Systematic revision of the arboreal Neotropical ‘Thorellii’ clade of Centruroides Marx, 1890 bark scorpions (Buthidae c.l. Koch, 1837) with descriptions of six new species

The arboreal Neotropical ‘thorellii’ clade of Centruroides Marx, 1890 bark scorpions (Buthidae C.L. Koch, 1837) is revised, using a novel approach to species delimitation. A phylogenetic analysis, based on 112 morphological characters and 1078 aligned DNA nucleotides from the mitochondrial Cytochrome c Oxidase Subunit I (COI) gene, provided the framework for placing singletons from geographically disparate localities (and often with suboptimal preservation) using COI minibarcodes, thereby enlarging the taxon sample for diagnosis and delimitation of morphological species. Six new species are described, tripling the known diversity in the clade to nine: Centruroides berstoni, sp. nov.; Centruroides catemacoensis, sp. nov.; Centruroides chanae, sp. nov.; Centruroides cuauhmapan, sp. nov.; Centruroides hamadryas, sp. nov.; Centruroides yucatanensis, sp. nov. Revised diagnoses are presented for Centruroides hoffmanni Armas, 1996, Centruroides rileyi Sissom, 1995, and Centruroides schmidti Sissom, 1995. Comparative images, a key and distribution maps for all species of the clade are provided, along with a summary of available data for their ecology.

Methods
Genomic DNA was extracted from muscle tissue from the fourth leg of well-preserved specimens using the spin column extraction protocol of the Qiagen DNeasy Blood & Tissue Kit (Valencia, CA). PCR amplification of complete mitochondrial and nuclear genes proved impossible for many older museum samples, due to degradation and fragmentation of the DNA. Therefore, a 125 base-pair (bp) hypervariable region of the Cytochrome c Oxidase Subunit I (COI) gene was partially amplified (Musnier et al., 2008). Newly generated sequences were edited, forward and reverse primers removed, and complementary strands assembled into consensus sequences using Geneious v. 11.0.4 (Kearse et al., 2012), by reference to a 1078 bp fragment of COI for C. rileyi from GenBank (Esposito and Prendini, 2019).