Phylogenomics, biogeography, and description of a new subfamily and genus of African characiform fishes (Teleostei: Alestidae)

The Congo River, with the highest diversity of riverine fishes in Africa, only recently established its contemporary outlet into the Atlantic around the Miocene-Pliocene transition (~5 millions of years ago; Ma). Yet, its role in shaping ichthyofaunal diversification across central Africa through interactions with adjacent Atlantic coastal rivers remains unexplored at both regional and local scales. The African characiform family Alestidae, with lineages distributed across the entire region, offers an ideal system to investigate inland-coastal biogeographic connections. However, phylogenetic relationships within Alestidae remain unresolved, particularly with respect to two key genera, Brachypetersius and Nannopetersius, which inhabit both regions of interest. Applying likelihood and species-tree inferences using 1,759 nuclear ultraconserved elements (UCEs) and 13 protein-coding genes of mitochondrial genomes from 42 alestid taxa, we resolve both Brachypetersius and Nannopetersius as pol..., Ultraconserved elements and phylogenomic analyses
For UCE sequencing of 40 taxa of Alestidae, total genomic DNA (gDNA) was extracted using the DNeasy tissue kit. Libraries were quantified and enriched with the myBaits Ostariophysan 2.7Kv1 probeset (overnight hybridization, 65 °C washes) and quantified with a spectrofluorimetric assay. Sequencing was performed on the Illumina NovaSeq 6000 platform (partial S4 PE150 lane), yielding ~14 Gbp in total. 
We used PHYLUCE v.1.7.3 to prepare sequences for all UCE-based downstream analyses. We removed adapters and low-quality bases using Illumiprocessor v.2.10 and Trimmomatic v.0.39, assembled contigs with SPAdes v.3.14.1 and Velvet v.1.2.10, and identified orthologous UCE loci using the myBaits Ostariophysan 2.7Kv1 probeset and excluded putative paralog regions in PHYLUCE. We extracted and aligned loci using an edge-trimming approach in MAFFT v.7.475 and constructed three matrices: a 65 % complete matrix (i.e., loci present in ≥ 26 ter..., # Phylogenomics, biogeography, and description of a new subfamily and genus of African characiform fishes (*Teleostei: Alestidae*)

## Description of the data and file structure

Data was collected from museum specimens. Ultraconserved elements from 40 taxa were sequenced using Illumina. Mitogenomes were extracted from 37 of these, with 5 additional taxa sequenced specifically for mitogenomes. Data were processed with PHYLUCE, Geneious, MitoFish, MitoAnnotator, and downstream analyses used IQ-TREE, SWSC-EN, PartitionFinder2, RAxML, ASTRAL, BEAST, Tracer, LogCombiner, TreeAnnotator. Geocoordinates of *Clavocharax* were mapped in QGIS to delimit bioregions for ancestral range estimation in RASP using BioGeoBEARS model testing.

## Files and variables

#### **File: alestidae-100.xml**

Description: Data file used for BEAST time-calibrated analysis.

#### **File: alestidae-MCC.tre**

Description: Maximum Clade Credibility timetree of Alestidae

#### **File: alestidae-mtdna.phy**

Descriptio...,