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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Molecular Phylogenetics and Evolution 69 (2013) 177–187 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev A new phylogeny of tetraodontiform fishes (Tetraodontiformes, Acanthomorpha) based on 22 loci Francesco Santini a,b,⇑, Laurie Sorenson a, Michael E. Alfaro a,⇑ a b University of California Los Angeles, Department of Ecology and Evolutionary Biology, 610 Charles E. Young Drive South, Los Angeles, CA 90095, USA Dipartimento di Scienze della Terra, Università degli Studi di Torino, Torino 10125, Italy a r t i c l e i n f o Article history: Received 5 February 2013 Revised 16 May 2013 Accepted 20 May 2013 Available online 31 May 2013 Keywords: Tetraodontiformes Phylogenomics Pufferfishes Coral reefs Morphology a b s t r a c t Tetraodontiform fishes represent one of the most peculiar radiations of teleost fishes. In spite of this, we do not currently have a consensus on the phylogenetic relationships among the major tetraodontiform lineages, with different morphological and molecular datasets all supporting contrasting relationships. In this paper we present the results of the analysis of tetraodontiform interrelationships based on two mitochondrial and 20 nuclear loci for 40 species of tetraodontiforms (representing all of the 10 currently recognized families), as well as three outgroups. Bayesian and maximum likelihood analyses of the concatenated dataset (18,682 nucleotides) strongly support novel relationships among the major tetraodontiform lineages. Our results recover two large clades already found in mitogenomic analyses (although the position of triacanthids differ), while they strongly conflict with hypotheses of tetraodontiform relationships inferred by previous studies based on morphology, as well as studies of higher-level teleost relationships based on nuclear loci, which included multiple tetraodontiform lineages. A parsimony gene-tree, species-tree analysis recovers relationships that are mostly congruent with the analyses of the concatenated dataset, with the significant exception of the position of the pufferfishes + porcupine fishes clade. Our findings suggest that while the phylogenetic placement of some tetraodontiform lineages (triacanthids, molids) remains problematic even after sequencing 22 loci, an overall molecular consensus is beginning to emerge regarding the existence of several major clades. This new hypothesis will require a re-evaluation of the phylogenetic usefulness of several morphological features, such as the fusion of several jaw bones into a parrot-like beak, or the reduction and loss of some of the fins, which may have occurred independently more times than previously thought. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The approximately 430 extant species of tetraodontiforms (Froese and Pauly, 2012), represent one of the most morphologically and ecologically diverse radiations of spiny-rayed fishes (Fig. 1) (Tyler, 1980; Santini and Stellwag, 2002). They exhibit a remarkable diversity in body forms and size, skeletal structures, ecology, and genome size (Tyler, 1980; Brainerd et al., 2001; Jaillon et al., 2004; Santini and Stellwag, 2002). Although half of the tetraodontiform diversity is reef-associated (Alfaro et al., 2007), the 10 recognized families inhabit a wide variety of habitats. In addition to clades found predominantly on coral reefs, sea grasses, and other tropical, shallow-water environments (Triacanthidae, the triplespines; Balistidae, the triggerfishes; Monacanthidae, the filefishes; ⇑ Corresponding authors. Address: University of California Los Angeles, Department of Ecology and Evolutionary Biology, 610 Charles E. Young Drive South, Los Angeles, CA 90095, USA. Fax: +1 310 206 3987. E-mail addresses: francesco.santini@alumni.utoronto.ca (F. Santini), michaelalfaro@ucla.edu (M.E. Alfaro). 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.05.014 Aracanidae, the boxfishes; Ostraciidae, the trunkfishes; Tetraodontidae, the pufferfishes; and Diodontidae, the porcupinefishes), Tetraodontiformes include deep-water taxa (Triacanthodidae, the spikefishes; and Triodontidae, the three-tooth puffer), and pelagic groups (Molidae, the ocean sunfishes). Because representatives of several families are found in shallow water in the Mediterranean, the tetraodontiforms were well known to ancient cultures, and the unusual appearance of several tetraodontiform members attracted the attention of natural historians and comparative anatomists since the time of Aristotle and Pliny the elder (Tyler, 1980). The smallest species, such as the Malabar pufferfish Carinotetraodon travancoricus or the minute filefish Rudarius minutus, are only a few centimeters long and weigh less than 50 g as adults, while the largest, the ocean sunfish Mola mola, is over 3 m long and can weigh more than 2.000 kg (Nelson, 2006). Some tetraodontiforms also exhibit pronounced phenotypic traits such as heavily armored scale plates, spiny dermal processes, greatly reduced or missing pelvic and spiny dorsal fins, aborted caudal regions, and fused jaw-bones that form a parrot-like beak (Tyler, 1980). Reduction and loss of skeletal elements and duplica- Author's personal copy 178 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 Aracanidae Balistidae Diodontidae Molidae Monacanthidae Ostraciidae Tetraodontidae Triacanthidae Triacanthodidae Triodontidae Fig. 1. Pie chart of species diversity of the 10 extant tetraodontiform families. Fish outlines modified from Fishbase.org. tion of trophic musculature are both important trends that contribute to the morphological diversification within this group (Winterbottom, 1974; Tyler, 1980; Friel and Wainwright, 1997, 1999; Santini and Stellwag, 2002; Santini and Tyler, 2003). More recently, after the discovery that several tetraodontiform lineages possess extremely compact genomes, among the smallest of all vertebrates (Hinegardner, 1968; Brainerd et al., 2001), pufferfishes became a major model organism in vertebrate genomics. The species Takifugu rubripes and Tetraodon nigroviridis became the first non-human vertebrates to have their entire genomes sequenced (Brenner et al., 1993; Jaillon et al., 2004). Tetraodontiforms have been the subject of numerous morphological phylogenetic studies over the last 40 years (Winterbottom, 1974; Tyler, 1980; Leis, 1984; Rosen, 1984; Santini and Tyler, 2003, 2004). These studies generally recognized three major lineages: the Triacanthoidei, the Balistoidei, and the Tetraodontoidei (Fig. 2). The Triacanthoidei traditionally includes the deep-sea triacanthodids and the shallow-water triacanthids, all of which possess a body plan not unlike that of most perch-like fishes, with a spiny dorsal fin, well developed pelvis with pelvic spines and soft rays, and epineural bones. The Balistoidei include the aracanid and ostraciid boxfishes, two groups characterized by the loss of the first dorsal fin and of the pelvic complex, as well as the evolution of a carapace formed by interdigitated scale plates; these two families together form the sister group to a clade formed by balistids and monacanthids, two lineages characterized by significant reductive trends in the dorsal and pelvic fins. The monophyly of these two subclades, as well as their sister group relationship, was very strongly supported by dozens of synapomorphies in Win- terbottom (1974) and Santini and Tyler (2003, 2004), as well as by a large number of characters in Tyler’s (1980) evolutionary taxonomic study. The third tetraodontiform group, the Tetraodontoidei or gymnodonts, is characterized by a strong articulation of the premaxillae, and in some cases also the dentaries, to one another through interdigitations, so as to form a parrot-like beak; this group contains the three-tooth puffer, the ocean sunfishes, the smooth pufferfishes and the porcupine fishes (also known as spiny puffers). In spite of the different types of characters and methods of data analyses, all morphological studies produced highly congruent topologies, with the differences between the various hypotheses primarily due to the placement of two lineages: the triacanthids, and the ostraciid + aracanid clade (Fig. 2). A number of disagreements exist among the morphological studies. In Winterbottom’s myological study (1974) the triacanthids were hypothesized to be the sister group to the triacanthodid spikefishes, although support for this topology was not strong. Santini and Tyler (2003, 2004) recovered the triacanthids as sister group to the balistoids (Fig. 2). Boxfishes appeared to be closely related to tetraodontoids in Leis’ (1984) analysis of larval characters, and Rosen’s (1984) osteological study of tetraodontiform and zeiform fishes. Tyler and Sorbini (1996) also recognized an entirely extinct lineage of heavily armoured Cretaceous fishes, the plectocretacicoids, which was shown to be a tetraodontiform stem group (Tyler and Sorbini, 1996; Santini and Tyler, 2003, 2004; Tyler and Santini, 2005). All of these studies were, however, performed before the current understanding of acanthomorph higher-level relationships changed, and lophiiforms, and not zeiforms, were found to be the Author's personal copy Table 1 List of taxa included in this study with tissue voucher and GenBank numbers. Tissues were from the personal collections of Michael E. Alfaro (MEA), Victor Brian Alfaro (VRA) and Francesco Santini (FS) at UCLA, and Peter Wainwright (PW) at UC Davis, as well as from the Natural History Museum in Victoria (NMV), South African Institute of Aquatic Biology (SAIAB), Natural History Museum of the University of Kansas (KU), Scripps Institute of Oceanography (SIO), Field Museum (FMNH) and Australian Museum (EBU). Common name Tissue # coxl Cytb EGR1 EGR3 ENC1 Gylt irbp KIAA2013 Antigonia capros – Caproidae Antennarius striatus – Antennaridae Ogcocephalus nasutus – Ogcocephalidae Anoplocapros lenticularis – Aracanidae Aracana aurita Balistapus undulatus – Balistidae Balistes vetula Rhinecanthus aculeatus Chilomycterus reticulatus – Diodontidae Chilomycterus schoepfii Diodon hystrix Mola mola – Molidae Ranzania laevis Aluterus scriptus – Monacanthidae Amanses scopas Cantherines pardalis Monacanthus chinensis Oxymonacanthus longirostris Paraluteres prionurus Stephanolepis cirrhifer Acanthostracion quadricornis – Ostraciidae Lactoria cornuta Ostracion cubicus Arothron stellatus – Tetraodontidae Canthigaster coronata Canthigaster rostrata Chelonodon patoca Lagocephalus laevigatus Marilyna darwinii Sphoeroides pachygaster Takifugu ocellatus Tetraodon leiurus Tetraodon mbu Tetraodon nigroviridis Torquigener pleurogramma Xenopterus naritus Pseudotriacanthus strigilifer – Triacanthidae Triacanthus nieuhofii Trixiphichthys weberi Parahollardia lineata – Triacanthodidae Triacanthodes ethiops Tydemania navigatoris Triodon macropterus – Triodontidae Deepbody boarfish Striated frogfish Shortnose batfish White-barred boxfish Striped cowfish Orange-lined triggerfish Queen triggerfish Blackbar triggerfish Spotfin burrfish Striped burrfish Spot-fin porcupinefish ocean sunfish Slender sunfish Scribbled leatherjacket Broom filefish Honeycomb filefish Fan-bellied leatherjacket Harlequin filefish False puffer Threadsail filefish Scrawled cowfish Longhorn cowfish Yellow boxfish Stellate puffer Crowned puffer Caribbean sharpnose-puffer Milkspotted puffer Smooth puffer Darwin Toadfish Blunthead puffer Ocellated Puffer Longnose Puffer Fresh water puffer fish Spotted green pufferfish Weeping toado Golden puffer Long-spined tripodfish Silver tripodfish Blacktip tripodfish Jambeau Shortsnout spikefish Fleshy-lipped spikefish Treetooth puffer KU 8290 VRA17 MEA 229 ME A 140 MEA 171 MEA 144 PW1181 MEA 146 SIO 3409-143 PW 1224 FS05 EBU 38997 SIO 3405-31 MEA 170 KU 4457 KU 8687 MEA 512 MEA 134 PW 1268 KU 8688 MEA169 PW 1270 FS02 VRA15 PW 1529 MEA 235 VRA9 KU 8370 MEA 274 SIO 3404-71 MEA 476 MEA 181 MEA 270 PW 1262 EBU 44123-019 PW 1263 PW 1276 MEA 430 EBU 22120 KU 5881 NMV 82004 NMV 82083 FMNH119629 KF027498 KF027499 KF027500 KF027501 KF027502 FJ582893 KF027503 KF027504 KF027505 KF027506 KF027507 KF027508 KF027509 KF027510 KF027511 KF027512 KF027513 KF027514 KF027515 KF027516 KF027517 KF027518 KF027519 KF027520 KF027521 KF027522 KF027523 KF027524 KF027525 KF027526 KF027527 KF027528 KF027529 JQ681838 KF027530 KF027531 KF027532 KF027533 KF027534 KF027535 KF027536 KF027537 KF027538 KF027539 AB282828 KF027540 KF027541 KF027542 KF027543 KF027544 KF027545 KF027546 KF027578 KF027579 KF027580 KF027581 KF027582 KF027583 KF027584 KF027585 KF027586 KF027587 KF027588 KF027589 KF027590 KF027591 KF027592 KF027593 KF027594 KF028326 KF028327 KF028328 KF028329 KF028330 KF028331 KF028332 KF028333 KF028334 KF028335 KF028336 KF028337 KF028338 KF028339 KF027630 KF028341 KF027632 KF028343 KF028344 KF028345 KF0276 KF028347 KF028348 KF028349 KF027640 KF028351 KF02772 KF028353 KF028354 KF028355 KF028356 KF027647 KF028358 KF028359 EF539254 KF027619 KF027620 KF027621 KF027622 KF027623 KF027624 EF539261 KF027625 KF0276 EF539262 KF027627 KF027628 KF027629 KF027630 KF027631 KF027632 KF027633 KF027634 KF028345 KF027636 KF027637 KF027638 KF027639 KF027640 KF027641 KF027642 KF027643 KF027604 KF027645 KF027646 KF027647 KF027648 CR638510 KF027649 KF027650 KF027651 KF027652 KF027657 KF027658 KF027659 KF027660 KF027661 KF027662 KF027663 KF027664 KF027665 KF027666 KF027667 KF027668 KF027669 KF027670 KF027671 KF027672 KF027673 KF027674 KF027675 KF0276 KF02767 KF027678 KF027679 KF027680 KF027681 KF027682 KF027683 KF027684 KF027685 KF027686 KF027687 KF02768 KF027689 KF027690 KF027691 KF027692 KF027693 KF027694 KF027695 KF027696 KF027697 KF027698 KF027699 KF02770 DQ168037 KF027701 KF027702 KF027703 KF027704 KF027705 KF027706 KF027707 KF027708 KF027709 KF027710 KF027711 KF027712 KF027713 KF027714 KF027715 KF027716 KF027717 KF027718 KF027719 KF027678 KF027721 KF027722 KF027723 KF027724 KF027725 KF027726 KF027727 KF027728 KF027729 KF027730 KF027731 KF027732 KF027733 KF027734 KF027735 KF027736 KF027779 KF027738 KF027739 KF027740 KF027741 KF027742 KF027743 KF027744 KF027745 KF027746 KF027747 KF027748 KF027749 KF027750 KF027751 KF027752 KF027753 KF027754 KF027755 KF027756 KF027757 KF027758 KF027759 KF027760 KF027761 KF027762 KF027763 KF027764 KF027765 MLL Myh6 Plagl2 Ptr Rag1 Rh RYR3 SH3PX3 sidkey Sreb2 Tbr1 UBE3A Zic1 znf503 EU638027 AY362215 KF027784 KF027785 KF027786 KF027787 KF027788 KF027821 KF027822 KF027823 KF027824 KF027825 KF027826 KF027827 EF536269 KF027863 KF027864 KF027865 KF027866 KF027867 KF027868 KF027899 KF027900 KF027901 KF027902 KF027903 KF027904 KF027905 AY308785 KF027941 KF027942 KF027943 KF027944 EU108869 AY700310 KF027970 KF028012 KF027971 KF027972 KF027973 KF027974 KF027975 KF028011 KF028052 KF028013 KF028014 KF028015 KF028016 KF028017 KF028051 KF028094 KF028053 KF028054 KF028055 KF028056 KF028057 KF028093 KF028136 KF028095 KF028096 KF028097 KF028098 KF028099 KF028135 KF028174 KF028137 KF028138 KF028139 KF028140 KF028141 KF028173 KF028211 KF028175 KF028176 KF028177 KF028178 KF028179 KF028210 KF028252 KF028212 KF028213 KF028214 KF028215 KF028216 EF533930 KF028290 KF028253 KF028254 KF028255 KF028256 KF028257 KF028289 KF027547 KF027548 EF392609 KF027549 KF027550 KF027551 KF027552 KF027553 KF027554 KF02755 KF027556 KF027557 KF027558 KF027559 KF027560 KF027561 KF027562 KF027563 KF027564 KF027561 KF027566 KF027567 KF027568 KF027569 KF027570 KF027571 KF027572 KF027573 KF027574 KF027575 KF027576 KF027577 AP009170 KF027595 KF027596 KF027597 KF027558 KF02759 KF027560 KF027601 KF027602 KF027603 KF027604 KF027605 KF027606 KF027607 KF027608 KF027609 KF027610 KF027611 KF027612 KF027613 KF027614 KF027615 KF027616 KF027617 KF027618 KF028360 KF028361 KF028362 KF028363 KF028364 KF028365 KF028366 KF028367 KF027653 KF027654 KF027655 KF027656 KF027766 KF027767 KF02768 KF027769 KF027728 KF027771 KF027772 KF02773 KF027774 KF027775 KF027776 KF0277 KF027778 KF027779 KF027780 KF027781 KF027782 KF027783 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 Scientific name KF028291 KF028292 KF028293 KF028294 179 (continued on next page) Author's personal copy 180 Table 1 (continued) MLL Myh6 Plagl2 Ptr Rag1 Rh RYR3 SH3PX3 sidkey Sreb2 Tbr1 UBE3A Zic1 KF027789 KF027790 KF027791 KF027792 AY362251 KF027793 KF027833 KF027794 KF027795 KF027836 KF027837 KF027796 KF027797 KF027798 KF027799 KF027800 KF027801 KF027802 KF027803 KF027804 KF027805 KF027806 KF027807 KF027808 KF027809 KF027810 KF027811 KF027812 KF027813 KF027814 KF027815 KF027816 KF027817 KF027818 KF027819 KF027820 KF027828 KF027829 KF027830 KF027831 KF027832 KF027872 KF027873 KF027834 KF027835 KF027876 KF027877 KF027838 KF027839 KF027840 KF027841 KF027842 KF027843 KF027844 KF027845 KF027846 KF027847 KF027848 KF027849 KF027850 KF027851 KF027852 KF027853 KF027854 KF027855 KF027856 KF027857 KF027858 KF027859 KF027860 KF027861 KF027862 EF536275 KF027869 KF027870 KF027909 KF027871 KF027948 KF027911 KF027874 KF027875 KF027914 KF027915 KF027878 KF027879 KF027880 KF027881 KF027882 KF027883 KF027884 KF027885 KF027886 KF027925 KF027926 KF027887 KF027888 KF027929 KF027891 KF027890 KF027889 KF027933 KF027892 KF027893 KF027894 KF027895 KF027896 KF027897 KF027898 KF027906 KF027907 KF027908 KF027946 KF027910 KF027981 AY700331 KF027912 KF027913 KF027950 KF027951 KF027916 KF027917 KF027918 KF027919 KF027920 KF027921 KF027922 KF027923 KF027924 KF027958 KF027959 KF027927 KF027928 KF027961 KF027930 KF027931 KF027932 KF027964 KF027934 KF027935 KF027936 KF027937 KF027938 KF027939 KF027940 AY308790 KF027945 AY700326 KF027979 KF027947 KF028020 KF027982 AY308793 KF027949 KF027984 KF027985 AY700336 KF027987 AY700345 KF027952 KF027953 KF027954 KF027955 KF027956 KF027957 KF027995 KF027996 KF027960 KF027998 KF027999 KF027962 KF027963 AY700360 KF028003 EF101314 KF027965 AY308789 KF027966 KF027967 KF027968 KF027969 KF027976 KF027977 KF027978 KF028061 KF027980 KF028063 KF028021 KF028022 KF027983 KF028024 KF028025 KF027986 KF028027 KF027988 KF027989 KF027990 KF027991 KF027992 KF027993 KF027994 KF028035 KF028036 KF027997 KF028038 KF028039 KF028000 KF028001 KF028002 KF028043 KF028004 KF028005 KF028006 KF028007 KF028008 KF028009 KF028010 KF028018 KF028059 KF028060 KF028103 KF028019 KF028105 KF028064 KF028065 KF028023 KF028067 KF028068 KF028026 KF028070 KF028028 KF028029 KF028030 KF028031 KF028032 KF028033 KF028034 KF028077 KF028078 KF028037 KF028080 KF028081 KF028040 KF028041 KF028042 KF028085 KF028044 KF028045 KF028046 KF028047 KF028048 KF028049 KF028050 KF028058 KF028101 KF028102 KF028145 KF028062 KF028147 KF028185 KF028106 KF028066 KF028108 KF028109 KF028069 KF028111 KF028071 KF028072 KF028073 KF028115 KF028074 KF028075 KF028076 KF028119 KF028120 KF028079 KF028122 KF028123 KF028082 KF028083 KF028084 KF028127 KF028086 KF028087 KF028088 KF028089 KF028090 KF028091 KF028092 KF028100 KF028143 KF028144 KF028183 KF028104 KF028221 KF028222 KF028223 KF028107 KF028186 KF028226 KF028110 KF028149 KF028112 KF028113 KF028114 KF028153 KF028116 KF028117 KF028118 KF028157 KF028158 KF028121 KF028160 KF028161 KF028124 KF028125 KF028126 KF028165 KF028128 KF028129 KF028130 KF028131 KF028132 KF028133 KF028134 KF028142 KF028181 KF028182 KF028219 KF028146 KF028261 KF028299 KF028180 KF028218 KF028259 EF533938 KF028184 KF028217 KF028258 KF028296 KF028297 KF028220 EF533937 KF028295 KF028148 KF028225 KF028264 KF028187 KF028228 KF028150 KF028151 KF028152 KF028191 KF028154 KF028155 KF028156 KF028236 KF028195 KF028159 KF028197 KF028198 KF028162 KF028163 KF028164 KF028202 KF028166 KF028167 KF028168 KF028169 KF028170 KF028171 KF028172 KF028224 KF028263 KF028302 KF028227 KF028266 KF028188 KF028189 KF028190 KF028232 KF028192 KF028193 KF028194 KF028274 KF028237 KF028196 KF028239 KF028240 KF028199 KF028200 KF028201 KF028244 KF028203 KF028204 KF028205 KF028206 KF028207 KF028208 KF028209 KF028262 KF028301 KF028300 KF028260 KF028267 KF028268 KF028269 KF028307 KF028271 KF028272 KF028273 KF028275 KF028238 KF028277 KF028278 KF028241 KF028242 KF028243 KF028282 KF028245 KF028246 KF028247 KF028248 KF028249 KF028250 KF028251 KF028311 KF028276 KF028313 KF028314 KF028279 KF028280 KF028281 KF028318 KF028283 KF028284 KF028321 KF028285 KF028286 KF028287 KF028288 KF028298 KF028303 KF028305 KF028306 KF028308 KF028309 KF028310 KF028312 KF028315 KF028316 KF028317 KF028319 KF028320 KF028322 KF028323 KF028324 KF028325 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 KF028265 KF028304 KF028229 KF028230 KF028231 KF028270 KF028233 KF028234 KF028235 znf503 Author's personal copy F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 181 Fig. 2. Summary of previous morphological hypotheses of tetraodontiform relationships. Fish outlines modified from Fishbase.org. likely sister group to the tetraodontiforms (Chen et al., 2003; Miya et al., 2003; Holcroft, 2004; Yamanoue et al., 2007; Li et al., 2008; Santini et al., 2009; Near et al., 2012), thus calling into question the phylogenetic position of these extinct taxa. More recent molecular studies of tetraodontiforms (Holcroft, 2005; Alfaro et al., 2007; Yamanoue et al., 2007, 2008) have generally supported the monophyly of families (Fig. 3), but conflict with some of the higher-level relationships suggested by morphological analyses (Fig. 2). Bayesian and likelihood analyses of the nuclear locus Rag1 and the mitochondrial 12S and 16S revealed boxfishes and ocean sunfishes as sister taxa (Holcroft, 2005; Alfaro et al., 2007), with this novel clade sister to the three-tooth puffer (Alfaro et al., 2007). In contrast to morphological analyses, these studies also recovered a clade formed by triggerfishes and filefishes, triplespines and spikefishes, and another that contained pufferfishes and porcupine fishes. Topologies based upon mitogenomic studies (Yamanoue et al., 2007, 2008) are at odds with both morphological and nuclear DNA-based analyses in placing a clade comprised of triodontid + (triacanthodids + ostracioids) as sister to all other tetraodontiforms (Fig. 3). Relationships within the remaining tetraodontiforms include the molids as the sister group to the clade comprising two subclades: triacanthids + (tetraodontids + diodontids) and balistids + monacanthids. To resolve these apparent conflicts regarding higher-level relationships within tetraodontiforms, we undertook a new molecularbased analysis. Our study samples the largest number of molecular markers to date within tetraodontiforms (20 nuclear and 2 mito- chondrial loci) and provides a well-supported framework for interpreting the evolutionary history of this spectacular radiation. 2. Materials and methods 2.1. Sampling We secured tissue samples for 40 species of tetraodontiforms, plus three outgroups, through loans from university or museum collections, or purchases through the pet trade (Table 1). The species included in this study were selected in order to include the phylogenetically most diverging lineages within each family on the basis of previously published studies (Santini and Tyler, 2002a, 2002b; Dornburg et al., 2011; Santini et al., 2013a, 2013b), thus recovering the whole crown of most families. Sequences already available in GenBank for markers used in this study were also added to our dataset (Table 1). To verify the identification of our tissue samples, all extractions were barcoded for cytochrome oxidase subunit I, and BLASTed using the barcoding of life identification search engine (http://www.barcodinglife.com/index.php/IDS_OpenIdEngine). In spite of the current uncertainty about percomorph higher-level relationships, a strong agreement exists among molecular studies for the presence of a clade formed by Tetraodontiformes, Lophiiformes and Caproidae (Chen et al., 2003; Miya et al., 2003; Holcroft, 2004; Li et al., 2008; Santini et al., 2009; Near et al., Author's personal copy 182 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 Fig. 3. Summary of previous molecular hypotheses of tetraodontiform relationships. Fish outlines modified from Fishbase.org. 2012). For this reason we included two lophiiforms (Antennarius, Ogcocephalus) and a caproid (Antigonia) as outgroups. 2.2. DNA extraction, PCR amplification, and sequencing DNA was extracted from muscle tissue or fin clips stored in 70% ethanol using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), following the protocol suggested by the manufacturer. We used the polymerase chain reaction (PCR) to amplify two mitochondrial genes (cytochrome oxidase subunit I, cox1, and cytochrome b, Cytb) and 20 nuclear genes (early growth response gene 1 (EGR1), early growth response gene 3 (EGR3), ectodermal–neural cortex 1 (ENC1), glycosyltransferase (Glyt), interphotoreceptor retinoid-binding protein (IRBP); ENSDARG00000059744 (KIAA2013), mixed-linked Leukemia-like gene (MLL); cardiac muscle myosin heavy chain 6 alpha (myh6), pleiomorphic adenoma gene-like 2 (plagl2), hypothetical protein LOC564097 (Ptr), recombination activating gene 1 (Rag1), rhodopsin (Rh), novel protein similar to vertebrate ryanodine receptor 3 (RYR3), protein similar to SH3 and PX domain containing 3 gene (SH3PX3), si:dkey-174m14.3 (sidkey), Super conserved receptor expressed in brain 2 (sreb2), T-box brain 1 (tbr1), zgc:92173(UBE3A), zic family member 1 (zic1), and Zinc finger protein 503 (znf503)). One to two microliters of genomic template was used per 25-lL reaction containing 5 lL of 5 Go-Taq Flexi PCR buffer (Promega), 2 lL MgCl2 (25 mM), 0.5 lL dNTPs (2.5 lM), 1.25 lL of each primer (10 lM), and 0.125 lL of Promega GoTaq Flexi DNA polymerase (5 U/lL). Primers and PCR conditions were obtained from the literature: Ward et al. (2005) for cox1; Sevilla et al. (2007) for Cytb; Chen et al. (2008) for EGR1 and EGR3; Li et al. (2009) for MLL; Li et al. (2007) for ENC1, Glyt, myh6, plagl2, Ptr, RYR3, SH3PX3, sreb2, tbr1, and zic1; Li et al. (2010) for KIAA2013, sidkey, UBE3A, and znf503; Lopez et al. (2004) for Rag1; and Chen et al. (2003) for Rh. PCRs were performed on MJ Research PTC-200 Peltier or Eppendorf Mastercycler ProS thermal cyclers. All products were stored at 20 °C after amplification. We used ExoSap (Amersham Biosciences) to remove the excess dNTPs and unincorporated primers from the PCR products; purified products were then cycle-sequenced using the BigDye Terminator v.3.1 cycle sequencing kit (1/8th reaction) (Applied Biosystems) with each gene’s original or additional internal primers used for amplification. The cycle sequencing protocol consisted of 25 cycles with a 10-s 94 °C denaturation, 5-s of 50 °C annealing, and a 4-min 60 °C extension stage. Sequencing was conducted at the Yale University DNA Analysis Facility using an ABI 3730xl DNA Genetic Analyzer (Applied Biosystems). 2.3. Phylogenetic analyses Chromatograms were checked and assembled into contigs using Geneious 5.4 (Drummond et al., 2011). The consensus sequences for each individual gene were aligned in Geneious using the MUSCLE software (Edgar, 2004), and the alignments subsequently checked by eye for accuracy. The sequences were trimmed to Author's personal copy F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 183 Fig. 4. Bayesian phylogenetic hypothesis of tetraodontiform relationships based on analysis of the concatenated dataset using Mrbayes 3.2. Black circles indicate nodes with posterior probability values equal or greater than 0.99; gray circles indicate posterior probability between 0.98 and 0.85, white circles indicate support lower than 0.85. Fish outlines modified from Fishbase.org. minimize missing characters, and our final data matrix consisted of 680 bp for cox1, 1089 bp for Cytb, 858 bp for EGR1, 799 for EGR3, 859 for ENC1, 747 for Glyt, 783 for IRBP, 717 for KIAA2013, 708 bp for MLL, 834 bp for myh6, 756 for plagl2, 774 for Ptr, 1407 bp for Rag1, 771 bp for Rh, 835 for RYR3, 749 for SH3PX3, 990 for sidkey, 872 for sreb2, 698 for tbr1, 674 for UBE3A, 834 for zic1, and 1248 for znf503, for a total of 18682 nucleotides used in the concatenated analyses. All sequences generated for this study were deposited in GenBank (Table 1). We used jModelTest (Posada, 2008) to select the best fitting model of sequence evolution from the candidate pool of models that can be utilized in MrBayes 3.2 (Ronquist et al., 2012) using corrected Akaike Information Criterion (AICc; Akaike, 1973). We did not include the proportion of invariant sites parameter in the candidate pool, as this parameter is already taken into consideration by the gamma parameter (Yang, 2006). The GTR + G model was selected as the most appropriate for cox1, Cytb, EGR1, EGR3, Glyt, KIAA, MLL, myh6, plagl2, Rag1, RYR3, sidkey, UBE3A, zic1, and znf503; HKY + G was selected as the best model for Ptr, Rh, and sreb2; TRN + G was selected as the best model forENC1, IRBP, SH3PX3, tbr1. The individual gene datasets were subject to maximum likelihood analyses using RAxML (Stamatakis, 2006) to test for incongruence between gene histories of different loci, and to identify potentially contaminated or mislabeled sequences. We assigned a GTR + G model to each individual gene partition, implementing the RAxML model closest to the jModelTest results, and ran 100 fast bootstrap replicates with the GTR + CAT model. The 22 individual gene datasets were then concatenated in Mesquite 2.75 (Maddison and Maddison, 2011), and the full dataset was partitioned by gene and subject to maximum likelihood analyses with RAxML (Stamatakis, 2006). Each partition was again assigned its own GTR + G model, the closest model to the jModelTest selection implemented in RAxML, and subject to a full maximum likelihood search. We also ran 1000 fast bootstrap replicates using the GTR + CAT model. We used MrBayes 3.2 (Ronquist et al., 2012) to perform Bayesian analyses; we partitioned the concatenated dataset by locus and assigned each gene the model selected by jModelTest. Each Bayesian analysis was run for 20 million generations, with four chains (one cold, three heated), sampling every 1000 generations. Trace files were checked in Tracer 1.5 (Rambaut and Drummond, 2009) to ensure that the chains had reached convergence, and the first 25% of trees was discarded as burnin. Post-burnin trees were combined to obtain a 50% majority rule consensus tree. We used iGTP (Chaudhary et al., 2010) to estimate a species tree using gene tree parsimony; the individual gene trees obtained with RAxML were rooted using the lophiiform outgroups (Capros was removed to reduce the number of taxa analyzed and speed up the analysis) and used as input files. We performed a heuristic Author's personal copy 184 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 Fig. 5. Species-tree hypothesis of tetraodontiform relationships based on parsimony gene-tree, species-tree analysis of molecular dataset. Nuclear genes supporting each clade, indicated by letters below branches, are provided in Table S1. search using a randomized hill climb in order to infer the species tree that minimized the cost for the deep coalescence, and mapped the individual loci that supported each node on the most parsimonious tree. 3. Results 3.1. Phylogenetic analyses Both maximum likelihood and Bayesian analyses of the concatenated dataset strongly support a very similar topology. Tetraodontiform monophyly is strongly supported (Bayesian posterior probability, PP, >0.99) in the Bayesian phylogeny (Fig. 4) and a bootstrap proportion (BSP) of 100% in the maximum likelihood phylogeny (Fig. S1). Both topologies infer two main clades: the first includes the Triodontidae sister to a group containing Ostraciidae + Aracanidae and Triacanthidae + Triacanthodidae. Support for the monophyly of this clade is high (>0.99 PP, 70% BSP). Both sets of analyses show weak support for the sister group relationships of boxfishes and the spikefishes + triplespines group, with 0.58 PP and 39% BSP, while all other nodes in the trees are very highly supported with >0.99 PP and 99% or 100% BSP. Within triacanthodids the Hollardinae Parahollardia is sister to a clade formed by the Triacanthodinae Triacanthodes and Tydemania, with high support (>0.99 PP, 100% BSP). The second tetraodontiform clade, supported by >0.99 PP, but only 72% BSP, is composed of the balistoids (Balistidae + Monacanthidae), sister to the bulk of the old gymnodonts, with Molidae sister to Diodontidae + Tetraodontidae. Support for every node in this clade is very high in the Bayesian tree (Fig. 4), with PP of at least 0.99 for all but that supporting two of the diodontid species. In the maximum likelihood tree support is not as high, with BSP below 70% for the sister group relationship between molids and diodontids + tetraodontids, and some of the tetraodontid subclades. Within diodontids Chilomycterus reticulatus appears as the sister group of Diodon to the exclusion of C. schoepfii. Within tetraodontids several subclades are identified, including a Marilyna + (Takifugu + Torquigener), a Lagocephalus + Sphoeroides, and a third large clade that includes three distinct lineages of Tetraodon, in addition to the reef-associated Arothron and Canthigaster. The species tree estimated using iGTP (Fig. 5) is largely congruent with the concatenated analyses results for the relationships Author's personal copy F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 among tetraodontiform groups, but with two exceptions: tetraodontids + diodontids form the sister group to all other tetraodontiforms, and Triodon is the sister group to the ostracioids, as opposed to the clade formed by triacanthids + triacanthodids and boxfishes. 4. Discussion 4.1. Tetraodontiform relationships Our new multi-locus phylogeny, based on the largest molecular dataset yet assembled for tetraodontiform fishes, supports the monophyly of the order, all the families, several of the currently recognized superfamilies (e.g., Balistoidea, triggerfishes + filefishes; Ostracioidea, boxfishes + trunkfishes), as well as the previously abandoned Triacanthoidea (Triacanthodidae + Triacanthidae, Winterbottom, 1974). Our study, however, infers several novel higher-level relationships not hypothesized in any previous study. The clade formed by Triodontidae + (Ostracioidea + Triacanthoidea) does not resemble any of the previous morphological topologies, but some of its components appeared in previous molecular studies. Holcroft (2005) and Alfaro et al. (2007) retrieved a close relationship of Triodontidae and Ostracioidea, but Triacanthoidea was not part of this group, which appeared to be sister group to the Molidae instead. Similarly, while this clade shares several components with one of the two main tetraodontiform lineages recovered by the Yamanoue et al. (2008) mitogenomic study, it differs in the position of the Triacanthidae, which appears to be deeply nested in this clade as opposed to being closely related to puffers + porcupine fishes as in the mitogenomic topology. It is important to point out that while Triodontidae usually appears in a much more derived position within Tetraodontiformes in all morphological studies, Triodon is the only extant tetraodontiform that possesses a number of morphological characters only found in stem tetraodontiforms, as well as other percomorphs, such as a ceratohyal bone with a beryciform foramen, a basisphenoid bone in the skull, true ribs, a parhyurapophysis, autogenous haemal arch and spine of PU3, and procurrent caudal-fin rays (Tyler, 1980; Santini and Tyler, 2003; Britz and Johnson, 2012). These features, together with the presence of a pelvic bone (well developed, although the pelvic fins have been lost), and a first (spiny) dorsal fin suggest that Triodon may be a more ancient lineage than previously thought. The second tetraodontiform clade recovered in our study is more congruent with previous morphological and molecular studies. A clade containing Balistoidea and the Molidae + (Tetraodontidae + Diodontidae) clade is recovered by most morphological studies (Fig. 3), although this clade always includes Triodontidae and Ostracioidea. Among the molecular studies, weak support for a close relationship between balistoid and tetraodontoid fishes is found in Holcroft (2005) and Alfaro et al. (2007), while greater support is present in the Yamanoue et al. (2009) mitogenomic study (Fig. 3). Near et al.’s (2012) maximum likelihood analysis of nine nuclear loci (most of which have been included in this study) infers a topology that differs significantly from our findings, with diodontids + tetraodontids appearing as sister taxa to all other tetraodontiforms, and triacanthids and triacanthodids appearing as close relatives of molid sunfishes. Most of the disagreements between the older studies and our new results stem from the position of Triacanthidae and Molidae. Triacanthidae and Triacanthodidae appear as sequential sister taxa to the balistoids, while Molidae belongs within a different tetraodontiform clade in Holcroft (2005) and Alfaro et al. (2007). In Yamanoue et al. (2008), Triacanthidae appears as the sister group to Tetraodontidae + Diodontidae, while Molidae is sister to this entire clade (Triacanthidae + (Tetraodontidae + Diodontidae)), rather than in their most traditional position as sister group to Tetraodontidae + Diodontidae. 185 Within some of the families, our results differ from a number of previous studies. Within triacanthids we recover Triacanthus nested within other triplespine genera, as opposed to finding this genus to be the sister to all other triacanthids as in morphological studies (Tyler, 1968; Santini and Tyler, 2002a). Within triggerfishes, we find Balistapus sister to a Balistes + Rhinecanthus clade, which conflicts with the findings of Dornburg et al. (2008, 2011) that inferred Balistes sister to the clade containing Balistapus + Rhinecanthus. Within Monacanthidae we find Aluterus sister to all remaining filefishes, while Oxymonacanthus appears deeply nested within this clade; this is in contrast with the mitogenomic study of Yamanoue et al. (2009) that found Oxymonacanthus sister taxon to all other species in our sampling, and Aluterus nested within the Monacanthidae. Within the Tetraodontidae we find high support (>0.99 PP) for a clade formed by the Indo-West Pacific Marilyna + (Takifugu + Torquigener) being sister to all other tetraodontids. This finding contrasts with a previous mitogenomic study that identified the pelagic genus Lagocephalus as the sister lineage to all other puffers (Yamanoue et al., 2011), but is supported by another study based on a subset of loci included in this study with a much denser taxonomic sampling within tetraodontids (Santini et al., 2013b). In other cases our findings corroborate previous hypotheses. Within Triacanthodidae the traditional split between Hollardinae and Triacanthodinae (Tyler, 1968; Santini and Tyler, 2003, 2004) is supported by Hollardia being sister taxon to the other species in our sampling. Similarly the relationships within the Ostracidae are in agreement with both morphological (Klassen, 1995) and densely sampled molecular studies (Santini et al., 2013a). 4.2. Novel tetraodontiform phylogeny and morphological evolution Our new phylogenetic hypothesis of tetraodontiform relationships suggests the need to re-evaluate a number of morphological features that have traditionally been considered robust indicators of tetraodontiform relationships. For example, the robust interdigitation of the premaxillae to form a beak-like structure has long been considered strong evidence in support of the monophyly of the gymnodonts, a clade formed by Triodontidae, Molidae, Diodontidae and Tetraodontidae. This clade has been recovered by all morphological analyses based on adult characters published to date (Winterbottom, 1974; Tyler, 1980; Santini and Tyler, 2003, 2004). Our topology, however, suggests that the interdigitation of the premaxillae may either have been acquired in the ancestor of all tetraodontiforms and subsequently lost at least twice (in balistoids and in ostracioids + triacanthids + triacanthodids), or may have occurred twice independently in the molid + diodontid + tetraodontid clade and then in triodontids. While the strong interdigitation of jaw bones to form a beak-like structure might be considered a rare event, thus suggesting that it should be a reliable indicator of phylogenetic relationships, the degree of jaw-bone articulation is distinct between Triodon, where it is highly incomplete, and the remaining gymnodonts (molids, tetraodontids and diodontids), where it is much stronger (Tyler, 1980; Santini and Tyler, 2003). This fact could also call into question the homology of these conditions. Traditionally, most of the disagreement among morphological studies was due to the position of the triacanthids and the ostracioid boxfishes (Leis, 1984; Santini and Tyler, 2003). Triacanthids appeared as either the sister to the triacanthodids, the sister to all other tetraodontiforms (excluding the triacanthodids), or the sister group to a clade formed by balistoids and ostracioids (Winterbottom, 1974; Santini and Tyler, 2003, 2004), but always appeared as one of the earliest diverging tetraodontiform lineages. Ostracioid boxfishes, a morphologically highly modified group that has experienced the complete loss of the first dorsal fin and the en- Author's personal copy 186 F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 tire pelvic complex (pelvic girdle plus fins), as well as the development of a massive carapace-like structure that encases the entire body and is formed by thickened and enlarged scale-plates, have traditionally been a more difficult lineage to place using morphological features (Fig. 2). While adult characters (both myological and osteological) recover a close relationship between boxfishes and balistoid trigger and filefishes, studies based on early life-history traits invariably fail to recover this grouping (Leis, 1984). Some recent larval studies have suggested a close relationship between boxfishes and ocean sunfishes on the basis of the fusion of the vertebral centra in the occipital region, and the presence of a thick band of cartilage on the side of the pterygiophores of the median fins (Britz and Johnson, 2005; Johnson and Britz, 2005). These studies, unfortunately, were based on examination of a very limited number of tetraodontiform species (two molids, one ostraciid), and did not produce any explicit phylogenetic analysis based on character matrices that could have allowed an evaluation of the phylogenetic distribution of these putative ostracioid + molid synapomorphies (or the presence and distribution of characters supporting alternative topologies), as was done in the explicitly phylogenetic studies by Winterbottom (1974), Leis (1984) and Santini and Tyler (2003, 2004). More recently a number of studies based on the ontogeny of various myological and skeletal features of tetraodontiforms relied on the morphological topology of Santini and Tyler (2003) for interpreting the evolution of adductor mandibulae muscles, the caudal complex, and the suspensorium and jaw apparatus (Konstantinidis and Harris, 2011; Konstantinidis and Johnson, 2012a, 2012b). We interpret this as an indication that larval materials do not provide sufficient evidence to overturn the tetraodontiform topology obtained from the analysis of the 219 morphological characters found in the Santini and Tyler (2013) datasets, and that the occipito-vertebral fusion and the thick layer of cartilage in the median-fin pterygiophores of ocean sunfishes and boxfishes are likely homoplastic features, potentially linked to the presence of carapace-like structures in these two groups (Tyler, 1980). This conclusion is also supported by the results of this study, which conclusively shows that ostracioid boxfishes and molids are not closely related. 5. Conclusion Bayesian and maximum likelihood analyses of the largest phylogenomic dataset currently assembled for tetraodontiforms reveal a novel topology, which corroborates monophyly of the order and all of its families, but suggests new relationships among the major lineages. Tetraodontiforms appear to split into two main groups: one formed by Triodon, the triacanthodids + triacanthids, and the ostracioid boxfishes; the second formed by balistids + monacanthids, sister to a clade formed by molids, diodontids and tetraodontids. Our new topology suggests that several of the morphological features that have long been thought to represent strong support of traditional groupings, such as the interdigitation of the premaxillae to form a parrot-like beak in gymnodonts, are in need of reevaluation. Our new topology may also allow us to re-evaluate the position of Triodon, one of the earliest diverging lineages in our tree, which possess several morphological features not found in any other extant tetraodontiforms, but present in stem tetraodontiforms and other percomorph lineages. Acknowledgments Funding for this project was provided by NSF Grant DEB 0842397 ‘‘Systematics and Influence of Coral Reefs on Diversification in Tetraodontiform Fishes’’ to MEA and FS. This research project was made possible by the generous loan or gift of tissues from a number of col- leagues and institutions: Victor Brian Alfaro (UCLA), Sean Kong (UCLA), Peter Wainwright (UC Davis), Andrew Bentley and Ed Wiley (University of Kansas), Mark McGrouther (Australian Museum), H.J. Walker and Phil Hastings (Scripps Institute of Oceanography), Leo Smith (Field Museum of Natural History). We thank Mai Nguyen and Vincent Wu for help with the labwork, and Giacomo Bernardi, Giorgio Carnevale and two anonymous reviewers for helpful comments to an earlier version of this manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2013.05. 014. References Akaike, H., 1973. Information theory as an extension of the maximum likelihood principle. In: Petrov, B.N., Caski, F. (Eds.), Second International Symposium on Information Theory. Akademiai Kiado, Budapest, pp. 267–281. Alfaro, M.E., Santini, F., Brock, C.D., 2007. Do reefs drive diversification in marine teleosts? Evidence from the pufferfishes and their allies (order Tetraodontiformes). Evolution 61, 2104–2126. Brainerd, E.L., Slutz, S.S., Hall, E.K., Phillis, R.W., 2001. Patterns of genome size evolution in tetraodontiform fishes. Evolution 55, 2363–2368. Brenner, S., Elgar, G., Sandford, R., Macrae, A., Venkatesh, B., Aparicio, S., 1993. Characterization of the pufferfish (fugu) genome as a compact model vertebrate genome. Nature 366, 265–268. Britz, R., Johnson, G.D., 2005. Occipito-vertebral fusion in ocean sunfishes (Teleostei: Tetraodontiformes: Molidae) and its phylogenetic implications. J. Morphol. 266, 74–79. Britz, R., Johnson, G.D., 2012. The caudal skeleton of a 20 mm Triodon and homology of its counterparts. Proc. Biol. Soc. Wash. 125, 66–73. Chaudhary, R., Bansal, M.S., Wehe, A., Fernández-Baca, D., Eulenstein, O., 2010. IGTP: a software package for large-scale gene tree parsimony analysis. BMC Bioinformatics 11, 574. Chen, W.J., Bonillo, C., Lecointre, G., 2003. Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Mol. Phylogenet. Evol. 26, 262–288. Chen, W.J., Miya, M., Saitoh, K., Mayden, R.L., 2008. Phylogenetic utility of two existing and four novel nuclear gene loci in reconstruction Tree of Life of rayfinned fishes: the order Cypriniformes (Ostariophysi) as a case study. Gene 423, 125–134. Dornburg, A., Santini, F., Alfaro, M.E., 2008. The influence of model averaging on clade posteriors: an example using the triggerfishes (Family: Balistidae). Syst. Biol. 57, 905–919. Dornburg, A., Sidlauskas, B., Sorenson, L., Santini, F., Near, T., Alfaro, M.E., 2011. The influence of an innovative locomotor strategy on the phenotypic diversification of triggerfish (Family: Balistidae). Evolution 65, 1912–1926. Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., 2011. Geneious v5.4. <http://www.geneious.com>. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Friel, J.P., Wainwright, P.C., 1997. A model system of structural duplication: homologies of adductor mandibulae muscles in tetraodontiform fishes. Syst. Biol. 46, 441–463. Friel, J.P., Wainwright, P.C., 1999. Evolution of complexity in motor patterns and jaw musculature of tetraodontiform fishes. J. Exp. Biol. 202, 867–880. Froese, R., Pauly, D., 2012. FishBase. <http://www.fishbase.org>. Hinegardner, R., 1968. Evolution of cellular DNA content in teleost fishes. Am. Nat. 102, 517–523. Holcroft, N.I., 2004. A molecular test of alternative hypotheses of tetraodontiform (Acanthomorpha: Tetraodontiformes) sister group relationships using data from the RAG1 gene. Mol. Phylogenet. Evol. 32, 749–760. Holcroft, N.I., 2005. A molecular analysis of the interrelationships of tetraodontiform fishes (Acanthomorpha: Tetraodontiformes). Mol. Phylogenet. Evol. 34, 525–544. Jaillon, O., Aury, J.M., Brunet, F., Petit, J.-L., Stange-Thomann, N., Mauceli, E., et al., 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431, 946–957. Johnson, G.D., Britz, R., 2005. Leis’ conundrum: homology of the clavus of the Ocean Sunfishes. 2. Ontogeny of the median fins and axial skeleton of Ranzania laevis (Teleostei, Tetraodontiformes, Molidae). J. Morphol. 266, 11–21. Klassen, G.J., 1995. Phylogeny and biogeography of the Ostraciinae (Tetraodontiformes:Ostraciidae). Bull. Mar. Sci. 57, 393–441. Konstantinidis, P., Harris, M.P., 2011. Same but different: ontogeny and evolution of the Musculus adductor mandibulae in the Tetraodontiformes. J. Exp. Zool. (Mol. Dev. Evol.) 316, 10–20. Konstantinidis, P., Johnson, G.D., 2012a. A comparative ontogenetic study of the tetraodontiform caudal complex. Acta Zool. 93, 98–114. Author's personal copy F. Santini et al. / Molecular Phylogenetics and Evolution 69 (2013) 177–187 Konstantinidis, P., Johnson, G.D., 2012b. Ontogeny of the jaw apparatus and suspensorium of the Tetraodontiformes. Acta Zool. 93, 351–366. Leis, J.M., 1984. Tetraodontiformes: relationships. In: Moser, H.G., Richards, W.J., Cohen, D.M., Fahay, M.P., Kendall Jr., A.W., Richardson, S.L. (Eds.), Ontogeny and Systematics of Fishes. Am. Soc. Ichthyol. Herp., pp. 459–463 (Spec. Publ. No. 1). Li, C., Orti, G., Zhang, G., Lu, G., 2007. A practical approach to phylogenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case study. BMC Evol. Biol. 7, 44–54. Li, C., Lu, G., Orti, G., 2008. Optimal data parititioning and a test case for ray-finned fishes (Actinopterygii) based on ten nuclear loci. Syst. Biol. 57, 519–539. Li, B., Dettaï, A., Cruaud, C., Couloux, A., Desoutter-Meniger, M., Lecointre, G., 2009. RNF213, a new nuclear marker for acanthomorph phylogeny. Mol. Phylogenet. Evol. 50, 345–363. Li, C., Orti, G., Zhao, J., 2010. The phylogenetic placement of sinipercid fishes (‘‘Perciformes’’) revealed by 11 nuclear genes. Mol. Phylogenet. Evol. 56, 1096– 1104. Lopez, J.A., Chen, W.J., Ortí, G., 2004. Esociform phylogeny. Copeia 2004, 449–464. Maddison, W.P., Maddison, D.R., 2011. Mesquite: A Modular System for Evolutionary Analysis, Version 2.75. <http://www.mesquiteproject.org>. Miya, M., Takeshima, H., Endo, H., Ishiguro, N.B., Inoue, J.G., Mukai, T., Satoh, T.P., Yamaguchi, M., Kawaguchi, A., Mabuchi, K., Shirai, S.M., Nishida, M., 2003. Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences. Mol. Phylogenet. Evol. 26, 121–138. Near, T.J., Eytan, R.I., Dornburg, A., Kuhn, K.L., Moore, J.A., Davis, M.P., Wainwright, P.C., Friedman, M., Smith, W.L., 2012. Resolution of ray-finned fish phylogeny and timing of diversification. Proc. Natl. Acad. Sci. USA 109, 13698–13703. Nelson, J.S., 2006. Fishes of the World, fourth ed. John Wiley & Sons, Inc., Hoboken, New Jersey, USA. Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Rambaut, A., Drummond, A.J., 2009. Tracer v1.5. <http://beast.bio.ed.ac.uk/Tracer>. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. Rosen, D.E., 1984. Zeiforms as primitive plectognath fishes. Am. Mus. Nov. 2782, 1– 38. Santini, F., Stellwag, E.J., 2002. Phylogeny, fossils, and model systems in the study of evolutionary developmental biology. Mol. Phylogenet. Evol. 24, 379–383. Santini, F., Tyler, J.C., 2002a. Phylogeny and biogeography of the extant species of triplespine fishes (Triacanthidae, Tetraodontiformes). Zool. Scr. 31, 321–330. Santini, F., Tyler, J.C., 2002b. Phylogeny of the ocean sunfishes (Molidae, Tetraodontiformes), a highly derived group of teleost fishes. Ital. J. Zool. 69, 37–43. Santini, F., Tyler, J.C., 2003. A phylogeny of the families of fossil and extant tetraodontiform fishes (Acanthomorpha, Tetraodontiformes), Upper Cretaceous to recent. Zool. J. Linn. Soc. 139, 565–617. Santini, F., Tyler, J.C., 2004. The importance of even highly incomplete fossil taxa in reconstructing the phylogenetic relationships of the Tetraodontiformes (Acanthomorpha: Pisces). Integr. Comp. Biol. 44, 349–357. 187 Santini, F., Harmon, L.J., Carnevale, G., Alfaro, M.E., 2009. Did genome duplication drive the origin of teleosts? A comparative study of diversification in ray-finned fishes. BMC Evol. Biol. 9, 164–178. Santini, F., Sorenson, L., Marcroft, T., Dornburg, A., Alfaro, M.E., 2013a. A multilocus molecular phylogeny of boxfishes (Aracanidae, Ostraciidae; Tetraodontiformes). Mol. Phylogenet. Evol. 66, 153–160. Santini, F., Nguyen, M., Sorenson, L., Alfaro, T.B., Eastman, J.M., Alfaro, M.E., 2013b. Do habitat shifts drive the diversity in teleost fishes? An example from the pufferfishes (Tetraodontidae). J. Evol. Biol. 66, 1003–1018. Sevilla, R.G., Diez, A., Noren, M., Mouchel, O., Jerome, M., Verrez-Bagnis, V., et al., 2007. Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes. Mol. Ecol. Resour. 7, 730–734. Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688– 2690. Tyler, J.C., 1968. A monograph on plectognath fishes of the superfamily Triacanthoidea. Monogr. Acad. Nat. Sci. Philad. 16, 1–364. Tyler, J.C., 1980. Osteology, phylogeny, and higher classification of the fishes of the order Plectognathi (Tetraodontiformes). NOAA Tech. Rep. NMFS Circ. 434, 1– 422. Tyler, J.C., Santini, F., 2005. A phylogeny of the fossil and extant zeiform-like fishes, Upper Cretaceous to Recent, with comments on the putative zeomorph clade (Acanthomorpha). Zool. Scr. 34, 157–175. Tyler, J.C., Sorbini, L., 1996. New superfamily and three new families of tetraodontiform fishes from the Upper Cretaceous: the earliest and most morphologically primitive plectognaths. Smithson. Contrib. Paleobiol. 82, 1–59. Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., Hebert, P.D., 2005. DNA barcoding Australia’s fish species. Philos. Trans. Roy. Soc. B 360, 1847–1857. Winterbottom, R., 1974. The familial phylogeny of the Tetraodontiformes (Acanthopterygii: Pisces) as evidenced by their comparative myology. Smithson. Contrib. Zool. 155, 1–201. Yamanoue, Y., Miya, M., Matsuura, K., Yagishita, N., Mabuchi, K., Sakai, H., Katoh, M., Nishida, M., 2007. Phylogenetic position of tetraodontiform fishes within the higher teleosts: Bayesian inferences based on 44 whole mitochondrial genome sequences. Mol. Phylogenet. Evol. 45, 89–101. Yamanoue, Y., Miya, M., Matsuura, K., Katoh, M., Sakai, H., Nishida, M., 2008. A new perspective on phylogeny and evolution of tetraodontiform fishes (Pisces: Acanthopterygii) based on whole mitochondrial genome sequences: basal ecological diversification? BMC Evol. Biol. 8, 212. Yamanoue, Y., Miya, M., Matsuura, K., Sakai, H., Katoh, M., Nishida, M., 2009. Unique patterns of pelvic fin evolution: a case study of balistoid fishes (Pisces: Tetraodontiforms) based on whole mitochondrial genome sequences. Mol. Phylogenet. Evol. 50, 179–189. Yamanoue, Y., Miya, M., Doi, H., Mabuchi, K., Sakai, H., Nishida, M., 2011. Multiple invasions into freshwater by pufferfishes (Teleostei: Tetraodontidae): a mitogenomic perspective. PLoS One 6, e17410. Yang, Z., 2006. Computational Molecular Evolution. Oxford University Press, Oxford.