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 suggest [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 largescale 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 nonmonophyly 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. 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