The nearly 150 species of Anostomidae comprise one of the most diverse and taxonomically dynamic families of Neotropical freshwater fishes. A recent revision of the enigmatic and poorly diagnosed genus Anostomoides demonstrated that it contains two valid species, each with complicated taxonomic histories; however, that study did not address their phylogenetic placement. Herein, we provide the integrated molecular and morphological data and scripts used to demonstrate their distant evolutionary relationship, and thus the polyphyly of Anostomoides. Results from analysis of these data reconstruct one of the species in a previously hypothesized placement within a clade also containing Laemolyta, Rhytiodus and Schizodon, and the other as a morphologically and genetically distinctive lineage that diverged early in the history of the family. The paper that these data support describes and illustrates the osteology of this remarkable species, discusses the evolutionary implications of its unique suite of features, and uses those characteristics to diagnose a new genus that evolved independently of all other known members of the family for approximately 37 Myr.

These notes represent excerpts from a longer and more detailed methods section available in the full publication. 

In broad strokes, we integrated new data on both nominal species of Anostomoides with a previously published morphological data matrix (Sidlauskas & Vari, 2008) and GenBank sequences for six commonly sequenced loci in characiform systematics. Much of the prior genetic data have been used to infer previous hypotheses of anostomid or anostomoid phylogeny (Ramirez et al., 2017; Melo et al., 2018; Burns & Sidlauskas, 2019, and others). Taxon sampling in the molecular partition prioritized sequences of the same species present in the morphological partition, but also included representatives of a few genetically distinctive lineages for which morphological data were not available. The final matrix includes 55 anostomid species and 11 outgroup species, most of which are represented in both data partitions.

Morphological data included the character matrix of Sidlauskas & Vari (2008), plus data from the extinct species †Leporinus scalabrinii (Bogan et al., 2012) and new data from examination of several skeletons of both nominal species of Anostomoides. The final morphological data matrix includes 158 characters coded for 46 living ingroup taxa, one extinct ingroup taxon, and 11 outgroup taxa. 

The molecular dataset includes the mitochondrial genes cytochrome oxidase c subunit I (COI, 630 bp), cytochrome b(Cytb, 1,005 bp) and 16S rRNA (16S, 599 bp) and the nuclear genes myosin heavy chain 6 gene (Myh6, 750 bp), recombination activating gene 1 (Rag1, 1,317 bp), and recombination activating gene 2 (Rag2, 1,020 bp). Gene sequences were aligned using Clustal Omega (Madeira et al., 2019) and trimmed in Mesquite v. 3.61 (Maddison & Maddison, 2017). Gene trees for each alignment were generated in MrBayes v.3.2.7a (Ronquist et al., 2012) to check for and remove contaminated sequences (those identical to a species in another genus, or those clustering a species in a wildly different placement from its congeners). 

Phylogenetic relationships and divergence times were investigated in a combined molecular and morphological analysis in BEAST v.2.6.2 (Bouckaert et al., 2014), using the CladeAge package (Matschiner et al., 2017; Barido-Sottani et al., 2018). Models of nucleotide evolution were estimated via the bModelTest package (Bouckaert & Drummond, 2017). The Mk model (Lewis, 2001) was used for the morphological dataset, assuming equal rates of character change. We patterned tip and node-based fossil calibrations on a set of fossils also used in two recent studies of characiform phylogeny (Burns & Sidlauskas, 2019; Kolmann et al., 2020). We used the birth-death tree prior and a relaxed lognormal clock model, setting the net diversification rate between 0.041 and 0.081 and turnover rate between 0.001 and 0.37, as recommended by Santini et al. (2009).  The sampling rate varied between 0.0066 and 0.01806, as suggested by Foote & Miller (2007) (see also Matschiner et al. (2017). All other parameters were left as default. PartitionFinder v1.1.1 (Lanfear et al., 2012) was implemented to select the best partitioning scheme for the molecular matrix using the corrected Akaike Information Criterion (AICc). The analysis was rooted on Brycon falcatus, a representative of the distant outgroup Bryconidae. 

We checked that each run had reached stationarity by trace inspection in Tracer v1.7.1 (Rambaut et al., 2018), removed a 10% burnin portion, and verified stationarity of each run by ensuring that the ESS values for all or most parameters in each run exceeded 200 (Rambaut et al., 2018). We verified convergence between the runs by ensuring that the 95% posterior densities for the overall likelihood values and the parameter values overlapped substantially, and that ESS values for all parameters still exceeded 200 for the pooled posterior distributions. The posterior distribution of trees were then used to construct a maximum clade credibility (MCC) tree in TreeAnnotator v1.8.4.

We reconstructed the evolutionary history of selected osteological characters using maximum likelihood on the maximum clade credibility tree via the ace command in the APE package v5.3 (Paradis and Schliep 2019). In these reconstructions, we used the Akaike Information Criterion (AIC) to compare the fits of an equal rates model (ER) and a model in which the transition rates between the character states were free to vary (the ARD, or All Rates Different model) using the fitMk command from phytools (Revell 2012). For each character, we used the model with the lowest AIC score to perform separate reconstructions with and without Anostomoides nattereri pruned from the phylogenies, thereby exploring the effect of the inclusion of that species on the reconstruction.

Please consult the attached Readme.rtf file for detailed information about the contents of each file in this data package.  We provide these data and scripts open access and encourage their reuse, though naturally we would appreciate credit and citation as the originators.