diversity Article Genetic Differentiation and Population Structure of Threatened Prunus africana Kalm. in Western Cameroon Using Molecular Markers Justine G. Nzweundji 1,2, Ulrike Huewe 3, Nicolas Niemenak 2,Néhémie T. Donfagsiteli 1 and Klaus Eimert 3,* 1 Institute of Medical Research and Medicinal Plants Studies, P.O. Box 1663 Yaounde, Cameroon; [email protected] (J.G.N.); [email protected] (N.T.D.) 2 Department of Biological Science, Higher Teacher’s Training College, P.O. Box 47 Yaounde, Cameroon; [email protected] 3 Institute of Molecular Plant Sciences, Geisenheim University, Von-Lade-Strasse 1, D-65366 Geisenheim, Germany; [email protected] * Correspondence: [email protected] Received: 30 October 2020; Accepted: 23 November 2020; Published: 26 November 2020 Abstract: Genetic diversity of species is an important baseline for the domestication process. In Cameroon, Prunus africana, an important and threatened medicinal tree, is among the priority species for domestication. The bark extract has been used to treat various diseases; mainly benign prostatic hyperplasia which affects men above the age of 50. As little is known about the genetic diversity of P. africana in Cameroon, we aimed to determine the genetic diversity and differentiation of several P. africana populations in the western provinces, using sets of chloroplast DNA markers and nuclear microsatellites previously developed for Prunus species. Genetic diversity in the observed populations was considerable and genetic differentiation between populations proved substantial with 21% of the total observed variation detected among populations, revealing a distinct genetic structure among certain populations. However, the lack of correlation between genetic and geographic distances does not support isolation by distance (IBD). The analysis of chloroplast DNA haplotypes revealed no strong phylogeographic component in the genetic structure observed in the western populations of P. africana in Cameroon. The outcome of this study will contribute to improve the genetic characterization of P. africana for its better domestication and conservation in the Cameroon agroforestry system. Keywords: microsatellite; SSR; chloroplast DNA; haplotypes; genetic diversity; Prunus africana 1. Introduction Prunus africana (Hook. f.) Kalkman, of the Rosaceae family, is a multipurpose tree species that grows in Afromontane forests and whose barks have been used for traditional healing for hundreds of years [1–3]. These barks have been used for generations in African traditional medicine to address prostatic hyperplasia that affects 50% of men above the age of 50. The bark extract has been shown to successfully treat prostatic hyperplasia in rats [4,5]. In 1997, at least 23 different companies sold brand-name herbal preparations made from P. africana barks; currently, at least 40 brand-name products containing P. africana bark extracts are marketed directly in 10 countries [6]. P. africana exploitation has moved from local to large-scale commercial use for national and international trade over the past decades [7]. The global demand for P. africana bark is estimated at 4000 t per year for a value of finished goods estimated at USD 220 million [6]. One of the main supply sources to satisfy such demand consists of natural populations in Cameroon [8]. However, the high demand and overexploitation of the species Diversity 2020, 12, 446; doi:10.3390/d12120446 www.mdpi.com/journal/diversity Diversity 2020, 12, 446 2 of 11 have led to its classification as endangered species under CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in 1995 restricting its exportation. Generally speaking, such massive losses of valuable plant species and the resulting adverse environmental and socioeconomic impacts have recently triggered efforts aimed at the conservation of plant resources. Therefore, designing strategies for improved management of species appears urgent. Such strategies include the characterization of plant species for improved domestication and conservation. Molecular characterization reveals the genetic variation of different accessions of plant species which is essential for plant improvement, selection and conservation [9,10]. Such characterization has been carried out for the identification and differentiation of species and the correlation between geographical and genetic distances between different accessions of species and within and between their populations [11–13]. While the characterization of agroforestry plants appears the best way to conserve many priority species for domestication, studies assessing the genetic diversity of plant species in Africa are scarce [14]. For instance, low differentiation and high gene flow have been shown among wild populations and between wild and cultivated populations of Dacryodes edulis in Cameroon [15]. In Irvingia gabonensis, pronounced phenological differences between observed populations suggest genetic differentiation within the taxa [16]. In the Prunus genus, molecular markers have been used for the characterization of genetic diversity; including isozyme markers for genetic identification [17]. Additionally, assessment of genetic variability using simple sequence repeats (SSR) markers on Prunus domestica L. has shown high diversity in terms of the degree of polymorphism [18–20]. Randomly amplified polymorphic DNA (RAPD) and SSR molecular markers have been applied for better knowledge of germplasm diversity in Prunus persica [21–23]. The genetic diversity of P. persica showed polymorphic patterns amongst the cultivars [24], with some markers possibly linked to commercial characteristics, which could be useful to fruit growers and breeding programs [24,25]. SSR and AFLP (Amplified Fragment Length Polymorphisms) molecular markers proved useful and highly informative for grouping and identifying Prunus avium (sweet cherry), which show diverse genetic variation [26–29]. Six microsatellite loci (nuclear simple sequence repeats (nSSRs)) initially characterized in P. avium were successfully transferred into P. africana [30–33]. In P. africana, nuclear and chloroplast molecular markers were used to assess genetic diversity and differentiation across several African countries, specifically to model conservation approaches considering threats by agricultural expansion and climate change [6]. Previous studies have shown high genetic diversity in P. africana, using randomly amplified polymorphic DNA (RAPD) [34] and microsatellite markers [35]. It has also been shown that accessions from Cameroon are genetically close to some accessions in West and East Africa [33]. However, the genetic structure of P. africana populations in East Africa showed low levels of diversity [10]. Thus, molecular diversity and differentiation of P. africana still need to be assessed in more detail [36]. To the best of our knowledge, few studies have been conducted to determine the degree of diversity within and between accessions of different agroecological areas in Cameroon. The main objective of this project is to assess the genetic diversity and differentiation of P. africana in Cameroon using microsatellite DNA markers and chloroplast haplotypes across three regions of Cameroon’s habitats. The study will help to characterize P. africana germplasm from different locations as well as to determine the degree of genetic diversity and differentiation found in the sampled ranges in Cameroon. Appropriate characterization of plant materials is essential for the successful conservation of plant resources, and to ensure their sustainable use. The study will contribute to improve the sustainable exploitation of P. africana’s natural resources, by providing information on genetic variation and differentiation within the species, supporting selective breeding programs for crop improvement or conservation measures at local and regional levels. Diversity 2020, 12, 446 3 of 11 2. Materials and Methods 2.1. Population Sampling and DNA Extraction Prunus africana leaves were sampled from local trees in February to early March 2018 in three regions of Cameroon where P. africana is native with the help of forest officers and farmers in the indicated rural areas. Leaves of P. africana were harvested in the South-West Region at Ekona (SW), in the Littoral Region at Koupe-Manengouba (LT), and finally, in the North-West Region at Oku (NW) (Table1). At least 20 trees (about 15 years of age) per region were sampled. Fresh leaves were pressed and dried in a stove at 39 ◦C for 48 h then stored in Ziploc bags (S.C. Johnson & Son Inc., Racine, WI, USA) until DNA extraction was performed. Table 1. Sampled populations of P. africana, geographic location of sampling areas, and sample sizes. a a Population Longitude◦ E Latitude◦ N Sample Size NW 10.480169 6.196875 20 LT 9.3054 4.213717 20 SW 9.6804 4.843833 25 a Geographic coordinates in decimal degrees (WGS84). South-West Region at Ekona (SW); Littoral Region at Koupe-Manengouba (LT); North-West Region at Oku (NW). DNA was extracted from 60 mg of dried leaves using the Invisorb®Spin Plant Mini Kit (STRATEC Molecular GmbH, Berlin, Germany). The supplier’s protocol was slightly modified by homogenizing the leaves without the use of liquid nitrogen in a Retsch mill (Retsch GmbH, Haan, Germany) for 3 min at the frequency of 25 Hz. DNA quantity was assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Darmstadt, Germany). 2.2. Microsatellite Analysis For SSR analysis, DNA was amplified using five primer pairs