Microsatellites facilitate species delimitation in Congolese Ancistrocladus (Ancistrocladaceae), a genus with pharmacologically potent naphthylisoquinoline alkaloids

Abstract

AbstractAncistrocladus (Ancistrocladaceae) is an Old World genus of woody lianas, occurring in tropical Africa and Southeast Asia. Its species produce a number of structurally unique naphthylisoquinoline alkaloids with promising activities against the pathogens of tropical infectious diseases and against HIV. Species identification for phytochemical and pharmacological research is difficult because diagnostic reproductive features are frequently not available. Using enrichment and subcloning, and a 454 shotgun sequencing technique with semi‐automatized SSR motif screening, we have successfully developed a set of primers that amplify microsatellite loci across most Ancistrocladus species. The primers are designed to support species identification of indeterminate samples. Twelve markers from a total of 99 tested loci were identified as suitable for cross‐species amplification. The markers produced distinct species clusters in a STRUCTURE analysis and a principal coordinate analysis (PCoA) based on a test sampling consisting of 21 samples from six different Ancistrocladus species. When applied to 74 samples of Ancistrocladus congolensis s.l., a complex of morphologically similar species from tropical Central Africa, these markers showed medium to high variability in ten populations. A STRUCTURE analysis of the A. congolensis s.l. samples revealed five distinct clusters, confirming three of the five taxonomically recognized units and also indicating the presence of a hitherto unrecognized species in the region. The taxonomic status of the recently described new species A. ileboensis is supported by our results. The novel microsatellite markers developed in this study enable rapid screening of samples and provide an efficient tool for species identification and detection of cryptic taxa.