We present a total evidence phylogenetic analysis of the Neotropical orb-weaving spider genus Wagneriana and discuss the phylogenetic impacts of methodological choices. We analysed 167 phenotypic characters and nine loci scored for 115 Wagneriana and outgroups, including 46 newly sequenced species. We compared total evidence analyses and molecular-only analyses to evaluate the impact of phenotypic evidence, and we performed analyses using the programs POY, TNT, RAxML, GARLI, IQ-TREE and MrBayes to evaluate the effects of multiple sequence alignment and optimality criteria. In all analyses, Wagneriana carimagua and Wagneriana uropygialis were nested in the genera Parawixia and Alpaida, respectively, and the remaining species of Wagneriana fell into three main clades, none of which formed a pair of sister taxa. However, sister-group relationships among the main clades and their internal relationships were strongly influenced by methodological choices. Alignment methods had comparable topological effects to those of optimality criteria in terms of 'subtree pruning and regrafting' moves. The inclusion of phenotypic evidence, 2.80-3.05% of the total evidence matrices, increased support irrespective of the optimality criterion used. The monophyly of some groups was recovered only after the addition of morphological characters. A new araneid genus, Popperaneus gen. nov., is erected, and Paraverrucosa is resurrected. Four new synonymies and seven new combinations are proposed.
