No abstract

Relative to the commonly used mitochondrial and nuclear protein‐coding genes, the noncoding intron sequences are a promising source of informative markers that have the potential to resolve difficult phylogenetic nodes such as rapid radiations and recent divergences. Yet many issues exist in the use of intron markers, which prevent their extensive application as conventional markers. We used the diverse group of snakes as an example to try paving the way for massive identification and application of intron markers. We performed a series of bioinformatics screenings which identified appropriate introns between single‐copy and conserved exons from two snake genomes, adding particular constraints on sequence length variability and sequence variability. A total of 1,273 candidate intron loci were retrieved. Primers for nested polymerase chain reaction (PCR) were designed for over a hundred candidates and tested in 16 snake representatives. 96 intron markers were developed that could be amplified across a broad range of snake taxa with high PCR successful rates. The markers were then applied to 49 snake samples. The large number of amplicons was subjected to next‐generation sequencing (NGS). An analytic strategy was developed to accurately recover the amplicon sequences, and approximately, 76% of the marker sequences were recovered. The average p‐distances of the intron markers at interfamily, intergenus, interspecies, and intraspecies levels were .168, .052, .015, and .004, respectively, suggesting that they were useful to study snake relationships of different evolutionary depths. A snake phylogeny was constructed with the intron markers, which produced concordant results with robust support at both interfamily and intragenus levels. The intron markers provide a convenient way to explore the signals in the noncoding regions to address the controversies on the snake tree. Our improved strategy of genome screening is effective and can be applied to other animal groups. NGS coupled with appropriate sequence processing can greatly facilitate the extensive application of molecular markers.

A: Phylogeny of 505 specimens inferred from IQTree and MrBayes using seven genetic markers. Support for each branch is shown at the nodes, with bootstrap percentages given first, followed by posterior probabilities. Different species delimitation methods are shown on the right. Photo by Ye-Jie Lin. B: Genitalia of females (a: Ectatosticta xuanzang ; b: Ectatosticta dapeng ; c: Ectatosticta davidi ; d: Ectatosticta baixiang sp. nov. ; e: Ectatosticta qingshi sp. nov. ; f: Ectatosticta helii sp. nov. ; g: Ectatosticta bajie ; h: Ectatosticta shaseng sp. nov. ; i: Ectatosticta rulai ; j: Ectatosticta yukuni ; k: Ectatosticta puxian sp. nov. ; l: Ectatosticta wukong ; m: Ectatosticta baima sp. nov. ; n: Ectatosticta deltshevi ; o: Ectatosticta wenshu sp. nov. ). C: Distribution of Ectatosticta spiders in China. Dark spots represent locations of samples and correspond to Supplemnetary Table S1. The asterisk represents the species Ectatosticta shennongjiaensis .