Table S1. Taxon sampling, habitat type, and Genbank numbers of specimens used in this study. Habitat is scored as 0=marine, 1=freshwater, 2=anadromous, and 3=catadromous. Taxa Habitat rag1 rag2 16s cytb Clupeidae Alosa aestivalis 2 NA DQ912146 DQ912076 EU552615 Alosa alabamae 2 NA KJ158109 KJ158129 KJ158091 Alosa alosa 0 NA NA NC_009575 NC_009575 Alosa chrysochloris 1 DQ912117 DQ912151 DQ912081 KJ158092 Alosa fallax 0 NA NA EU552737 EU552574 Alosa mediocris 2 KJ158146 KJ158110 KJ158130 KJ158093 Alosa pseudoharengus 2 DQ912115 DQ912149 DQ912079 AP009132 Alosa sapidissima 2 DQ912116 DQ912150 DQ912080 EU552616 Anodontostoma chacunda 0 NA NA AP011614 AP011614 Brevoortia aurea 0 NA NA NA EF564665 Brevoortia patronus 0 DQ912105 DQ912138 DQ912068 EU552618 Brevoortia smithi 0 KJ158148 KJ158112 KJ158131 KJ158094 Brevoortia tyrannus 0 DQ912106 DQ912139 DQ912069 EU552614 Clupea harengus 0 DQ912114 DQ912148 DQ912078 EU552606 Clupea pallasii 0 DQ912118 DQ912152 DQ912082 EU552599 Clupeichthys aesarnensis 1 NA NA AP011584 AP011584 Clupeichthys perakensis 1 NA NA AP011585 AP011585 Clupeoides borneensis 1 NA NA AP011586 AP011586 Clupeonella cultriventris 2 NA NA NC_015109 NC_015109 Corica laciniata 1 NA NA AP011589 AP011589 Dorosoma cepedianum 1 DQ912099 DQ912132 DQ912062 EU552586 Dorosoma petenense 1 KJ158147 KJ158111 NC_009580 EU552581 Ehirava fluviatilis 0 NA NA AP011588 AP011588 Escualosa thoracata 0 NA NA AP011601 AP011601 Ethmidium maculatum 0 NA NA AP011602 AP011602 Ethmalosa fimbriata 0 NA NA NC_009582 NC_009582 Etrumeus teres 0 DQ912110 DQ912143 DQ912073 EU552621 Etrumeus whiteheadi 0 NA NA EU552730 EU552567 Gilchristella aestuaria 1 NA NA EU552741 EU552578 Gudusia chapra 1 KJ158145 KJ158108 KJ158128 KJ158090 Harengula humeralis 0 KJ158135 KJ158098 KJ158116 NA Harengula jaguana 0 DQ912122 DQ912156 DQ912086 EU552617 Hilsa kelee 0 NA NA AP011613 AP011613 Hyperlophus vittatus 0 NA NA EU552750 EU552587 Jenkinsia lamprotaenia 0 DQ912107 DQ912140 DQ912070 EU552613 Konosirus punctatus 0 NA NA NA AB548682 Lile stolifera 0 KJ158137 KJ158100 KJ158119 KJ158080 Limnothrissa miodon 1 NA NA EU552720 EU552553 Microthrissa congica 1 NA NA EU552789 EU552625 Microthrissa royauxi 1 NA NA EU552790 EU552626 Nematalosa erebi 1 NA NA EU552755 EU552592 Nematalosa japonica 0 NA NA NC_009586 NC_009586 Odaxothrissa vittata 1 DQ912131 DQ912167 DQ912096 NC_009590 Opisthonema libertate 0 NA KJ158101 KJ158120 KJ158081 Opisthonema oglinum 0 DQ912111 DQ912144 DQ912074 EU552620 Pellonula vorax 1 NA NA EU552792 EU552628 Pellonula leonensis 1 DQ912130 DQ912166 DQ912095 EU552624 Potamalosa richmondia 1 NA NA AP011594 AP011594 Potamothrissa obtusirostris 1 NA NA EU552787 EU552623 Ramnogaster sp. 0 NA NA NA GQ890211 Rhinosardinia amazonica 1 NA NA EU552713 EU552550 Rhinosardinia bahiensis 1 KJ158149 KJ158113 KJ158132 KJ158095 Sardina pilchardus 0 NA DQ912158 NC_009592 AF472582 Sardinella aurita 0 DQ912104 DQ912137 DQ912067 EU552619 Sardinella lemuru 0 KJ158136 NA KJ158117 KJ158078 Sardinella maderensis 0 NA NA NC_009587 NC_009587 Sardinops sagax 0 NA NA NC_002616 EU552565 Sauvagella madagascariensis 1 NA NA EU552773 EU552610 Sauvagella robusta 1 NA NA EU552770 EU552608 Sierrathrissa leonensis 1 NA NA EU552756 EU552593 Spratelloides delicatulus 0 DQ912128 DQ912164 NC_009588 NC_009588 Spratelloides gracilis 0 DQ912129 DQ912165 NC_009589 NC_009589 Spratelloides robustus 0 NA NA EU552786 NA Sprattus antipodum 0 NA NA AP011608 AP011608 Sprattus muelleri 0 NA NA AP011607 AP011607 Sprattus sprattus 0 NA NA AP009234 AP009234 Stolothrissa tanganicae 1 NA NA EU552719 EU552552 Tenualosa ilisha 2 NA NA DQ400344 EU552622 Tenualosa thibaudeaui 1 NA NA AP011604 AP011604 Chirocentridae Chirocentrus dorab 0 DQ912127 DQ912163 AP006229 NC_006913 Denticipitidae Denticeps clupeoides 1 DQ912100 DQ912133 DQ912063 EU552629 Engraulidae Amazonsprattus scintilla 1 JQ012538 JQ012667 JQ012456 JQ012351 Anchoa cubana 0 JQ012550 JQ012705 JQ012447 JQ012342 Anchoa lyolepis 0 JQ012573 JQ012688 JQ012449 JQ012344 Anchoa cayorum 0 JQ012555 JQ012700 JQ012451 JQ012347 Anchoa mitchilli 0 JQ012552 JQ012698 JQ012462 JQ012357 Anchoa walkeri 0 JQ012568 JQ012713 JQ012474 JQ012369 Anchoa nasus 0 JQ012575 JQ012690 JQ012478 JQ012373 Anchoa chamensis 0 JQ012563 JQ012718 JQ012480 JQ012375 Anchoa parva 0 JQ012558 JQ012702 JQ012482 JQ012377 Anchoa lamprotaenia 0 JQ012630 JQ012696 JQ012484 JQ012379 Anchoa colonensis 0 JQ012559 JQ012716 JQ012488 JQ012383 Anchoa panamensis 0 JQ012570 JQ012712 JQ012497 JQ012392 Anchoa mundeoloides 0 JQ012565 JQ012715 JQ012524 JQ012419 Anchoa delicatissima 0 JQ012557 JQ012704 JQ012453 JQ012348 Anchoa schofieldi 0 JQ012571 JQ012711 JQ012454 JQ012349 Anchoa filfera 0 JQ012542 JQ012722 JQ012492 JQ012387 Anchoa spinifer 0 KJ158140 KJ158104 KJ158123 KJ158085 Anchoa sp. 0 KJ158141 KJ158105 KJ158124 KJ158086 Anchovia macrolepidota 0 JQ012561 JQ012709 JQ012499 JQ012394 Anchovia surinamensis 1 JQ012613 JQ012665 JQ012507 JQ012402 Anchovia clupeoides 0 KJ158142 KJ158106 KJ158125 KJ158087 Anchoviella guianensis 1 JQ012606 JQ012657 JQ012429 JQ012324 Anchoviella alleni 1 JQ012598 JQ012655 JQ012438 JQ012333 Anchoviella alleni 1 JQ012628 JQ012669 JQ012440 JQ012335 Anchoviella sp. 2 1 JQ012581 JQ012648 JQ012442 JQ012337 Anchoviella carrikeri 1 JQ012605 JQ012659 JQ012444 JQ012339 Anchoviella sp. 1 1 JQ012589 JQ012677 JQ012458 JQ012353 Anchoviella balboae 0 JQ012566 JQ012720 JQ012476 JQ012371 Anchoviella elongata 0 JQ012548 JQ012707 JQ012486 JQ012381 Anchoviella cf. guianensis 1 JQ012585 JQ012673 JQ012505 JQ012400 Anchoviella sp. 3 1 JQ012593 JQ012632 JQ012509 JQ012404 Anchoviella guianensis 1 JQ012591 JQ012652 JQ012511 JQ012406 Anchoviella brevirostris 0 JQ012608 JQ012686 JQ012517 JQ012412 Anchoviella lepidentistole 0 JQ012596 JQ012634 JQ012519 JQ012414 Anchoviella carrikeri 1 JQ012583 JQ012650 JQ012528 JQ012422 Anchoviella carrikeri 1 JQ012599 JQ012663 JQ012530 JQ012424 Anchoviella cf. guianensis 1 JQ012544 JQ012646 JQ012432 JQ012327 Anchoviella jamesi 1 KJ158138 KJ158102 NA KJ158083 Anchoviella manamensis 1 KJ158139 KJ158103 NA KJ158084 Cetengraulis edentulus 0 JQ012577 JQ012692 JQ012490 JQ012385 Cetengraulis mysticetus 0 JQ012579 JQ012694 JQ012495 JQ012390 Coilia brachygnathus 1 DQ912124 DQ912159 DQ912089 EU694410 Coilia lyndmani 1 NA NA NC_014271 NC_014271 Coilia mystus 0 DQ912126 DQ912162 DQ912092 EU694407 Coilia nasus 0 DQ912123 DQ912157 AP009135 AP009135 Coilia reynaldi 0 NA NA NC_014276 NC_014276 Encrasicholina devisi 0 JQ012626 JQ012684 JQ012469 JQ012364 Encrasicholina punctifer 0 NA NA AP011561 AP011561 Engraulis anchoita 0 NA NA JQ012521 JQ012416 Engraulis ringens 0 JQ012533 JQ012731 JQ012532 JQ012426 Engraulis eurystole 0 DQ912121 DQ912155 DQ912085 JQ012427 Engraulis japonicus 0 AY430205 NA NC003097 NC_003097 Engraulis mordax 0 JQ012546 JQ012728 JQ012455 JQ012350 Engraulis encrasicolus 0 JQ012540 JQ012726 JQ012464 JQ012359 Jurengraulis juruensis 1 JQ012610 JQ012732 JQ012434 JQ012329 Lycengraulis poeyi 0 JQ012621 JQ012642 JQ012475 JQ012370 Lycengraulis grossidens 0 JQ012622 JQ012639 JQ012501 JQ012396 Lycengraulis batesii 1 JQ012619 JQ012643 JQ012515 JQ012411 Lycothrissa crocodilus 0 JQ012534 JQ012683 JQ012526 JQ012420 Pterengraulis atherinoides 1 JQ012616 JQ012636 JQ012428 JQ012323 Setipinna melanochir 0 NA NA AP011565 AP011565 Setipinna taty 0 NA JQ012681 JQ012470 JQ012365 Setipinna cf. tenuifilis 0 NA JQ012682 JQ012503 JQ012398 Stolephorus cf. chinensis 0 NA NA AP011566 AP011566 Stolephorus cf. waitei 0 NA NA AP011567 AP011567 Stolephorus sp. 0 JQ012536 JQ012671 JQ012466 JQ012361 Thryssa baelama 0 NA NA NC_014264 NC_014264 Thryssa c.f. dussumieri 0 JQ012535 JQ012678 JQ012468 JQ012363 Thryssa mystax 0 JQ012537 JQ012680 JQ012471 JQ012366 Pristigasteridae Chirocentrodon bleekerianus NA NA KJ158121 NA Ilisha africana 0 NA NA NC_009584 NC_009584 Ilisha amazonica 1 KJ158151 KJ158115 KJ158134 KJ158097 Ilisha elongata 0 NA DQ912160 DQ912090 AP009141 Ilisha megaloptera 0 NA KJ158099 KJ158118 KJ158079 Odontognathus mucronatus 0 NA NA KJ158122 KJ158082 Pellona castelnaeana 1 DQ912102 DQ912135 DQ912065 EU552554 Pellona flavipinnis 1 DQ912101 DQ912134 DQ912064 EU552551 Pellona harroweri 0 KJ158143 KJ158107 KJ158126 KJ158088 Pristigaster cayana 1 KJ158150 KJ158114 KJ158133 KJ158096 Pristigaster whiteheadi 1 KJ158144 NA KJ158127 KJ158089 Sundasalangidae Sundasalanx mekongensis 1 NA NA AP006232 AP006232 Outgroups Salmonidae Oncorhynchus mykiss 2 U15663 U31670 NC_001717 NC_001717 Cyprinidae Danio rerio 1 U71093 DRU71094 NC_002333 NC_002333 Cyprinidae Cyprinus carpio 1 AY787040 DQ366994 NC_001606 NC_001606 Ictuluridae Ictalurus punctatus 1 AY423859 AY184245 NC003489 NC_003489 Hepsetidae Hepsetus odoe 1 DQ912097 AY804086 NC_015819 NC_015819 Chanidae Chanos chanos 0 AY430207 NA NC_004693 NC_004693 Table S2. Models of evolution and partitioning strategy selected by PartitionFinder and implemented in RAxML. Partition Model selected Model used rag1, 1st position SYM+G GTR+G rag1, 2nd position, rag2 2nd position SYM+I+G GTR+G rag1, 3rd position SYM+I+G GTR+G rag2, 1st position GTR+I+G GTR+G rag2, 3rd position K80+I+G HKY+G cytb, 1st position SYM+I+G GTR+G cytb 2nd position GTR+I+G GTR+G cytb, 3rd position GTR+G GTR+G 16s GTR+G GTR+G Fossil Calibrations To determine absolute divergence times, we used seven fossil and biogeographic age calibrations with exponential priors to set a hard minimum and soft maximum bound [1]. Several of the fossil calibrations have been used in recent studies [2-4], however we include additional clupeiform fossils that have not yet been included in diversification time analysis of this group.