Results

Phylogenetic relationships. Our concatenated Bayesian and maximum likelihood analyses based upon the combined nDNA and mtDNA dataset resulted in largely congruent topologies (Fig. 1; S 2 – available only as online supplementary file accessed with the online version of the article at http://www.scielo.br/ni). Both analyses produced phylogenies with poor support for higher-level relationships. In both phylogenies, Clupeoidei (all clupeiforms except the monotypic clupeiform family Denticipitidae containing Denticeps clupeoides) was monophyletic. Denticeps clupeoides was consistently placed among the outgroup taxa, rendering Clupeiformes paraphyletic. Anchovies (Engraulidae) and longfin herrings (Pristigasteridae) were recovered as monophyletic. The herring and sardine family (Clupeidae) was not recovered as monophyletic in either our Bayesian or likelihood analyses. Our Bayesian analysis placed Clupeinae in a lineage with Dussumieriinae that was sister to a lineage containing Pristigasteridae + Chirocentridae + Engraulidae +- the remaining clupeid lineages. Our maximum likelihood phylogeny placed Clupeinae as sister to a lineage containing Pristigasteridae + Chirocentridae + Engraulidae in the maximum likelihood phylogeny. Both analyses recovered Spratelloidinae, a subfamily of Dussumieriidae (sensu Lavoué et al., 2014) as sister to all remaining clupeoids.

We found discordance between mtDNA and nDNA in tree topology, particularly at the highest taxonomic levels (Figs. 1-2). We only discuss maximum likelihood and Bayesian results separately if they produced conflicting results. The nDNA dataset was largely consistent with the combined nDNA + mtDNA topology with a few notable exceptions. The nDNA Bayesian phylogeny resolved Chirocentridae as sister to Pristigasteridae (PP = 0.70) and the maximum likelihood analysis resolved Chirocentridae as sister to a lineage containing Pristigasteridae, Clupeinae, and Dussumieriinae (BS = 0.82). Bayesian analyses of nDNA placed Clupeinae sister to Pristigasteridae + Chirocentridae (PP = 1.0) and maximum likelihood analyses of nDNA placed Clupeinae sister to Dussumieriinae (BS = 0.67). mtDNA placed Pristigasteridae within Clupeidae sister to Alosinae (PP = 1.0, BS = 0.33), but analyses of nDNA recovered prisigasteridae as sister to a clade including Engraulidae, Chirocentridae, Clupeinae, and Dussumieriinae. In contrast to the nDNA, our mtDNA phylogeny placed Alosinae, Pristigasteridae, Dorosomatinae, Clupeinae, and Dussumierinae in a single clade. nDNA placed Spratelloidinae sister to all remaining Clupeoidei, but mtDNA placed Spratelloidinae outside of Clupeiformes. There was also incongruence between nDNA and mtDNA at lower taxonomic levels. For example, in New World anchovies Anchoa spinifer + Jurengraulis juruensis were either sister to all other members of the Amazonian anchovy clade (nDNA) or nested well within this clade (mtDNA). The placement of a clade that included Engraulis encrasicolus, E. eurystole, and E. japonicas was resolved as sister to all other New World marine anchovies except Anchoa filifera (nDNA) or nested deeply within New World marine anchovies (mtDNA).

Divergence times. The combined nDNA+mtDNA, the nDNA, and the mtDNA datasets all estimated an early to middle Cretaceous MRCA of Clupeoidei (Figs. 1-4). Branching events between major clupeiform lineages were estimated to occur during the late Cretaceous and early Cenozoic: Spratelloidinae (nDNA: MRCA = 100 Ma; mtDNA: MRCA = 109 Ma), Clupeidae (excluding Clupeinae) (nDNA: MRCA = 79 Ma; mtDNA: MRCA = 33 Ma), Engraulidae (nDNA: MRCA = 50 Ma; mtDNA: MRCA = 51 Ma), and Pristigasteridae (nDNA: MRCA = 42 Ma; mtDNA: MRCA = 21 Ma).

Comparative analyses. The LTT plot for Clupeoidei shows constant lineage accumulation through time (Fig. 5). The calculated gamma value was -0.61 but not significant if missing taxon sampling is as low as 25% (P = 0.89); the Bloom, Lovejoy (2014) dataset included approximately 37% of described Clupeoidei. The best-supported model of diversification was a pure birth model, in which lineages accumulate constantly over time regardless of the number of species in the clade. Clupeidae LTT plots show a constant accumulation of lineages over time. The MCCR test showed the negative gamma value of -1.34 for this clade was not significant (P = 0.93) at threshold of 67% missing data, which represents described clupeid diversity and is likely an underestimate of the actual number of species in this clade. The chosen diversification model was a pure birth model, followed closely by a diversity-dependent model. The LTT plot for the subclade Dorosomatinae (sensu Lavoué et al., 2014) showed a slow-down in lineage accumulation and a corresponding highly negative gamma value (-3.08). The negative gamma value for Dorosomatinae was significant at a threshold of 50% missing data (P = 0.01) but not when missing data was set at 75% (P = 0.13). The best-fit model for Dorosomatinae was a diversity dependent logistic growth model in which lineages accumulate as a function of clade diversity. The LTT plot of the New World marine anchovy clade showed a slow-down in lineage accumulation rate and had a gamma value of -2.51. The negative gamma is significant with 25% missing taxa (P = 0.02), but only marginally significant with 50% missing data (P = 0.51). Our taxon sampling (28 species) likely represents more than 50% of the species in this clade, thus we interpret this clade as experiencing a slow-down. The chosen diversification model for New World marine anchovies was diversity dependent growth. The LTT plot for South American freshwater anchovies reveals a slow-down in lineage accumulation over time and the gamma value of -3.59 is highly negative and significant at a threshold of 75% missing taxa (P = 0.01). The best-fit model for New World freshwater anchovies was a diversity dependent model.