S1 Supp Tables

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. Our calibrations include:

1) For the MRCA of ostarioclupeomorpha we used Tischlingerichthys viohli from the

Thitonian (early Jurrasic; 149ma), the oldest known crown species of this group [5]. We used Anaethalion, the oldest stem elopomorph from the late Kimmeridgian (Jurassic;

152ma) [6] for the 95% upper boundary.

2) For the MRCA of Clupeidae+Sundasalangidae we used the oldest crown Clupeidae,

Nolfia riachuelensis from Albian (late Lower Cretaceous, 99ma) marine deposits in northeastern Brazil [7]. The 95% upper boundary was determined by the oldest stem clupeomorphs [7-9] from the Barremian (Lower Cretaceous; 125ma).

3) For the MRCA of Pristigasterids, we used Gasteroclupea branisai, the oldest crown

Pristigasteridae from the late Cretaceous (67ma) [10]. The 95% upper boundary was determined by the oldest stem clupeomorphs from the Barremian [7-9].

4) A fossil of Dorosoma petenense from the Gatuña Formation in southeastern New

Mexico dated to the Pliocene-Pleistocene boundary (2.5ma) was used for the MRCA of Dorosoma petenense and Dorosoma cepedianum [11]. The 95% upper boundary for this node was determined by Nolfia riachuelensis from the Albian (late Lower Creteaceous,

99ma, the oldest known crown Clupeidae [7].

5-7) The following pairs of anchovies are each sister taxa separated by the Isthmus of

Panama: 1) Cetengraulis edentulus / C. mysticetus, 2) Anchovia macrolepidota /

Anchovia clupeoides, and 3) Lycengraulis grossidens / L. poeyi [12-14]. For each species pair we used a minimum age of 3.0ma. For a 95% upper bound we used the oldest crown

Pristigasteridae, Gasteroclupea branisai, from the late Cretaceous (67ma).

EXPANDED PHYLOGENETIC RESULTS and DISCUSSION

Phylogenetic relationships

The best ML tree (-lnL = -147977.650367) with bootstrap support values is shown in Figure S1. Our results show that Clupeiformes are not monophyletic because

Denticeps clupeoides was sister to all remaining Ostarioclupeomorphs. However, clupeoidei (all Clupeiformes except Denticeps) was well supported as monophyletic. Our higher-level relationships among clupeiforms indicate that Chirocentridae is sister to

Engraulidae with a bootstrap of 93% and a posterior probability of 1.0. Pristigasteridae was well supported as monophyletic and our ML tree places this clade as sister to the

Engraulidae + Chirocentridae clade (but bootstrap <70%), while our BEAST analysis

(relationships discussed are from the MCC tree throughout) recovers Pristigasteridae as sister to Clupeidae (but with no statistical support). We found that Clupeidae is not monophyletic because Spratelloidini

(Spratelloides + Jenkinsia) [10] was sister to all other Clupeiformes, rather than part of the Clupeidae clade in both the ML and BEAST trees. This is a novel placement for

Spratelloidini, which was previously considered sister to Dussumieriini, forming the clade Dussumieriinae. Sundasalanx mekongensis (Sundasalangidae) was nested within

Clupeidae as a member of the Ehiravini clade [10, 15] in the both the ML tree and

BEAST tree. We also did not find support for the monophyly of Alosinae, Clupeinae,

Dorosomatinae, or Pellonulinae; instead the members of these previously recognized groups were spread throughout Clupeidae. Our analyses provide the first molecular support for Pellonulini; however, our analyses indicate that this clade is not sister to

Ehiravini as previously suggested [10].

Engraulidae was well supported as a monophyletic clade with a bootstrap of

100% and posterior probability of 1.0. The relationships among engraulids were well resolved and supported the monophyly of both Engraulinae, comprised of New World anchovies and Indo-Pacific genera Stolephorus and Encrasicholina, and Coilinae, the remaining Indo-Pacific genera. Engraulini (New World anchovies) are well supported as a monophyletic group.

Diversification times

The diversification time estimates for Clupeiformes are shown in S2. The mean posterior age for the MRCA of Clupeiformes dated to the Late Jurassic (150 Ma).

Clupeidae was the oldest major lineage dating to the Cretaceous with a mean posterior age for the MRCA of 105 Ma. The mean posterior age for the MRCA’s of Engraulidae

(89 Ma) and Pristigasteridae (71 Ma) dated to the Late Cretaceous.

DISCUSSION

Phylogenetic relationships and divergence times in Clupeiformes

The higher-level relationships among Clupeiformes have proven difficult to resolve.

Even the monophyly of Clupeiformes has been questioned recently because Denticeps clupeoides, the lone extant representative of Denticepitoidei, has been grouped with

Ostariophysi [16] or as the sister to Ostariophysi and all other clupeiforms [4]. Our study is consistent with the latter arrangement, however bootstrap and posterior probabilities were low. The inclusion of Denticeps in Clupeiformes has been supported by mitogenomic data [17, 18] and by the presence of a recessus lateralis, a definitive synapomorphy for Clupeiformes [10, 19]. Li and Orti [16] argued that the exclusion of

Denticeps from Clupeiformes is a result of GC compositional bias in rag genes. A large- scale study on actinopterygian phylogenetics including nine nuclear genes also supported the position of Denticeps as a clupeiform (Near et al. 2012).

Several studies have yielded conflicting evidence for the phylogenetic placement of Chirocentridae within Clupeiformes. Chirocentridae is comprised of two species

(Chirocentrus dorab & C. nudus) commonly referred to as wolf herring because they are pelagic predators with large jaws and fang-like canines, an elongate body, and deeply forked caudal fin. Determining the position of this lineage has proved challenging in part because of this anomalous morphology for a clupeiform. Grande [10] grouped Chirocentridae with Clupeidae based on a high ratio of ribs to preural vertebrae, although the interpretation of this character was later shown to be ambiguous [20]. However,

Patterson and Johnson [21] support the Chirocentridae+Clupeidae relationship based on the fusion of epicentrals with anterior ribs, and Miyashita [22] argued for a

Pristigasteridae+Clupeidae+Chirocentridae clade based on a W-shaped occipital articulation in the first vertebra. Several molecular studies have indicated that

Chirocentridae is sister to Spratelloidini (the latter clade discussed below), and nested within Clupeidae [4, 16-18, 23]. Di Dario [20] proposed that Chirocentridae is sister to

Engraulidae (anchovies); this novel phylogenetic hypothesis was based on seven morphological characters from the suspensorium, branchial arches, and infraorbitals. Our study provides the first molecular support for Chirocentridae and Engraulidae as sister taxa and the weight of the evidence supports the decision to include Chirocentridae in

Engrauloidea [20].

The affinity of Pristigasteridae continues to be one of the most uncertain elements of higher-level clupeiform phylogenetics. Morphological studies have placed pristigasterids either in a polytomy with engraulids and clupeids, or as sister to all other clupeiforms except Denticeps [10, 20, 24]. Molecular evidence has pointed towards a close relationship between Clupeidae and Pristigasteridae; studies based on mitochondridal data suggest Clupeidae is sister to Pristigasteridae [4, 17, 18, 23], while a combined nuclear and mitochondrial study found Pristigasteridae was nested within

Clupeidae [16], but neither study had statistical support for these respective topologies.

Our ML analysis recovered Pristigasteridae as sister to Engrauloidea

(Engraulidae+Chirocentridae), but this was not statistically supported. Meanwhile, our BEAST analysis supports Pristigasteridae as sister to Clupeidae, although with no statistical support (PP =0.49). Thus, the phylogenetic placement of pristigaserids remains elusive.

The monophyly of Clupeidae has been questioned because studies have shown that Chirocentridae, Pristigasteridae, and Sundasalangidae are nested within this group.

Our results only support the latter as a member of Clupeidae (see above). However we also find the Spratelloidini are sister to all remaining clupeiforms, rather than sister to the

Dussmieriini. The proposed Spratelloidini+Dussmieriini relationship forms the

Dussumieriinae, and is supported by the presence of a W-shaped, un-keeled pelvic scute unique to these lineages [10]. Based on morphological data, Nelson [25] suggested that

Dussumieriinae is a basal clupeid, implicitly recognizing they are distinct from other clupeids. Moreover, Li and Orti [16] reported a low GC composition in Spratelloides delicatulus and the presence of an undescribed intron in both rag1 and rag2 for

Spratelloides gracilis. Thus, there is concordant evidence that these taxa have a unique evolutionary history among clupeids. We excluded the introns from our dataset and other members of Spratelloidini have nucleotide compositions comparable to other clupeiforms, suggesting this is not biasing our results. Nonetheless, Dussumierinae

(including Spratelloidini) possess two long, rod-like postcleithra, the only morphological synapomorphy proposed for Clupeidae [10]. The conflicting evidence discussed here suggests future studies are needed to confirm the exclusion of Spratelloidini from

Clupeidae.

Members of Clupeidae have traditionally been divided into five subfamilies:

Alosinae, Dorosomatinae, Clupeinae, Dussumieriinae, and Pellonulinae. However, there has been little evidence that any of these proposed groups are monophyletic. Grande acknowledged that Alosinae, Dorosomatinae, and Clupeinae were simply “groups of convenience” with no supporting morphological characters. While previous molecular studies had limited taxon sampling to test the monophyly of these groups, no study has found convincing support for these three clades. The Ehiravini and Pellonulini were thought to be sister taxa that together comprised the Pellonulinae, based on the loss of anterior supramaxilla [10]. However, this character state also occurs in Dussumieriinae,

Dorosomatinae, and Engrauloidea [10] and molecular studies show there is no evidence for the Ehiravini+Pellonulini relationship [4; this study]. Our study corroborates the non- monophyly of Alosinae, Dorosomatinae, and Pellonulinae, and Dussumieriinae and suggests these taxonomic names need to be redefined.

While the clades recognized as subfamilies within Clupeidae are clearly in need of revision, we do find support for the monophyly of Pellonulini, but not Ehirvani.

Pellonulini is a freshwater clade restricted to western and central Africa, and includes

Pellonula, Odaxothrissa, Microthrissa, Limnothrissa, Stolothrissa, Potamothrissa,

Sierrathrissa, Nanothrissa, Poecilothrissa, Laeviscutella, Thrattidion, Congothrissa, and

Cynothrissa. The Pellonulini are diagnosed by the articulation of the postcleithrum with the supracleithrum occurring well behind the cleithrum [10]. This character state was apparently independently derived in the Ehiravini genera Clupeichthys and Ehirava [10], although Ehirava has never been included in a molecular study. Ehiravini is a freshwater clade from southern Africa, Madagascar, and southeastern Asia, comprised of

Spratellomorpha, Sauvagella, Dayella, Ehirava, Clupeichthys, Corica, Spratellomorpha, and Gilchristella; this clade is diagnosed by the unique elements in sensory canals and caudal fin structure [10, 15]. Our ML and BEAST results recover these taxa as a clade

(although ML with no bootstrap support), but also find that Sundasalanx mekongensis

(Sundasalangidae) is nested within this clade, a relationship also found in a recent mitogenomic study [18].

Our results were largely consistent with a recent study on the phylogenetics of engraulids [12]. The Indo-Pacific anchovies in Coilinae, which include Coilia, Thryssa,

Lycothrissa, and Setipinna, are well supported as monophyletic. Coilinae is sister to

Engraulinae, which includes the Indo-Pacific genera Stolephorus and Encrasicolina, and the New World anchovy clade Engraulini. In this study, our analyses indicate that

Stolephorus may be paraphyletic, which has not previously been reported [12].

Engraulini is well supported as a monophyletic clade. Within Engraulini, Engraulis ringens and E. anchoita are sister to two large clades, the first comprised of New World marine anchovies and the second comprised mostly of South American freshwater anchovies.

Our diversification time estimates indicate that the MRCA of Clupeiformes dates to the Late Jurassic. The major clupeiform clades, pristigasterids, engraulids, and clupeids originated during the Middle to Late Cretaceous. Our age estimates are older than those of Wilson et al. [4], who estimated a date of ~80 Ma for the MRCA of

Clupeiformes and ~64 Ma for Clupeidae and slightly older than those of Lavoue et al.

[18] (precise ages not available). The taxon sampling for engraulids and pristigasterids in

Wilson et al.’s study was too low to estimate ages for these groups. However, a recent study [26] on higher-level fish relationships and ages suggested that crown Clupeiformes date at least to 175 Ma, but within the bounds of our age ranges (120-180 Ma) from the 95% HPD of this node. Our chronogram indicates that much of the diversity of clupeiforms originated during the Eocene to more recent, which is consistent with the diversity of Clupeiformes in the fossil record [7-10, 27-29]. Our chronogram for

Clupeiformes provides a temporal context for future studies to investigate how paleogeographic events shaped the evolutionary history of this ecologically and economically important group.

REFERENCES

1. Ho S.Y.W., Phillips M.J. 2009 Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times. Syst Biol 58(3), 367-380. (doi:10.1093/sysbio/syp035). 2. Alfaro M.E., Santini F., Brock C., Alamillo H., Dornburg A., Rabosky D.L., Carnevale G., Harmon L.J. 2009 Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates. Proceedings of the National Academy of Sciences of the United States of America 106(32), 13410-13414. (doi:10.1073/pnas.0811087106). 3. 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, 194. (doi:194 10.1186/1471-2148-9-194). 4. Wilson A.B., Teugels G.G., Meyer A. 2008 Marine Incursion: The Freshwater Herring of Lake Tanganyika Are the Product of a Marine Invasion into West Africa. PLoS ONE 3(4), e1979. doi:1910.1371/journal.pone.0001979. 5. Arratia G. 2004 Basal teleosts and teleostean phylogeny. Paleo Ichthyologica 7, 5-168. 6. Arratia G. 2004 Mesozoic halecostomes and the early radiation of teleosts. In Mesozoic fishes 3 - Systematics, paleoenviroments, and biodiversity (eds. Arratia G., Tintori A.), pp. 279-315. Munchen, Verlag Dr. Friedrich Pfeil. 7. Figueiredo F.J. 2009 A new marine clupeoid fish from the Lower Cretaceous of the Sergipe-Alagoas Basin, northeastern Brazil. Zootaxa (2164), 21-32. 8. Figueiredo F.J. 2009 A New Clupeiform Fish from the Lower Cretaceous (Barremian) of Sergipe-Alagoas Basin, Northeastern Brazil. J Vert Paleontol 29(4), 993- 1005. 9. Murray A.M., Simons E.L., Attia Y.S. 2005 A new clupeid fish (Clupeomorpha) from the oligocene of Fayum, Egypt, with notes on some other fossil clupeomorphs. J Vert Paleontol 25(2), 300-308. (doi:10.1671/0272-4634(2005)025[0300:ancfcf]2.0.co;2). 10. Grande L. 1985 Recent and fossil clupeomorph fishes with materials for revision of the subgroups of clupeoids. Bull Am Mus Nat Hist 181(2), 231-372. 11. Miller R.R. 1982 First Fossil Record (Plio-Pleistocene) of Threadfin Shad, Dorosoma petenense, from the Gatuna Formation of Southeastern New Mexico. J Paleontol 56(2), 423-425. 12. Bloom D.D., Lovejoy N.R. 2012 Molecular phylogenetics reveals a pattern of biome conservatism in New World anchovies (Family Engraulidae). J Evol Biol 25(4), 701-715. 13. Nelson G. 1984 Identity of the Anchovy Hildebrandichthys setiger with Notes on Relationships and Biogeography of the Genera Engraulis and Cetengraulis. Copeia (2), 422-427. 14. Whitehead P.J.P., Nelson G.J., Wongratana T. 1988 FAO species catalogue, vol. 7. Clupeoid fishes of the world (Suborder Clupeoidei). Rome, UNDP FAO. 15. Stiassny M.L.J. 2002 Revision of Sauvagella Bertin (Clupeidae; Pellonulinae; Ehiravini) with a description of a new species from the freshwaters of Madagascar and diagnosis of the Ehiravini. Copeia (1), 67-76. 16. Li C.H., Orti G. 2007 Molecular phylogeny of clupeiformes (Actinopterygii) inferred from nuclear and mitochondrial DNA sequences. Mol Phylogen Evol 44(1), 386- 398. (doi:10.1016/j.ympev.2006.10.030). 17. Lavoué S., Miya M., Saitoh K., Ishiguro N.B., Nishida M. 2007 Phylogenetic relationships among anchovies, sardines, herrings and their relatives (Clupeiformes), inferred from whole mitogenome sequences. Mol Phylogen Evol 43(3), 1096-1105. (doi:10.1016/j.ympev.2006.09.018). 18. Lavoué S., Miya M., Musikasinthorn P., Chen W.-J., Nishida M. 2013 Mitogenomic Evidence for an Indo-West Pacific Origin of the Clupeoidei (Teleostei: Clupeiformes). PLoS ONE 8(2), e56485. (doi:10.1371/journal.pone.0056485). 19. Di Dario F. 2004 Homology between the recessus lateralis and cephalic sensory canals, with the proposition of additional synapomorphies for the Clupeiformes and the Clupeoidei. Zool J Linn Soc 141(2), 257-270. 20. Di Dario F. 2009 Chirocentrids as engrauloids: evidence from suspensorium, branchial arches, and infraorbital bones (Clupeomorpha, Teleostei). Zool J Linn Soc 156(2), 363-383. 21. Patterson C., Johnson G.D. 1995 The intermusclular bones and ligaments of teleostean fishes. Smithson Contrib Zool 559, 1-78. 22. Miyashita T. 2010 Unique occipital articulation with the first vertebra found in pristigasterids, chirocentrids, and clupeids (Teleostei: Clupeiformes: Clupeoidei). Ichthyol Res 57, 121-132. 23. Lavoué S., Miya M., Nishida M. 2010 Mitochondrial phylogenomics of anchovies (family Engraulidae) and recurrent origins of pronounced miniaturization in the order Clupeiformes. Mol Phylogen Evol 56(1), 480-485. (doi:10.1016/j.ympev.2009.11.022). 24. Di Dario F. 2002 Evidence supporting a sister-group relationship between Clupeoidea and Engrauloidea (Clupeomorpha). Copeia (2), 496-503. 25. Nelson G. 1970 The Hyobranchial Apparatus of Teleostean Fishes of the Families Engraulidae and Chirocentridae. Am Mus Novit (2410), 1-30. 26. 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. Proceedings of the National Academy of Sciences 109(34), 13698-13703. (doi:10.1073/pnas.1206625109). 27. Grande L. 1982 A revision of the fossil genus Knightia, with a description of a new genus from the Green River Formation (Teloeostei, Clupeidae). Am Mus Novit 2731, 1-22. 28. Grande L. 1984 Paleontology of the Green River Formation with a review of the fish fauna. Wyoming Geological Survey 63, 1-334. 29. Grande L., Nelson G. 1985 Interrelationships of Fossil and Recent Anchovies (Teleostei: Engrauloidea) and Description of a New Species from the Miocene of Cyprus Am Mus Novit (2826), 1-16.

Fig. 1. Maximum likelihood phylogeny of Clupeiformes with bootstrap values (1000 replicates) shown at each node. Previously proposed subclades within clupeiformes are color coded to demonstrate the lack of monophyly of most of these groups.

Fig. 2. Chronogram of Clupeiformes resulting from our BEAST analysis. The bars at nodes indicate the 95% HPD of the posterior age estimates.

Oncorhynchus mykiss Denticeps clupeoides Chanos chanos 100 100 Hepsetus odoe 9 7 Ictalurus punctatus 100 Danio rerio Cyprinus carpio 9 8 Spratelloides robustus Spratelloidini 9 5 Spratelloides delicatulus Spratelloides gracilis Jenkinsia lamprotaenia Dussmieriini 100 Etrumeus whiteheadi 8 4 Etrumeus teres 8 5 Potamalosa richmondia Hyperlophus vittatus Ethmidium maculatum 7 9 100 9 7 Ramnogaster sp. Sprattus antipodum 100 100 Sprattus muelleri Sprattus sprattus 9 4 Clupea harengus 9 9 Clupea pallasii Clupeonella cultriventris Ehirava fluviatilis 9 1 Gilchristella aestuaria 8 8 Sauvagella madagascariensis Sauvagella robusta Ehiravini 9 7 Sundasalanx mekongensis 9 5 Clupeoides borneensis 100 Clupeichthys perakensis 100 100 Clupeichthys aesarnensis Corica laciniata 8 5 Sardina pilchardus Dussumieriinae Sardinops sagax Pellonulinae 9 9 9 9 Brevoortia tyrannus Brevoortia patronus Clupeinae 9 9 Brevoortia aurea 9 7 9 9 Brevoortia smithi Alosinae 7 6 Alosa chrysochloris Clupeidae Alosa mediocris Dorosomatinae 9 9 Alosa pseudoharengus Sundasalangidae 9 8 Alosa aestivalis 8 9 9 2 Alosa sapidissima Pristigasteridae Alosa alabamae 9 8 Alosa alosa Chirocentridae 9 8 Alosa fallax Engraulidae Konosirus punctatus 100 Sardinella maderensis Sardinella cf. lemuru 7 3 Hilsa kelee 9 2 Lile stolifera 9 2 100 Rhinosardinia amazonica Rhinosardinia 9 5 Sardinella aurita 8 2 100 Harengula humeralis Harengula jaguana 9 9 Opisthonema libertae Opisthonema oglinum 8 3 100 Dorosoma petenense Dorosoma cepedianum Escualosa thoracata 9 8 Nematalosa erebi Nematalosa japonica Anodontostoma chacunda 100 Gudusia chapra 100 Tenualosa thibaudeaui Tenualosa ilisha Ethmalosa fimbriata 9 9 Pellonula leonensis 8 8 Odaxothrissa vittata Pellonulini Pellonula vorax Microthrissa congica 8 9 9 3 100 Limnothrissa miodon Stolothrissa tanganicae Potamothrissa obtusirostris Sierrathrissa leonensis Microthrissa royauxi Ilisha africana 100 Chirocentrodon bleekerianus 9 7 Pellona harroweri 100 Pellona castelnaeana 8 4 100 Ilisha amazonica Pellona flavipinnis 7 1 100 Pristigaster cayana Pristigaster whiteheadi 100 Odontagnathus mucronatus 100 Ilisha cf. megaloptera Ilisha elongata Chirocentrus dorab 100 Setipinna cf. tenuifilis 9 2 Setipinna taty Setipinna melanochir 9 4 8 0 Lycothrissa crocodilus 100 Thryssa mystax Thryssa cf. dussumieri 8 1 Thryssa baelama 100 Coilia reynaldi 100 Coilia mystus 100 Coilia lindmani 8 8 Coilia nasus 8 4 Coilia brachygnathus 100 Stolephorus chinensis Stolephorus waitei 8 0 Stolephorus sp. 9 7 Encrasicholina devisi Encrasicholina punctifer 0.3 8 6 100 Engraulis ringens Engraulis anchoita 9 9 Jurengraulis juruensis 9 7 Anchoa spinifer 100 Anchoviella manamensis Anchoviella brevirostris Anchoviella cf. guianensis 9 6 100 Anchoviella n sp. 2 100 Anchoviella carrikeri 9 7 Anchoviella guianensis 100 Anchoviella carrikeri Anchoviella carrikeri 100 Anchoviella guianensis Anchoviella guianensis 9 2 Anchoviella alleni 8 3 Anchoviella alleni Amazonsprattus scintilla 8 0 Anchoviella sp. 3 8 5 Anchoviella cf. guianensis 8 6 8 0 100 Anchoviella n sp. Anchoviella jamesi Anchovia surinamensis Anchoviella lepidentostole Pterengraulis atherinoides 9 1 7 9 Lycengraulis batesii 100 Lycengraulis grossidens 9 9 Lycengraulis poeyi Anchoa filifera Engraulis mordax 100 Cetengraulis edentulus Cetengraulis mysticetus 100 Engraulis japonicus 100 Engraulis eurystole 8 6 Engraulis encrasicolus 7 9 Anchoa nasus Anchoa lyolepis Anchoa lamprotaenia 100 Anchoa cayorum 9 2 Anchoa cubana Anchoa mitchilli 7 0 Anchoa parva 9 2 100 Anchoa delicatissima Anchoviella elongata 7 7 Anchoa chamensis 8 5 100 Anchovia clupeoides Anchovia macrolepidota 7 2 Anchoa colonensis Anchoa sp. 7 2 9 8 Anchoa mundeoloides 8 9 Anchoviella balboae Anchoa panamensis 9 3 Anchoa walkeri 9 9 Anchoa schofieldi Lycengraulis grossidens Lycengraulis poeyi Lycengraulis batesii Pterengraulis atherinoides Anchoviella lepidentostole Anchovia surinamensis Anchoviella jamesi Anchoviella sp.1 Anchoviella cf. guianensis Anchoviella sp. 3 Anchoviella alleni Amazonsprattus scintilla Anchoviella guianensis Anchoviella guianensis Anchoviella alleni Anchoviella carrikeri Anchoviella carrikeri Anchoviella guianensis Anchoviella sp. 2 Anchoviella carrikeri Anchoviella cf. guianensis Anchoviella manamensis Anchoviella brevirostris Jurengraulis juruensis Anchoa spinifer Anchoa filifera Engraulis japonicus Engraulis eurystole Engraulis encrasicolus Engraulis mordax Cetengraulis edentulus Cetengraulis mysticetus Anchoa nasus Anchoa lyolepis Anchoa lamprotaenia Anchoa cayorum Anchoa cubana Anchoa mitchilli Anchoa delicatissima Anchoa parva Anchoviella elongata Anchoa chamensis Anchovia macrolepidota Anchovia clupeoides Anchoa sp. Anchoa colonensis Anchoviella balboae Anchoa mundeoloides Anchoa panamensis Anchoa walkeri Anchoa schofieldi Engraulis anchoita Engraulis ringens Encrasicholina devisi Encrasicholina punctifer Stolephorus sp. Stolephorus waitei Stolephorus chinensis Setipinna cf. tenuifilis Setipinna taty Lycothrissa crocodilus Setipinna melanochir Thryssa mystax Thryssa cf. dussumieri Coilia nasus Coilia brachygnathus Coilia lindmani Coilia mystus Coilia reynaldi Thryssa baelama Chirocentrus dorab Ilisha africana Chirocentrodon bleekerianus Pellona harroweri Pellona castelnaeana Pellona flavipinnis Ilisha amazonica Pristigaster cayana Pristigaster whiteheadi Odontagnathus mucronatus Ilisha elongata Ilisha cf. megaloptera Etrumeus teres Etrumeus whiteheadi Hyperlophus vittatus Potamalosa richmondia Ethmidium maculatum Sprattus sprattus Clupea harengus Clupea pallasii Ramnogaster sp. Sprattus antipodum Sprattus muelleri Clupeonella cultriventris Ehirava fluviatilis Gilchristella aestuaria Sauvagella robusta Sauvagella madagascariensis Sundasalanx mekongensis Clupeoides borneensis Clupeichthys perakensis Corica laciniata Clupeichthys aesarnensis Sardina pilchardus Sardinops sagax Brevoortia tyrannus Brevoortia patronus Brevoortia smithi Brevoortia aurea Alosa pseudoharengus Alosa aestivalis Alosa chrysochloris Alosa mediocris Alosa alosa Alosa fallax Alosa alabamae Alosa sapidissima Sardinella cf. lemuru Sardinella maderensis Konosirus punctatus Ethmalosa fimbriata Sierrathrissa leonensis Potamothrissa obtusirostris Stolothrissa tanganicae Limnothrissa miodon Microthrissa royauxi Microthrissa congica Pellonula vorax Odaxothrissa vittata Pellonula leonensis Escualosa thoracata Nematalosa japonica Nematalosa erebi Anodontostoma chacunda Gudusia chapra Tenualosa ilisha Tenualosa thibaudeaui Hilsa kelee Lile stolifera Rhinosardinia amazonica Rhinosardinia sp. Sardinella aurita Harengula jaguana Harengula humeralis Opisthonema libertae Opisthonema oglinum Dorosoma cepedianum Dorosoma petenense Spratelloides robustus Spratelloides delicatulus Jenkinsia lamprotaenia Spratelloides gracilis Cyprinus carpio Danio rerio Ictalurus punctatus Hepsetus odoe Chanos chanos Denticeps clupeoides Oncorhynchus mykiss

250.0 225.0 200.0 175.0 150.0 125.0 100.0 75.0 50.0 25.0 0.0 millions of years ago