A versatile and highly efficient toolkit including 102 nuclear markers for vertebrate phylogenomics, tested by resolving the higher level relationships of the caudata.

Resolving difficult nodes for any part of the vertebrate tree of life often requires analyzing a large number of loci. Developing molecular markers that are workable for the groups of interest is often a bottleneck in phylogenetic research. Here, on the basis of a nested polymerase chain reaction (PCR) strategy, we present a universal toolkit including 102 nuclear protein-coding locus (NPCL) markers for vertebrate phylogenomics. The 102 NPCL markers have a broad range of evolutionary rates, which makes them useful for a wide range of time depths. The new NPCL toolkit has three important advantages compared with all previously developed NPCL sets: 1) the kit is universally applicable across vertebrates, with a PCR success rate of 94.6% in 16 widely divergent tested vertebrate species; 2) more than 90% of PCR reactions produce strong and single bands of the expected sizes that can be directly sequenced; and 3) all cleanup PCR reactions can be sequenced with only two specific universal primers. To test its actual phylogenetic utility, 30 NPCLs from this toolkit were used to address the higher level relationships of living salamanders. Of the 639 target PCR reactions performed on 19 salamanders and several outgroup species, 632 (98.9%) were successful, and 602 (94.1%) were directly sequenced. Concatenation and species-tree analyses on this 30-locus data set produced a fully resolved phylogeny and showed that Cryptobranchoidea (Cryptobranchidae + Hynobiidae) branches first within the salamander tree, followed by Sirenidae. Our experimental tests and our demonstration for a particular case show that our NPCL toolkit is a highly reliable, fast, and cost-effective approach for vertebrate phylogenomic studies and thus has the potential to accelerate the completion of many parts of the vertebrate tree of life.