Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species

Objective Angelica heterocarpa (Apiaceae) is a wild endemic French species with special conservation interest in the European Union. It belongs to Angelica complex genus which is widespread throughout the north temperate zone, and is sympatric with other congeneric species. The objective of this work is to develop and characterize microsatellite markers as a new tool for understanding the ecology and evolution of Angelica species complex. Results We identified simple sequence repeat (SSR) regions in a microsatellite‐enriched library from A. heterocarpa and A. sylvestris. All 16 selected SSR regions were found to amplify in these species and were highly polymorphic. Marker transferability was validated in A. razulii and A. archangelica. These markers will help us to better understand the evolutionary dynamic between rare endemics and widespread sister species, and be useful for conservation of the endemic species. Moreover, they can provide new tools for studying the numerous traditional medicinal herbs of the Angelica genus.


Introduction
Angelica L. is a large complex genus comprising approximately 110 species confined in the northern hemisphere, with the majority in Eurasia [1]. In this paper, four French native congeneric Angelica species are considered. Angelica heterocarpa Lloyd is endemic to southwestern French estuary banks. It is protected at national level in France and is listed as priority species in the Habitats Directive of European Union [2]. Angelica sylvestris L. is common in open and forest habitats and is widely distributed among Europe and Asia. Despite different ecological niches, A. heterocarpa and A. sylvestris can live in neighboring riverbank locations. The observation of morphological intermediates questions the taxonomic relationship and potential for hybridization between these two species. Two other species are present in southwestern France: Angelica razulii Gouan, a Pyrenean endemic that shares hydrographic zones with A. heterocarpa and A. sylvestris, and Angelica archangelica L. that occurs naturally in estuaries from northern France and Europe and is cultivated in southwestern France for aromatic and pharmacological interests.
For the effective conservation of A. heterocarpa, genetic markers providing resolution at the population level are essential although, until now, not available. Here, we report the characterization of 16 new polymorphic microsatellite markers for A. heterocarpa and A. Revardel

Methods
Plant material was collected across natural populations in France or Germany: two for A. heterocarpa (N = 78), three for A. sylvestris (N = 98) and one for A. razulii (N = 50) and A. archangelica (N = 3) ( Table 4 in Appendix 1). One or two leaflets were collected from each plant and preserved dried in silica gel. DNA was extracted with the Invitek extraction kit (Invitek, Berlin, Germany). Microsatellites markers were developed from sequences obtained from A. heterocarpa and A. sylvestris after enrichment by both traditional cloning and high throughput sequencing (GenoScreen, Lille, France) of microsatellite-enriched library [3]. Sequences containing microsatellites were identified using the QDD software [4] and primers were designed using the Primer 3 software [5] using default parameters with 56 °C as annealing temperature. A total of 119 primers pairs for these SSR loci were tested for amplification and genotyping of 4 of each A. heterocarpa and A. sylvestris individuals using primer extended with M13 sequence for fluorescent labeling [6]. All of them were found to amplify in the both species and among them, 16 showing polymorphisms and consistent peak profile were selected in the final genotyping protocol (Table 1).

Results and discussion
Overall, the developed microsatellite loci were highly polymorphic with a number of alleles per locus ranged from 2 to 21 with a mean of 8 alleles per locus in the five studied A. heterocarpa and A. sylvestris populations ( Table 2). At the population level, the observed heterozygosity ranged from 0.54 to 0.60, and the expected heterozygosity ranged from 0.66 to 0.74. Four out of the 16 loci showed significant deviations from Hardy-Weinberg equilibrium (HWE) in more than one population after correcting for multiple testing (P < 0.001; Table 2).
The cross-species transferability and polymorphism of the developed SSR markers were further tested in the two other congeneric species (Table 3). In A. razulii, only    Table 3). These new polymorphic markers will allow investigating population genetic structure, reproductive system, and potential hybridization within the Angelica species complex. Moreover, as numerous Angelica species are traditional medicine herbs all over the Eurasia continent, including A. sylvestris and A. archangelica [7], these microsatellite markers complement the molecular markers previously developed for other congeneric species of particular pharmacological interest [8][9][10].