The taxonomic significance of ddRADseq based microsatellite markers in the closely related species of Heracleum (Apiaceae).

Mehdi Daemi-Saeidabad,Adam Vivian-Smith,Mohsen Falahati-Anbaran,Hans K Stenøien,Abdolali Shojaeiyan,Tzen-Yuh Chiang

doi:10.1371/journal.pone.0232471

Abstract

Many studies on Heracleum have shown poor correspondence between observed molecular clusters and established taxonomic classification amongst closely related species. This might reflect both unresolved taxonomy but perhaps also a lack of good genetic markers. This lack of appropriate and cost effective species-specific genetic markers hinders a resolved relationship for the species complex, and this in turn causes profound management challenges for a genus that contains both endemic species, with important ecological roles, and species with an invasive potential. Microsatellites are traditionally considered markers of choice for comprehensive, yet inexpensive, analyses of genetic variation, including examination of population structure, species identity, linkage map construction and cryptic speciation. In this study, we have used double digest restriction site associated DNA sequencing (ddRADseq) to develop microsatellite markers in Heracleum rechingeri. Genomic DNA from three individuals were digested with Sbf1 and Nde1 and size selected for library construction. The size-selected fragments were sequenced on an Ion Torrent sequencer and a total of 54 microsatellite sequences were bioinformatically confirmed. Twenty five loci were then tested for amplification, resulting in 19 of these being successfully amplified across eight species, comprising both the so-called thick-stemmed species (H. persicum, H. rechingeri, H. gorganicum and H. lasiopetalum), and thin-stemmed species (H. anisactis, H. pastinasifolium and H. transcaucasicum). Both Bayesian and distance-based clustering, and principal coordinate analyses clearly separated these into two groups. Surprisingly, three H. pastinacifolium populations were not separated from populations of the morphologically similar endemic species, H. anisactis, suggesting lack of genetic differentiation. Likewise, high genetic similarity was found between H. persicum and H. rechingeri populations, questioning taxonomic separation at the species level between these taxa. Further analyses are needed to re-evaluate the taxonomic significance of observed morphological variability currently applied to distinguish these sister taxa. Nevertheless, our results represent progress in the effort to develop cost-efficient molecular tools for species discrimination in this genus.

Highlights

In many plant species complexes, factors such as hybridizations, phenotypic plasticity and high variability across the geographical distribution range makes species identification based on morphological characters challenging [1,2,3]
H. rechingeri has been treated as a separate species with a few distinct morphological traits, including the shape of mericarp and stylopodium compared to the widespread sister taxa H. persicum
Libraries were quantified on a Bioanalyzer High Sensitivity chip (Agilent Technologies), and a concentration of 35 pM selected for the library to be Species H. rechingeri H. rechingeri H. persicum H. persicum H. persicum H. persicum H. anisactis H. anisactis H. anisactis H. trasnscaucasicum H. pastinacifolium H. pastinacifolium H. pastinacifolium H. antasiaticum H. lasiopetalum H. gorganicum H. sphondylium

Summary

Introduction

In many plant species complexes, factors such as hybridizations, phenotypic plasticity and high variability across the geographical distribution range makes species identification based on morphological characters challenging [1,2,3]. Lack of appropriate species-specific genetic markers hinders progress to untangle clear relationships within a species complex in a cost-effective way [2,3,4, 6]. This is challenging for a genus with species having important ecological roles, or which are rare, or alternatively invasive, or of special interest for conservation management. The ecological significance of the Heracleum in terms of the negative effect on the local biodiversity has recently been reported in its invasive range in northern Europe [7]. The eradication of noxious Heracleum species in human populated areas is considered resourcedemanding [10]

Methods

Results

Discussion

Conclusion