlantic S.E. Charleston, South Carolina Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Durban, South Africa Plettenberg Bay, South Africa Ponta do Ouro, Mozambique Kenton-on-Sea, South Africa Fiumicino Fish Market, Italy Chesapeake Bay
Centracanthidae Spicara alta Spicara maena
AF247434 AF247435
ODU 2793 ODU 2788
Angola Fiumicino Fish Market, Italy
2791 2784 2785 2792
Note. GenBank Accession No., museum collection numbers, and collection locality are given. Museum acronyms are following Leviton et al. (1985) and Leviton and Gibbs (1988).
stops during electrophoreses, 7-deaza-dGTP was used during chain building. The flourescently labeled termination reactions were electrophoresed through a 66 cm, 0.25 mm thick, 4% LongRanger (FMC BioProducts) acrylamide gel on a Li-Cor 4000L automated sequencer. The resulting electronic gel image was analyzed using BaseImage V2.3 software (Li-Cor). The 16S nucleic acid sequences were aligned by the Clustal feature of Gene Jockey II (Biosoft, Cambridge, UK) and by CLUSTAL W (Thompson et al., 1994). Sequences were further aligned according to putative stem and loop structure following Wiley et al. (1998). Putative stem structures were those where base pairing might be expected. All other areas (loops, bulges, and non-paired regions) were classified as putative loops. A teleost model of the 16S fragment (Ortı et al., 1996) and a primate structural model (Horovitz and Meyer, 1995) were used to locate conserved loops and stems and then secondary-structure was folded by eye. Stem and loop regions were examined for base-pair complementarity and gaps were added to the alignment between distantly related taxa. The16S data set and the cyt b data set from Orrell et al. (2002) were assigned partitions in a single PAUP NEXUS file. Uncorrected sequence divergence was determined for pairwise comparisons of all taxa, ingroup taxa, and outgroup taxa. Gaps were treated as
missing data during calculations of sequence divergence. Stem and loop structures were examined for site saturation by plotting the total number of mutations as a function sequence divergence and the stem and loop transitions as a function of sequence divergence. Incongruence length difference (ILD) tests (Mickevich and Farris, 1981; Farris et al., 1994) were conducted on the 16S and cyt b data partitioned data using the partition homogeneity function in PAUP. Parsimony and maximum likelihood (ML) analyses were executed in PAUP* version 4.0b10 (Swofford, 2003) and performed on a 533 MHz, Alpha 64 bit processor running RedHat Linux version 7.1 Alpha. Parsimony analyses used heuristic searches of 1000 random taxon addition sequences with 10 trees held at each stepwise addition employing TBR branch swapping. No topological restraints were enforced and all characters were included and of equal weight. Gaps were coded either as a fifth character or as missing (uninformative). TreeRot.v2 (Sorensen, 1999) was used to calculate total Bremer support (BS) values at each node (Bremer, 1988) and to determine partitioned Bremer support (PBS) values (Baker and DeSalle, 1997; Baker et al., 1998) for each data partition in the combined parsimony tree. Bremer support values were calculated using 20 unrestricted random addition sequences per node. Jackknife
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support (JS) of 1000 replicates (37% character deletions, five random addition replicates following Farris et al., 1996) were also calculated to assess clade support for parsimony trees. Wilcoxon sign-ranks, two-tailed probability tests (Templeton, 1983) were used to determine if unconstrained most-parsimonious 16S and combined trees (gaps treated as fifth character in both) were significantly different (at P < 0:05) from constrained trees. Test constraints included the Sparidae, sensu strictu, the Sparidae + Spicara, each sparid subfamily and any ingroup genera that had multiple species in a tree. Modeltest Version 3.06 (Posada and Crandall, 1998) was used to select best-fit ML models for the 16S partitioned and combined data sets. A heuristic search of 10 replicates was used to determine the best-fit ML tree and bootstrap analysis (BA) of 100 replicates provided an estimate of nodal support.
3. Results Detailed sequence characteristics, sequence divergence, and mutational analysis for cyt b data are reported in Orrell et al. (2002). Sequencing of the 16S mtDNA gene produced an average of 574 (range 559–585) nucleotide base pairs per taxon. Multiple alignments resulted in a consensus length of 621 positions (base pairs and gaps). On average more regions sequenced were designated to the loop category (381 sites) than to the stem category (240 sites). Of the 621 positions, 40% were constant and 22% were parsimony uninformative. Most (80%) of the parsimony informative variable characters were from loop regions. The largest uncorrected sequence divergence was 26% between Centropomus undecimalis and Scolopsis ciliatus and the smallest was 1.2% between Pachymetopon aeneum and Polyamblyodon germanum. Mean pairwise sequence divergence between all pairwise comparisons of taxa was 9.70% (SD ¼ 0.048, n ¼ 1540). Average divergence between comparisons of outgroup taxa was 14.49% (SD ¼ 0.052, n ¼ 120) and between pairwise comparisons of ingroup taxa (including Spicara) 6.21% (SD ¼ 0.019, n ¼ 780). A plot of all substitutions as a function of uncorrected sequence divergence showed that an approximate of 70 substitutions was reached at greater than 25% sequence divergence. Transitional substitutions were found to be saturated at nearly 15% sequence divergence. Loop and stem transitions plotted as a function of uncorrected sequence divergence revealed that loop transitions saturated at nearly 15% sequence divergence. Stem transitions were not saturated as sequence divergence increased and reached a maximum of approximately 20 substitutions at greater than 25% sequence divergence. No incongruence was found between the 16S and cyt b data set partitions (observed distance D ¼ 7416, randomly selected distances S ¼ 39, P ¼ 0:61).
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3.1. 16S The heuristic search of the 16S data partition (gaps treated as fifth character) resulted in 276 equally parsimonious trees (tree length ¼ 1527). Of the 621 sites (base pairs and gaps), 251 were constant, 137 variable characters were parsimony uninformative, and 233 variable characters were parsimony informative. The best-fit ML model for the16S data was the complex, parameter rich, general time-reversible model with unequal base frequencies, six rate classes and a c rate heterogeneity parameter (GTR + I+ C). The ML analysis had the following statistics: estimated nucleotide frequencies A ¼ 0:289, C ¼ 0:274, G ¼ 0:208, T ¼ 0:229; nucleotide substitution rate matrix A $ C ¼ 1:80, A $ G ¼ 7:69, A $ T ¼ 2:85, C $ G ¼ 0:79, and C $ T ¼ 11:35; assumed proportion of invariable sites 0.23; and the shape of the estimated gamma parameter (a) ¼ 0.46. The heuristic search produced a single tree with a negative log likelihood score ()ln L) of 7066.30. The 16S parsimony and ML analyses supported two major clades within Sparidae, but the 16S parsimony was unable to fully resolve interrelationships of sparid genera and the internal nodes had very short branch lengths in the ML analysis. Templeton test results of constrained trees are given in Table 2. There was no significant difference between the most parsimonious trees (EPTs) and a constrained Sparidae sensu strictu (P ¼ 1:00), but there was a significant difference between a constrained Sparidae + Spicara and the EPTs. Within the Sparidae + Spicara, a clade comprising Boops boops + Sarpa salpa was basal to all other sparids. Pagrus, Pagellus, and Spicara were not monophyletic and the latter two genera were not significantly different when constrained and compared to the EPTs (P ¼ 0:005). None of the six subfamilies of Sparidae were monophyletic in this analysis. Templeton tests of constrained subfamilies showed that Boopsinae (P ¼ 0:004), Denticinae (P ¼ 0:016), Pagellinae (P ¼ 0:012), and Sparinae (P < 0:001) were significantly different than the unconstrained equally parsimonious trees. Diplodinae (P ¼ 0:191) and Pagrinae (P ¼ 0:464) were not significantly different from the unconstrained trees. There was no support from this analysis for Sparoidea; neither lethrinids nor nemipterids were sister to the sparids. When gap characters were treated as missing using the same parsimony parameters outlined above, the number of informative characters under parsimony was reduced to 218. A total of 344 equally parsimonious trees were recovered with length of 1377. The resulting consensus tree was similar to the tree with gaps coded as a fifth character, but there was less resolution for terminal sparid nodes. 3.2. Combined 16S and cyt b The combined data analysis (gaps treated as fifth character) yielded three equally parsimonious trees (tree
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Table 2 Results of Wilcoxon sign-ranks, two-tailed probability tests (Templeton, 1983) Constraint
Sparidae, sensu strictu Sparidae + Spicara Boopsinae Denticinae Diplodinae Pagellinae Pagrinae Sparinae Spicara All subfamilies Diplodus Pagrus Pagellus
16S
Combined
TL
n trees
Average P value
SD
TL
n trees
Average P value
SD
1527 1556 1554 1540 1539 1550 1530 1559 1556 1628 1527 1530 1552
276 140 1436 888 24 1003 8 2 212 6 24 356 418
n/a 0.007 0.004 0.016 0.191 0.012 0.464 <0.001 0.002 <0.001 n/a 0.492 0.005
n/a 0.004 0.003 0.008 0.023 0.006 0.014 0.001 0.001 0 n/a 0.054 0.003
7451 7528 7536 7491 7494 7531 7479 7545 7535 7773 7451 7500 7519
2 2 2 2 2 1 1 3 4 12 3 20 3
n/a <0.001 <0.001 0.006 0.003 <0.001 0.0231 <0.000 <0.001 <0.001 n/a 0.002 <0.001
n/a 0 0 0.001 0.001 0 0 0 0 0 n/a 0.001 0
Note. Unconstrained tree lengths:16S parsimony analysis (gaps as fifth character) TL ¼ 1527; Combined parsimony (gaps as fifth character) TL ¼ 7451. Constrained trees generated by a heuristic search of 100 random addition sequences with 5 trees held at each step. P values are averaged across all comparisons of unconstrained and constrained trees. * Indicates significant difference at P < 0:05.
length ¼ 7451). Of 1761 total characters, 753 (43%) characters were constant, 251 (14%) variable characters were parsimony uninformative, and 757 (43%) variable characters were parsimony informative characters. Jackknife support, total and partitioned Bremer support values are shown on a strict consensus of three equally parsimonious trees (Fig. 1). A monophyletic Sparidae + Spicara was recovered (JS ¼ 100, BS ¼ 35) with high Bremer support values from each of the data partition (16S PBS ¼ 11 and cyt b PBS ¼ 24). As in the 16S analysis, two major clades were found in the combined analysis (clades A and B, Fig. 1) with the most partitioned Bremer support coming from the cyt b partition (cyt b PBS ¼ 11.5 for clade A and PBS ¼ 12.6 for clade B). Contrary to the 16S parsimony tree, the clade containing Boops boops and Sarpa salpa was no longer basal to other Sparidae + Spicara, but was nested within clade B. All support for this node came from the cyt b partition (PBS ¼ 2.2). None of the sparid subfamilies were monophyletic in the combined parsimony tree, nor were Pagrus, Pagellus, or Spicara. Templeton tests of all subfamilies and of Pagrus, Pagellus, and Spicara were significantly different than the unconstrained tree. As in the 16S tree, there was no support from this analysis for Sparoidea. When gaps were treated as missing during parsimony analysis, three equally parsimonious trees were recovered, but the number of parsimony informative characters was reduced from 757 to 742 and the tree length was reduced to 7299 steps. The topology of the resulting consensus tree was identical to the consensus tree where gaps were treated as a fifth character. As with the 16S data, the best-fit ML model for the combined data was GTR + I+ C. The combined data sets had the following statistics: estimated nucleotide frequencies A ¼ 0:302, C ¼ 0:370, G ¼ 0:105, and T ¼ 0:223; nu-
cleotide substitution rate matrix A $ C ¼ 0:29, A $ G ¼ 6:02, A $ T ¼ 0:67, C $ G ¼ 0:66, and C $ T ¼ 5:19; assumed proportion of invariable sites ¼ 0.40 and the shape of the estimated c parameter (a) ¼ 0.57. The heuristic search produced a single tree with a negative log likelihood score ()ln L) 30575.90 (Fig. 2). The combined ML tree supported a monophyletic Sparidae + Spicara. Two major sparid clades were found in the analysis (clades A and B, Fig 2). The addition of the cyt b data to the 16S data in the combined ML tree greatly strengthened nodal support for sparid generic relationships and for clade A (BA ¼ 97) and clade B (BA ¼ 95). Neither Sparoidea, the six sparid subfamilies, nor Pagrus, Pagellus, or Spicara were monophyletic in the combined ML analysis.
4. Discussion Pairwise sequence divergences between outgroup taxa were similar to those reported for 16S in other teleosts. Farias et al. (1999) found a maximum uncorrected pairwise divergence of 20% (average 17%) in cichlids, Wiley et al. (1998) found a maximum of >25% TamuraNei distance in acanthomorph relationships, and Tang et al. (1999) found <20% Tamura-Nei distance in Acanthuroidei. On average, the mean sequence divergence for all pairwise comparisons of taxa in our study (9.7%) was well below the level at which loop substitutions appeared to reach saturation. Putative loop mutations deviated from a linear relationship when genetic distance approached approximately 15% sequence divergence. The average pairwise divergence for ingroup species (6.21%) was well below the apparent level of loop saturation. Average mean pairwise divergence for
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Fig. 1. A strict consensus of 3 equally parsimonious trees derived from parsimony analysis of combined data. Subfamilies are labeled as follows: BO, boopsinae; DE, denticinae; DI, diplodinae; PA, pagrinae; PE, pagellinae; and SP, sparinae. At each node are support values: leftmost is the jackknife value, within parentheses are partitioned decay values for 16S then cyt b and the rightmost is the total decay value. The two major sparid clades are designated A, B.
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Fig. 2. Maximum likelihood tree derived from combined data. Subfamilies are labeled as in Fig. 1. Values at nodes represent bootstrap support (given for bootstrap values P50%). The two major sparid clades are designated A, B.
outgroup species was 14.49%, nearly at the same level at which apparent saturation occurred in loops. Because neither stem nor loop characters were saturated for ingroup species, all 16S characters were potentially informative and were treated with equal weight. An equal
weight of all characters was defendable as a means to infer sparid relationships, but potentially inconsistent as a weight to infer outgroup relationships. The pairwise sequence divergence between Sparidae and outgroup taxa appeared saturated for loop transitions, and the
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relationships inferred from these characters could be mis-informative. Therefore, the reliability of 16S to infer the deep node separating Lethrinidae and Nemipteridae from the Aother percoids and Sparidae + Spicara is questionable because all node support comes from the loop characters and not from the more conserved stem characters. Both the 16S data and combined data analyses supported a monophyletic Sparidae + Spicara. The sister relationships of Dentex tumifrons with Spicara alta and of Spondyliosoma cantharus with Spicara maena were also found in Orrell et al. (2002). The overall position of Spicara and Centracanthidae is problematic without a more complete understanding of both genera and all species of Centracanthidae. This study included two species of Spicara but none of Centracanthus. The 16S parsimony and ML analyses supported two major clades within Sparidae, but the 16S parsimony was unable to fully resolve interrelationships of sparid genera and the internal nodes had very short branch lengths in the ML analysis. Of the five ingroup genera that contained multiple representatives, only Diplodus was monophyletic. Hanel and Sturmbauer (2000) found Pagellus, Pagrus, and Dentex non-monophyletic and Orrell et al. (2002) found Pagellus, Pagrus, Dentex, and Spicara, non-monophyletic using cyt b. There was no support from the 16S sequence data for any of the subfamilies proposed by Smith (1938); Akazaki (1962), and Smith and Smith (1986). The monophyly of the Denticinae and Pagrinae could not be overwhelmingly challenged based on the 16S data as the constrained trees were not significantly different from the unconstrained equally parsimonious trees. Once the more robust cyt b data was combined with the 16S data, the constrained trees for all sparid subfamilies were significantly different from unconstrained trees. Clearly, based on the combined sequence data, the subfamilies as currently defined are artificial, in agreement with the findings of Hanel and Sturmbauer (2000). Despite the regional nature of Hanel and SturmbauerÕs (2000) study and an unfortunate choice of Spicara outgroup, the authors had a broad enough sampling of sparid species to conclude that trophic types evolved more than once in sparid fishes. The subfamilies as currently defined are most useful as categories that aid in field identification of sparids. Dentition characters are easily recognizable and generally aid in separating regional species. These subfamilies are not based on phylogenetic information, as demonstrated in the present and previous studies (Hanel and Sturmbauer, 2000; Day, 2002; Orrell et al., 2002), and should not be considered valid or a means by which to classify sparids. The 16S sequence analyses generally lacked the ability to resolve relationships between Sparidae and other percoid families. Neither AkazakiÕs spariform fishes or JohnsonÕs Sparoidea were reconstructed. This is in
433
contrast to the recent findings of Carpenter and Johnson (2002) with significant osteological evidence for a Sparidae–Lethrinidae sister group relationship. Orrell et al. (2002) found the Lethrinidae sister to Sparidae using cyt b, but only a weighted cyt b tree supported the superfamily Sparoidea (Sparidae, Centracanthidae, Lethrinidae, and Nemipteridae). There was only minimal support in the weighted cyt b data for the Sparoidea; most support was from the third and first codon positions. In the present study, there was no support for a lethrinid–sparid sister relationship nor for a monophyletic Sparoidea from either the combined parsimony or ML trees. Although no attempt was made to account for cyt b saturation in third positional substitutions in the combined parsimony analysis, the ML combined model (GTR + I+ C) was sensitive to this saturation. Yet, it failed to reconstruct a monophyletic Sparoidea. Since 16S is more conservative than cyt b (less overall changes between taxa), it should be informative for deeper relationships (i.e., within Perciformes). However, there was no support from the 16S analysis for deeper relationships found in Carpenter and Johnson (2002) and Orrell et al. (2002). The apparent saturation of transitions in loop characters found in comparisons between outgroup species might account for the inability of the 16S sequence analyses to recover a monophyletic Sparoidea and for the low overall support for outgroup relationships. This study and Orrell et al. (2002) found that cyt b and 16S alone and combined where useful for intrafamilial sparid relationships. Due to the inability of the combined mtDNA analysis to reconstruct a monophyletic Sparoidea and the lack of support for deeper relationships, we question the use of mtDNA as an appropriate marker for phylogenetic inference among perciform families. A future analysis of sparoid relationships might utilize a nuclear marker which could be better suited for perciform interfamilial relationships.
Acknowledgments We thank the following individuals and organizations for their assistance in collecting specimens, without whose help this work would not have been possible: S. Almatar, L. Beckley, B.B. Collette, F. Crock, N. DeAngelis, M. DeGravelle, D. Etnier, H. Ishihara, J. Gelsleichter, A. Graham, R. Grubbs, K. Harada, Y. Iwatsuki, J. Jenke, R. Kraus, E. Massuti, K. Matsuura, L. Ter Morshuizen, P. Oliver, A.W. Paterson, J. Paxton, J. Scialdone, D. Scherrer, G. Sedberry, M. Smale, W. F. Smith-Vaniz, K. Utsugi, G. Yearsley, T. Wasaff, J. T. Williams, and the VIMS Trawl Survey. We acknowledge the following people for laboratory assistance at Virginia Institute of Marine Science (VIMS): J. McDowell, D. Carlini, V. Buonaccorsi, C. Morrison, and K.
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Macdonald. We thank the help and the advice from J. Musick, J. Graves, K. Reece, P. Van Veld, and G. D. Johnson. We thank Jeff Bates, Laboratory of Molecular Systematics for analysis support. We thank B.B. Collette and E.O. Wiley for manuscript review and comments. This research was supported by grants to TMO from: the Fisheries Department, Food and Agriculture Organization of the United Nations, the Lerner Gray Fund for Marine Research from the American Museum of Natural History, an E.C. and C.E. Raney Award of the American Society of Ichthyologists and Herpetologists, and through the VIMS Graduate Deans Office. This paper is Contribution No. 2574 of the Virginia Institute of Marine Science, The College of William and Mary.
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Kocher, T.D., Thomas, W.K., Meyer, A., Edward, S.V., P€aa€bo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 6196–6200. Leviton, A.E., Gibbs Jr., R.H., 1988. Standards in herpetology and ichthyology standard symbolic codes for institution resource collections in herpetology and ichthyology. Supplement no. 1: additions and corrections. Copeia, 280–282. Leviton, A.E., Gibbs Jr., R.H., Heal, E., Dawson, C.E., 1985. Standards in ichthyology and herpetology: Part 1. Standard symbolic codes for institutional resource collections in herpetology and ichthyology. Copeia, 802–832. Mickevich, M.F., Farris, J.S., 1981. The implications of congruence in Menidia. Systematic Zoology 30, 351–369. Orrell, T.M., Carpenter, K.E., Musick, J.A., Graves, J.E., 2002. A phylogenetic and biogeographic analysis of the Sparidae (Perciformes: Percoidei) based on cytochrome b sequences. Copeia, 618–631. Ortı, G., Petry, P., Porto, J.I.R., Jegu, M., Meyer, A., 1996. Patterns of nucleotide change in mitochondrial ribosomal RNA genes and the phylogeny of piranhas. J. Mol. Evol. 42, 169–182. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L., Grabowski, G., 1991. The Simple Fools Guide to PCR. Department of Zoology and Kewalo Marine Laboratory, University of Hawaii, Honolulu, HI. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Regan, C.T., 1913. On the classification of the percoid fishes. Ann. Mag. Nat. Hist. 8, 111–145. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning : A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1-999.. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463– 5467. Schultz, L.P., 1953. Family Lutjanidae: snappers. In: Fishes of the Marshall and Marianas Islands, Vol. I Families from Asymmetrontidae through Siganidae. Bull. U.S. Natl. Mus. 202, pp. 1–685. Seutin, G., White, B.N., Boag, P.T., 1990. Preservation of avian blood and tissue samples for DNA analysis. Can. J. Zool. 69, 82–90. Smith, J.L.B., 1938. The South African fishes of the families Sparidae and Denticidae. Trans. R. Soc. S. Africa. 26, 225–305. Smith, J.L.B., Smith, M.M., 1986. Family No. 183: Sparidae. In: Smith, M.M., Heemstra, P.C. (Eds.), SmithÕs Sea Fishes. Macmillian, Johannesburg, South Africa, pp. 1–1050. Sorensen, M.D., 1999. TreeRot, version2. Boston University, Boston, MA. Swofford, D.L., 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Tang, K.L., Berendzen, P.B., Wiley, E.O., Morrissey, J.F., Winterbottom, R., Johnson, G.D., 1999. The phylogenetic relationships of the suborder Acanthuroidei (Teleostei: Perciformes) based on molecular and morphological evidence. Mol. Phylogenet. Evol. 11, 415–425. Templeton, A.R., 1983. Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to humans and apes. Evolution 37, 221–244. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wiley, E.O., Johnson, G.D., Dimmick, W.W., 1998. The phylogenetic relationships of lampridiform fishes (Teleosteoi: Acanthomorpha), based on a total-evidence analysis of morphological and molecular data. Mol. Phylogenet. Evol. 10, 417–425.
A phylogeny of the fish family Sparidae (porgies) inferred from mitochondrial sequence dataq Thomas M. Orrella,* and Kent E. Carpenterb a
NOAA/NMFS Systematics Laboratory, Smithsonian Institution, NHB MRC-153, P.O. Box 37012, Washington, DC 20013-7012, USA b Department of Biology, Old Dominion University, Norfolk, VA 23529, USA Received 25 September 2001; revised 15 January 2004 Available online 5 March 2004
Abstract The porgies (Sparidae) comprise a diverse group of neritic fishes with a broad geographic distribution. We used mitochondrial DNA sequences from partial 16S ribosomal RNA and cytochrome b genes to reconstruct the phylogenetic history of these fishes. Sequences from 38 sparid species, 10 species in outgroups closely related to sparids, seven basal percoid species, and a non-perciform outgroup species were analyzed with parsimony and maximum likelihood. The Sparidae were monophyletic with the inclusion of Spicara, which is currently placed in the Centracanthidae. The genera Spicara, Pagrus, and Pagellus, were not monophyletic indicating a need for revision. Two main sparid lineages were recovered in all analyses, but the previously proposed six sparid subfamilies (Boopsinae, Denticinae, Diplodinae, Pagellinae, Pagrinae, and Sparinae) were not monophyletic. This suggests that dentition and feeding modes, upon which these subfamilies are based, were independently derived multiple times within sparid fishes. There was no evidence from the 16S or combined analyses for a monophyletic Sparoidea. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Perciformes; Percoidei; Sparoidea; Sparidae; phylogeny; mtDNA; 16S; rRNA; cyt b
1. Introduction The porgies (Percoidei: Sparidae) are primarily coastal fishes with about 110 species and 33 genera. Smith (1938) and Smith and Smith (1986) placed the genera of Sparidae in four subfamilies (Boopsinae, Denticinae, Pagellinae, and Sparinae) based primarily on dentition. Members of Boopsinae are herbivores and have compressed outer incisiform teeth and Denticinae are piscivores with enlarged canines in front and smaller conical teeth behind. Pagellinae lack canines, have small conical outer teeth, small inner molars, and are carnivorous on small invertebrates. Sparinae have jaws with bluntly rounded posterior molars and enlarged front teeth, and are carnivorous on crustaceans, mollusks, and small fishes. Akazaki (1962) further subdivided Sparinae q The authors would be happy to provided their 16S aligned data on request. * Corresponding author. Fax: 1-202-357-2986. E-mail address: [email protected] (T.M. Orrell).
1055-7903/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.01.012
by erecting two new subfamilies, Diplodinae and Pagrinae. Diplodinae have six to eight anterior teeth in the jaws and obliquely projecting incisors. Pagrinae have four canines on the upper jaw, four to six canines on the lower jaw, scales on the head extending to the interorbital region, and molar teeth in two series. Hanel and Sturmbauer (2000) used 16S sequences to estimate a phylogeny of northeastern Atlantic and Mediterranean sparids on which they mapped sparid trophic types. They examined a 486 bp fragment for 24 sparid fishes covering 10 sparid genera, and used the centracanthid Spicara as the outgroup. Their findings showed no support for three of the currently defined subfamilies (Boopsinae, Denticinae, and Sparinae) and that members of the genus Dentex are polyphyletic. Because Hanel and Sturmbauer (2000) used only the centracanthid Spicara as an outgroup, their study could not examine the placement of the sparids to closely related families. Orrell et al. (2002) inferred a phylogeny of representatives of all 33 sparid genera and a number of percoid outgroups using complete mitochondrial cytochrome b (cyt b) sequences.
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They also found the currently defined subfamilies to be non-monophyletic and found Spicara to be a member of the sparid ingroup. Day (2002) in a morphological analysis of most Sparidae genera found no support for the previously defined subfamilies and found Spicara to be nested deeply within the Sparidae. The Sparidae have variously been placed together with Caesionidae, Haemulidae, Lethrinidae, Lutjanidae, and Nemipteridae (Jordan and Fesler, 1893; Schultz, 1953). Akazaki (1962) used comparative morphology to define ‘‘spariform’’ fishes as including Lethrinidae, Nemipteridae, and Sparidae. Akazaki suggested that spariform fishes had three ‘‘stems’’: the primitive Nemipteridaestem; the intermediate Sparidae-stem; and the highly specialized Lethrinidae-stem. Johnson (1980) included AkazakiÕs three spariform families in his superfamily Sparoidea and he added Centracanthidae based on similarity with Sparidae of the maxillary–premaxillary distal articulation and other osteological characters. Centracanthidae were considered members of Sparidae by Jordan and Fesler (1893) and very closely related to Sparidae based on jaw morphology by Regan (1913) and Smith (1938). Johnson (1980) noted the close relationship of Centracanthidae to Sparidae, but he retained the family status of Centracanthidae, Apending a more complete understanding of sparoid interrelationships. Carpenter and Johnson (2002) concluded from a cladistic analysis of 54 morphological characters that the centracanthids were unresolved with respect to the Sparidae. They proposed the phylogenetic order to be Nemipteridae, Lethrinidae, Sparidae plus Centracanthidae. Here we report the results of a phylogenetic analysis of the sequences of partial mitochondrial 16S rRNA for 38 sparid taxa, 17 closely related percoid taxa, and one non-perciform outgroup. In addition, we report phylogenetic relationships estimated from combining the 16S sequences with cyt b sequences reported in Orrell et al. (2002). Parsimony and maximum likelihood analyses were used to test the monophyly of Sparidae, validity of the six subfamilies of Sparidae, evolutionary relationships of the 33 genera of the Sparidae, monophyly of Sparoidea, and relationship of sparoid fishes to other classically related percoids.
2. Materials and methods Materials examined, tissue preservation, DNA isolation, amplification, cloning, and sequencing for cyt b sequences are given in Orrell et al. (2002). Where possible the same voucher was used as a source of DNA for sequences of both cyt b and 16S. Sampling included all 33 recognized genera of the family Sparidae, other members of the superfamily Sparoidea (Centracanthidae, Lethrinidae, and Nemipteridae), and possible close outgroups in the Percoidei
(Caesionidae, Haemulidae, and Lutjanidae). The percoids, Centropomus and Moronidae + Lateolabrax, were used to root the resulting trees. The ostariophysin, Cyprinus carpio, was used as a distant outgroup. GenBank 16S sequences were used for C. carpio and Morone chrysops. Voucher designations and collection data are provided in Table 1. When possible, vouchers specimens were saved and deposited in regional or national museums. In some cases photographic vouchers were taken. In many cases, vouchers were too large to be preserved or preservation was not possible. All specimens were identified by co-authors or by regional taxonomic experts. Gill tissue or white muscle tissue was dissected from fresh or frozen samples and placed into a buffer solution of 0.25 M disodium ethylenediaminetetra-acetate (EDTA), 20% dimethyl sulfoxide (DMSO), saturated sodium chloride (NaCl), at pH 8.0 (Seutin et al., 1990), and stored at room temperature. The 16S primer sequences used for PCR amplification in this study were the 16Sar-50 (Palumbi et al., 1991) and 16Sbr-30 (Kocher et al., 1989). Primers were ordered from Genosys (Genosys Biotechnologies, The Woodlands, TX). A 50 ll PCR amplification of 16S was performed with 5–10 ng of each template DNA. The following reagents from the PCR Reagent System (Gibco-BRL Life Technologies) were used in each reaction: 5 ll 10 PCR Buffer plus Mg (200 mM Tris–HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2 ); 1 ll 10 mM dNTP Mix (10 mM each dATP, dCTP, dGTP, and dTTP); 50 pmols of each primer, 0.25 ll Platinum Taq DNA polymerase (5 U/ll). Platinum Taq was used to reduce the possibility of PCR miss-incorporations. All amplifications were performed on a MJ Research PTC200 thermocycler (Watertown, MA) with the following cycle parameters: initial denaturation of 95 °C for 4.0 min; 35 cycles of [denaturation 94 °C for 1.0 min, annealing 45–47 °C (depending on sample) for 1.0 min; extension 65 °C for 3.0 min]; final extension of 65 °C for 10 min; hold at 4 °C indefinitely. Amplified target sequences were cloned using the Invitrogen TA Cloning Kit (Invitrogen) and plasmid DNA was obtained by either standard plasmid preparation protocols Sambrook et al. (1989) or by using a PERFECTprep kit (50 – 30 ). Pure plasmid DNA was suspended in 65 ll diH2 0. At least two plasmids preparations were sequenced for each exemplar (both light and heavy strands) using dideoxynucleotide chain termination Sanger et al. (1977). Plasmid DNA was quantified using a DyNAQuant 200 flourometer (Amersham–Pharmacia Biotech) and approximately 300 fmol of plasmid DNA was used in each cycle sequencing reaction. Forward and reverse IRD800 flourescently labeled M13 primers (Li-Cor) were used for sequencing. A heat-stable DNA polymerase, Thermo Sequenase TM (Amersham–Pharmacia Biotech) was used to incorporate the IRD800 flourescently labeled primer during cycle sequencing. To relax structural
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Table 1 Collection data for samples used in this study Species
GenBank Accession No.
Voucher Number
Collection locality
Outgroup Taxa Centropomidae Centropomus undecimalis
AF247437
No voucher
Gulf of Mexico, Florida
Cyprinidae Cyprinus carpio
X61010
Sequences From GenBank (Chang et al., 1994)
Haemulidae Haemulon sciurus Pomadasys maculatus
AF247442 AF247443
No voucher No voucher
Florida, Big Pine Key, W. of Bridge Manila Fish Market, Luzon, Manila, Philippines
Lateolabracidae Lateolabrax japonicus Lateolabrax latus
AF247438 AF247439
VIMS 10381 MTUF 27451
Picture voucher, Market Sample, Japan Sasebo, Nagasaki Prefecture, Japan
Lethrinidae Lethrinus ornatus Lethrinus rubrioperculatus
AF247446 AF247447
USNM 345259 No voucher
Bolinao, Luzon, Philippines W. Australia CSIRO SS 8/95/45, Australia
Lutjanidae Caesio cuning Lutjanus decussatus
AF247444 AF247445
USNM 345193 USNM 346695
Fish market, Iloilo Panay, Philippines Fish market, northern Negros, Philippines
Moronidae Dicentrarchus punctatus Morone americana
AF247437 AF247440
No voucher No voucher
Fish market, Spain VIMS Trawl Survey, Chesapeake Bay, Virginia
Morone chrysops
AF055610
Morone saxatilis
AF247441
Sequences From GenBank (Tang et al., 1999) VIMS Uncat
AF247448
USNM 345202
AF247449
USNM 346853
Manila Fish Market, Luzon, Manila, Philippines Guimaras Island, Philippines
Sparidae Boopsinae Boops boops Crenidens crenidens Gymnocrotaphus curvidens Oblada melanura Pachymetopon aeneum Polyamblyodon germanum Sarpa salpa Spondyliosoma cantharus
AF247396 AF247397 AF247398 AF247399 AF247400 AF247401 AF247402 AF247403
No voucher No voucher RUSI 49447 No voucher RUSI 49672 RUSI 49690 RUSI 49456 ODU 2782
Fiumicino Fish Market, Italy Qatif Market, eastern Saudi Arabia Kenton-on-Sea, South Africa Spain, Azohia, Bay of Cartagena Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Fiumicino Fish Market, Italy
Denticinae Argyrozona argyrozona Cheimerius nufar Dentex tumifrons Petrus rupestris Polysteganus praeorbitalis Sparidentex hasta
AF247404 AF247405 AF247406 AF247407 AF247408 AF247431
RUSI 58449 RUSI 49443 AMS I.36450-002 RUSI 49684 RUSI 49686 ODU 2783
Durban Fish Market, South Africa Kenton-on-Sea, South Africa Nelson Bay, Australia Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Shuwaik Market, Kuwait City, Kuwait
Diplodinae Archosargus probatocephalus Diplodus bermudensis Diplodus cervinus Diplodus holbrooki Lagodon rhomboides
AF247414 AF247419 AF247420 AF247421 AF247423
VIMS 010192 No voucher RUSI 49680 ODU 2789 VIMS Uncat
Chesapeake Bay, Virginia Bermuda Kenton-on-Sea, South Africa Atlantic, South Carolina Florida Keys, Bahia Honda Ocean Side, Florida
Nemipteridae Nemipterus marginatus Scolopsis ciliatus
VIMS Trawl Survey, Chesapeake Bay, Virginia
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Table 1 (continued) Species
GenBank Accession No.
Voucher Number
Collection locality
Outgroup Taxa Pagellinae Boopsoidea inornata Lithognathus mormyrus Pagellus bogaraveo Pagellus bellottii
AF247409 AF247410 AF247411 AF247412
ODU ODU ODU ODU
St. Sebastian Bay, South Africa Fiumicino Fish Market, Italy Fiumicino Fish Market, Italy R/V African, Station 17491, South Africa
Pagrinae Argyrops spinifer Evynnis japonica Pagrus auratus Pagrus auriga Pagrus pagrus
AF247415 AF247422 AF247424 AF247425 AF247426
AMS I.36447-001 NSMT-P 47497 No voucher ODU 2786 ODU 2790
N. Territory, Australia Miyazaki, Kyushu Prefecture, Japan Sydney Fish Market, New Zealand V. Emmanul Fish Market, Rome, Italy Atlantic, South Carolina
Sparinae Acanthopagrus berda Calamus nodosus Chrysoblephus cristiceps Cymatoceps nasutus Porcostoma dentata Pterogymnus laniarius Rhabdosargus thorpei Sparodon durbanensis Sparus auratus Stenotomus chrysops
AF247413 AF247416 AF247417 AF247418 AF247427 AF247428 AF247429 AF247430 AF247432 AF247433
USNM 345989 No voucher RUSI 49441 RUSI 49445 RUSI 58450 No voucher RUSI 49683 RUSI 49673 ODU 2787 No voucher
Philippines, Manila, Market Atlantic S.E. Charleston, South Carolina Kenton-on-Sea, South Africa Kenton-on-Sea, South Africa Durban, South Africa Plettenberg Bay, South Africa Ponta do Ouro, Mozambique Kenton-on-Sea, South Africa Fiumicino Fish Market, Italy Chesapeake Bay
Centracanthidae Spicara alta Spicara maena
AF247434 AF247435
ODU 2793 ODU 2788
Angola Fiumicino Fish Market, Italy
2791 2784 2785 2792
Note. GenBank Accession No., museum collection numbers, and collection locality are given. Museum acronyms are following Leviton et al. (1985) and Leviton and Gibbs (1988).
stops during electrophoreses, 7-deaza-dGTP was used during chain building. The flourescently labeled termination reactions were electrophoresed through a 66 cm, 0.25 mm thick, 4% LongRanger (FMC BioProducts) acrylamide gel on a Li-Cor 4000L automated sequencer. The resulting electronic gel image was analyzed using BaseImage V2.3 software (Li-Cor). The 16S nucleic acid sequences were aligned by the Clustal feature of Gene Jockey II (Biosoft, Cambridge, UK) and by CLUSTAL W (Thompson et al., 1994). Sequences were further aligned according to putative stem and loop structure following Wiley et al. (1998). Putative stem structures were those where base pairing might be expected. All other areas (loops, bulges, and non-paired regions) were classified as putative loops. A teleost model of the 16S fragment (Ortı et al., 1996) and a primate structural model (Horovitz and Meyer, 1995) were used to locate conserved loops and stems and then secondary-structure was folded by eye. Stem and loop regions were examined for base-pair complementarity and gaps were added to the alignment between distantly related taxa. The16S data set and the cyt b data set from Orrell et al. (2002) were assigned partitions in a single PAUP NEXUS file. Uncorrected sequence divergence was determined for pairwise comparisons of all taxa, ingroup taxa, and outgroup taxa. Gaps were treated as
missing data during calculations of sequence divergence. Stem and loop structures were examined for site saturation by plotting the total number of mutations as a function sequence divergence and the stem and loop transitions as a function of sequence divergence. Incongruence length difference (ILD) tests (Mickevich and Farris, 1981; Farris et al., 1994) were conducted on the 16S and cyt b data partitioned data using the partition homogeneity function in PAUP. Parsimony and maximum likelihood (ML) analyses were executed in PAUP* version 4.0b10 (Swofford, 2003) and performed on a 533 MHz, Alpha 64 bit processor running RedHat Linux version 7.1 Alpha. Parsimony analyses used heuristic searches of 1000 random taxon addition sequences with 10 trees held at each stepwise addition employing TBR branch swapping. No topological restraints were enforced and all characters were included and of equal weight. Gaps were coded either as a fifth character or as missing (uninformative). TreeRot.v2 (Sorensen, 1999) was used to calculate total Bremer support (BS) values at each node (Bremer, 1988) and to determine partitioned Bremer support (PBS) values (Baker and DeSalle, 1997; Baker et al., 1998) for each data partition in the combined parsimony tree. Bremer support values were calculated using 20 unrestricted random addition sequences per node. Jackknife
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support (JS) of 1000 replicates (37% character deletions, five random addition replicates following Farris et al., 1996) were also calculated to assess clade support for parsimony trees. Wilcoxon sign-ranks, two-tailed probability tests (Templeton, 1983) were used to determine if unconstrained most-parsimonious 16S and combined trees (gaps treated as fifth character in both) were significantly different (at P < 0:05) from constrained trees. Test constraints included the Sparidae, sensu strictu, the Sparidae + Spicara, each sparid subfamily and any ingroup genera that had multiple species in a tree. Modeltest Version 3.06 (Posada and Crandall, 1998) was used to select best-fit ML models for the 16S partitioned and combined data sets. A heuristic search of 10 replicates was used to determine the best-fit ML tree and bootstrap analysis (BA) of 100 replicates provided an estimate of nodal support.
3. Results Detailed sequence characteristics, sequence divergence, and mutational analysis for cyt b data are reported in Orrell et al. (2002). Sequencing of the 16S mtDNA gene produced an average of 574 (range 559–585) nucleotide base pairs per taxon. Multiple alignments resulted in a consensus length of 621 positions (base pairs and gaps). On average more regions sequenced were designated to the loop category (381 sites) than to the stem category (240 sites). Of the 621 positions, 40% were constant and 22% were parsimony uninformative. Most (80%) of the parsimony informative variable characters were from loop regions. The largest uncorrected sequence divergence was 26% between Centropomus undecimalis and Scolopsis ciliatus and the smallest was 1.2% between Pachymetopon aeneum and Polyamblyodon germanum. Mean pairwise sequence divergence between all pairwise comparisons of taxa was 9.70% (SD ¼ 0.048, n ¼ 1540). Average divergence between comparisons of outgroup taxa was 14.49% (SD ¼ 0.052, n ¼ 120) and between pairwise comparisons of ingroup taxa (including Spicara) 6.21% (SD ¼ 0.019, n ¼ 780). A plot of all substitutions as a function of uncorrected sequence divergence showed that an approximate of 70 substitutions was reached at greater than 25% sequence divergence. Transitional substitutions were found to be saturated at nearly 15% sequence divergence. Loop and stem transitions plotted as a function of uncorrected sequence divergence revealed that loop transitions saturated at nearly 15% sequence divergence. Stem transitions were not saturated as sequence divergence increased and reached a maximum of approximately 20 substitutions at greater than 25% sequence divergence. No incongruence was found between the 16S and cyt b data set partitions (observed distance D ¼ 7416, randomly selected distances S ¼ 39, P ¼ 0:61).
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3.1. 16S The heuristic search of the 16S data partition (gaps treated as fifth character) resulted in 276 equally parsimonious trees (tree length ¼ 1527). Of the 621 sites (base pairs and gaps), 251 were constant, 137 variable characters were parsimony uninformative, and 233 variable characters were parsimony informative. The best-fit ML model for the16S data was the complex, parameter rich, general time-reversible model with unequal base frequencies, six rate classes and a c rate heterogeneity parameter (GTR + I+ C). The ML analysis had the following statistics: estimated nucleotide frequencies A ¼ 0:289, C ¼ 0:274, G ¼ 0:208, T ¼ 0:229; nucleotide substitution rate matrix A $ C ¼ 1:80, A $ G ¼ 7:69, A $ T ¼ 2:85, C $ G ¼ 0:79, and C $ T ¼ 11:35; assumed proportion of invariable sites 0.23; and the shape of the estimated gamma parameter (a) ¼ 0.46. The heuristic search produced a single tree with a negative log likelihood score ()ln L) of 7066.30. The 16S parsimony and ML analyses supported two major clades within Sparidae, but the 16S parsimony was unable to fully resolve interrelationships of sparid genera and the internal nodes had very short branch lengths in the ML analysis. Templeton test results of constrained trees are given in Table 2. There was no significant difference between the most parsimonious trees (EPTs) and a constrained Sparidae sensu strictu (P ¼ 1:00), but there was a significant difference between a constrained Sparidae + Spicara and the EPTs. Within the Sparidae + Spicara, a clade comprising Boops boops + Sarpa salpa was basal to all other sparids. Pagrus, Pagellus, and Spicara were not monophyletic and the latter two genera were not significantly different when constrained and compared to the EPTs (P ¼ 0:005). None of the six subfamilies of Sparidae were monophyletic in this analysis. Templeton tests of constrained subfamilies showed that Boopsinae (P ¼ 0:004), Denticinae (P ¼ 0:016), Pagellinae (P ¼ 0:012), and Sparinae (P < 0:001) were significantly different than the unconstrained equally parsimonious trees. Diplodinae (P ¼ 0:191) and Pagrinae (P ¼ 0:464) were not significantly different from the unconstrained trees. There was no support from this analysis for Sparoidea; neither lethrinids nor nemipterids were sister to the sparids. When gap characters were treated as missing using the same parsimony parameters outlined above, the number of informative characters under parsimony was reduced to 218. A total of 344 equally parsimonious trees were recovered with length of 1377. The resulting consensus tree was similar to the tree with gaps coded as a fifth character, but there was less resolution for terminal sparid nodes. 3.2. Combined 16S and cyt b The combined data analysis (gaps treated as fifth character) yielded three equally parsimonious trees (tree
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Table 2 Results of Wilcoxon sign-ranks, two-tailed probability tests (Templeton, 1983) Constraint
Sparidae, sensu strictu Sparidae + Spicara Boopsinae Denticinae Diplodinae Pagellinae Pagrinae Sparinae Spicara All subfamilies Diplodus Pagrus Pagellus
16S
Combined
TL
n trees
Average P value
SD
TL
n trees
Average P value
SD
1527 1556 1554 1540 1539 1550 1530 1559 1556 1628 1527 1530 1552
276 140 1436 888 24 1003 8 2 212 6 24 356 418
n/a 0.007 0.004 0.016 0.191 0.012 0.464 <0.001 0.002 <0.001 n/a 0.492 0.005
n/a 0.004 0.003 0.008 0.023 0.006 0.014 0.001 0.001 0 n/a 0.054 0.003
7451 7528 7536 7491 7494 7531 7479 7545 7535 7773 7451 7500 7519
2 2 2 2 2 1 1 3 4 12 3 20 3
n/a <0.001 <0.001 0.006 0.003 <0.001 0.0231 <0.000 <0.001 <0.001 n/a 0.002 <0.001
n/a 0 0 0.001 0.001 0 0 0 0 0 n/a 0.001 0
Note. Unconstrained tree lengths:16S parsimony analysis (gaps as fifth character) TL ¼ 1527; Combined parsimony (gaps as fifth character) TL ¼ 7451. Constrained trees generated by a heuristic search of 100 random addition sequences with 5 trees held at each step. P values are averaged across all comparisons of unconstrained and constrained trees. * Indicates significant difference at P < 0:05.
length ¼ 7451). Of 1761 total characters, 753 (43%) characters were constant, 251 (14%) variable characters were parsimony uninformative, and 757 (43%) variable characters were parsimony informative characters. Jackknife support, total and partitioned Bremer support values are shown on a strict consensus of three equally parsimonious trees (Fig. 1). A monophyletic Sparidae + Spicara was recovered (JS ¼ 100, BS ¼ 35) with high Bremer support values from each of the data partition (16S PBS ¼ 11 and cyt b PBS ¼ 24). As in the 16S analysis, two major clades were found in the combined analysis (clades A and B, Fig. 1) with the most partitioned Bremer support coming from the cyt b partition (cyt b PBS ¼ 11.5 for clade A and PBS ¼ 12.6 for clade B). Contrary to the 16S parsimony tree, the clade containing Boops boops and Sarpa salpa was no longer basal to other Sparidae + Spicara, but was nested within clade B. All support for this node came from the cyt b partition (PBS ¼ 2.2). None of the sparid subfamilies were monophyletic in the combined parsimony tree, nor were Pagrus, Pagellus, or Spicara. Templeton tests of all subfamilies and of Pagrus, Pagellus, and Spicara were significantly different than the unconstrained tree. As in the 16S tree, there was no support from this analysis for Sparoidea. When gaps were treated as missing during parsimony analysis, three equally parsimonious trees were recovered, but the number of parsimony informative characters was reduced from 757 to 742 and the tree length was reduced to 7299 steps. The topology of the resulting consensus tree was identical to the consensus tree where gaps were treated as a fifth character. As with the 16S data, the best-fit ML model for the combined data was GTR + I+ C. The combined data sets had the following statistics: estimated nucleotide frequencies A ¼ 0:302, C ¼ 0:370, G ¼ 0:105, and T ¼ 0:223; nu-
cleotide substitution rate matrix A $ C ¼ 0:29, A $ G ¼ 6:02, A $ T ¼ 0:67, C $ G ¼ 0:66, and C $ T ¼ 5:19; assumed proportion of invariable sites ¼ 0.40 and the shape of the estimated c parameter (a) ¼ 0.57. The heuristic search produced a single tree with a negative log likelihood score ()ln L) 30575.90 (Fig. 2). The combined ML tree supported a monophyletic Sparidae + Spicara. Two major sparid clades were found in the analysis (clades A and B, Fig 2). The addition of the cyt b data to the 16S data in the combined ML tree greatly strengthened nodal support for sparid generic relationships and for clade A (BA ¼ 97) and clade B (BA ¼ 95). Neither Sparoidea, the six sparid subfamilies, nor Pagrus, Pagellus, or Spicara were monophyletic in the combined ML analysis.
4. Discussion Pairwise sequence divergences between outgroup taxa were similar to those reported for 16S in other teleosts. Farias et al. (1999) found a maximum uncorrected pairwise divergence of 20% (average 17%) in cichlids, Wiley et al. (1998) found a maximum of >25% TamuraNei distance in acanthomorph relationships, and Tang et al. (1999) found <20% Tamura-Nei distance in Acanthuroidei. On average, the mean sequence divergence for all pairwise comparisons of taxa in our study (9.7%) was well below the level at which loop substitutions appeared to reach saturation. Putative loop mutations deviated from a linear relationship when genetic distance approached approximately 15% sequence divergence. The average pairwise divergence for ingroup species (6.21%) was well below the apparent level of loop saturation. Average mean pairwise divergence for
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Fig. 1. A strict consensus of 3 equally parsimonious trees derived from parsimony analysis of combined data. Subfamilies are labeled as follows: BO, boopsinae; DE, denticinae; DI, diplodinae; PA, pagrinae; PE, pagellinae; and SP, sparinae. At each node are support values: leftmost is the jackknife value, within parentheses are partitioned decay values for 16S then cyt b and the rightmost is the total decay value. The two major sparid clades are designated A, B.
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Fig. 2. Maximum likelihood tree derived from combined data. Subfamilies are labeled as in Fig. 1. Values at nodes represent bootstrap support (given for bootstrap values P50%). The two major sparid clades are designated A, B.
outgroup species was 14.49%, nearly at the same level at which apparent saturation occurred in loops. Because neither stem nor loop characters were saturated for ingroup species, all 16S characters were potentially informative and were treated with equal weight. An equal
weight of all characters was defendable as a means to infer sparid relationships, but potentially inconsistent as a weight to infer outgroup relationships. The pairwise sequence divergence between Sparidae and outgroup taxa appeared saturated for loop transitions, and the
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relationships inferred from these characters could be mis-informative. Therefore, the reliability of 16S to infer the deep node separating Lethrinidae and Nemipteridae from the Aother percoids and Sparidae + Spicara is questionable because all node support comes from the loop characters and not from the more conserved stem characters. Both the 16S data and combined data analyses supported a monophyletic Sparidae + Spicara. The sister relationships of Dentex tumifrons with Spicara alta and of Spondyliosoma cantharus with Spicara maena were also found in Orrell et al. (2002). The overall position of Spicara and Centracanthidae is problematic without a more complete understanding of both genera and all species of Centracanthidae. This study included two species of Spicara but none of Centracanthus. The 16S parsimony and ML analyses supported two major clades within Sparidae, but the 16S parsimony was unable to fully resolve interrelationships of sparid genera and the internal nodes had very short branch lengths in the ML analysis. Of the five ingroup genera that contained multiple representatives, only Diplodus was monophyletic. Hanel and Sturmbauer (2000) found Pagellus, Pagrus, and Dentex non-monophyletic and Orrell et al. (2002) found Pagellus, Pagrus, Dentex, and Spicara, non-monophyletic using cyt b. There was no support from the 16S sequence data for any of the subfamilies proposed by Smith (1938); Akazaki (1962), and Smith and Smith (1986). The monophyly of the Denticinae and Pagrinae could not be overwhelmingly challenged based on the 16S data as the constrained trees were not significantly different from the unconstrained equally parsimonious trees. Once the more robust cyt b data was combined with the 16S data, the constrained trees for all sparid subfamilies were significantly different from unconstrained trees. Clearly, based on the combined sequence data, the subfamilies as currently defined are artificial, in agreement with the findings of Hanel and Sturmbauer (2000). Despite the regional nature of Hanel and SturmbauerÕs (2000) study and an unfortunate choice of Spicara outgroup, the authors had a broad enough sampling of sparid species to conclude that trophic types evolved more than once in sparid fishes. The subfamilies as currently defined are most useful as categories that aid in field identification of sparids. Dentition characters are easily recognizable and generally aid in separating regional species. These subfamilies are not based on phylogenetic information, as demonstrated in the present and previous studies (Hanel and Sturmbauer, 2000; Day, 2002; Orrell et al., 2002), and should not be considered valid or a means by which to classify sparids. The 16S sequence analyses generally lacked the ability to resolve relationships between Sparidae and other percoid families. Neither AkazakiÕs spariform fishes or JohnsonÕs Sparoidea were reconstructed. This is in
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contrast to the recent findings of Carpenter and Johnson (2002) with significant osteological evidence for a Sparidae–Lethrinidae sister group relationship. Orrell et al. (2002) found the Lethrinidae sister to Sparidae using cyt b, but only a weighted cyt b tree supported the superfamily Sparoidea (Sparidae, Centracanthidae, Lethrinidae, and Nemipteridae). There was only minimal support in the weighted cyt b data for the Sparoidea; most support was from the third and first codon positions. In the present study, there was no support for a lethrinid–sparid sister relationship nor for a monophyletic Sparoidea from either the combined parsimony or ML trees. Although no attempt was made to account for cyt b saturation in third positional substitutions in the combined parsimony analysis, the ML combined model (GTR + I+ C) was sensitive to this saturation. Yet, it failed to reconstruct a monophyletic Sparoidea. Since 16S is more conservative than cyt b (less overall changes between taxa), it should be informative for deeper relationships (i.e., within Perciformes). However, there was no support from the 16S analysis for deeper relationships found in Carpenter and Johnson (2002) and Orrell et al. (2002). The apparent saturation of transitions in loop characters found in comparisons between outgroup species might account for the inability of the 16S sequence analyses to recover a monophyletic Sparoidea and for the low overall support for outgroup relationships. This study and Orrell et al. (2002) found that cyt b and 16S alone and combined where useful for intrafamilial sparid relationships. Due to the inability of the combined mtDNA analysis to reconstruct a monophyletic Sparoidea and the lack of support for deeper relationships, we question the use of mtDNA as an appropriate marker for phylogenetic inference among perciform families. A future analysis of sparoid relationships might utilize a nuclear marker which could be better suited for perciform interfamilial relationships.
Acknowledgments We thank the following individuals and organizations for their assistance in collecting specimens, without whose help this work would not have been possible: S. Almatar, L. Beckley, B.B. Collette, F. Crock, N. DeAngelis, M. DeGravelle, D. Etnier, H. Ishihara, J. Gelsleichter, A. Graham, R. Grubbs, K. Harada, Y. Iwatsuki, J. Jenke, R. Kraus, E. Massuti, K. Matsuura, L. Ter Morshuizen, P. Oliver, A.W. Paterson, J. Paxton, J. Scialdone, D. Scherrer, G. Sedberry, M. Smale, W. F. Smith-Vaniz, K. Utsugi, G. Yearsley, T. Wasaff, J. T. Williams, and the VIMS Trawl Survey. We acknowledge the following people for laboratory assistance at Virginia Institute of Marine Science (VIMS): J. McDowell, D. Carlini, V. Buonaccorsi, C. Morrison, and K.
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Macdonald. We thank the help and the advice from J. Musick, J. Graves, K. Reece, P. Van Veld, and G. D. Johnson. We thank Jeff Bates, Laboratory of Molecular Systematics for analysis support. We thank B.B. Collette and E.O. Wiley for manuscript review and comments. This research was supported by grants to TMO from: the Fisheries Department, Food and Agriculture Organization of the United Nations, the Lerner Gray Fund for Marine Research from the American Museum of Natural History, an E.C. and C.E. Raney Award of the American Society of Ichthyologists and Herpetologists, and through the VIMS Graduate Deans Office. This paper is Contribution No. 2574 of the Virginia Institute of Marine Science, The College of William and Mary.
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