According to a World Health Organization (WHO) 2021 report, 241 million cases of malaria were reported in 2020 (WHO, 2021). Most of these cases occurred in the African region (228 million), followed by the Eastern Mediterranean (5.75 million), and the South-East Asian region (5 million). Globally, malaria deaths reached 627,000, out of which, 96% are accounted for in sub-Saharan Africa (WHO, 2021). In addition, children under five accounted for 80% of the malaria deaths in Africa (WHO, 2021). Although efforts have been made towards reducing the global burden of malaria, drug resistance, accessibility, and the affordability of antimalarial medications continue to threaten this effort. To mitigate these issues, alternative approaches to using traditional medicine could provide more sustainable and cost-effective treatment options.

An estimated 80% of the world’s population almost exclusively depends on local medicinal plants for the prevention and treatment of various diseases because they are readily accessible and affordable (Kirby, 1996; WHO, 2002). Several efficacious medicines such as aspirin (an analgesic) derived from Salix alba L., Digoxin (cadiotonic) from Digitalis lantana Ehrh., Artemisinin (anti-malarial) from Artemisia annua L., and Quinine from Cinchona ledgeriana Moens ex Trimen. are used for the of malaria treatment (Luo et al., 1998) and originate from plants (Licciardi and Underwood, 2011; Veeresham, 2012). In the fight against malaria, medicinal plants with a history of use as potential antimalarials in resource-poor communities should be considered in the development of intervention approaches (Abebe and Garedew, 2019). In that capacity, steps to conserve and promote sustainable, resilient, and climate-smart agro-ecosystems must be encouraged while documenting and preserving traditional medicinal plants knowledge limited to local healers and elders in these communities. Abebe and Garedew (2019) identified 25 plant species from 22 families that had been used in traditional treatments of malaria in Ethiopia with Cyperus spp., Allium sativum L., Lepidium sativum L., and Echinops kebericho Mesfin. are utilized most extensively.

With an increase in anthropogenic activities, there is an urgent need to prioritize the protection of wild populations and the cultivation of useful medicinal plants to ensure future availability for healthcare needs. Cryptolepis sanguinolenta, locally known in Ghana as ‘Nibima’ in Twi, ‘Gangnamau’ in Hausa, or ‘Kadze’ in Ewe, is a perennial vine belonging to the Apocynaceae family with a history of use in Africa (Luo et al., 1998; Osafo et al., 2017). It grows in rainforests, deciduous forest belts, and secondary forest clearings along the west coast of Africa from Nigeria through Ghana to Senegal (Dokosi, 1998). In Ghana, however, it is located along the slopes of the Akwapim and Kwahu mountain ranges (Addae-Kyereme, 2004) with a long-standing history of use in the treatment of malaria.

Roots, the portion of the plant of commercial and medicinal value, contain the highest concentration of indoloquinoline and cryptolepine, which have the most potent antiplasmodial activity (Dwuma-Badu et al., 1978; Tackie et al., 1991; Cimanga et al., 1997).

Minor alkaloids including hydroquinone, quindoline, cryptoquindoline, cryptoheptine and 11-hydroxy derivatives of cryptolepine with biological/pharmacological activities have also been isolated (Dokosi, 1998). The combined presence of these alkaloids accounts for the biological and/or pharmacological activity of C. sanguinolenta. The effectiveness of its extracts is comparable to chloroquine when taken orally (Boye, 1989). Apart from its antimalarial properties (Ansah et al., 2005; Tempesta, 2010; Osei-Djarbeng et al., 2015), it is recognized for its anticancer (Ansah and Mensah, 2013), antihyperglycemic (WHO, 2002), antimicrobial (Sawer et al., 2005) and antimycrobacterial (Gibbons et al., 2003) potential. Preparations from C. sanguinolenta were approved in February 2021 for clinical trials for the treatment of COVID 19 by the Food and Drugs Authority of Ghana (FDA, 2021) and has been shown to inhibit hepatitis B virus replication (Domfeh et al., 2021).

In Ghana, C. sanguinolenta harvesting is done by smallholder farmers (plant collectors) who use the roots to make antimalarial decoctions. “Nibima” is one of the popular decoctions sold in local markets and is produced by the Centre for Plant Medicine Research (CPMR) (https://www.cpmr.org.gh/product-category/decoctions). Overtime, the plants have been overharvested and these plant collectors must travel further into the forest, often with overnight stays, to gather C. sanguinolenta (Amissah, personal communication 2021). Destructive harvesting of the entire plant, along with its root system, is not sustainable in the long term and has already resulted in a substantial decrease in wild populations (Jansen and Schmelzer, 2010; Amissah et al., 2016a).

Over collection and habitat loss poses a significant threat to the conservation efforts of endangered plant species, especially those used for medicinal purposes (Kala, 2000; Reed and Frankham, 2003). Human activities, such as illegal poaching or disruptive harvesting of endemic, medicinal plants, can negatively impact biodiversity at the gene, species, community and ecosystem levels (Meffe and Carroll, 1997; Templeton et al., 2001). This can result in habitat fragmentation, erosion of natural and adaptive genetic diversity, reduction in effective population size, lower evolutionary potential and ultimately contribute to species extinction (Templeton et al., 2001; Soares et al., 2019). Understanding the population genetics of a plant species provides insight into the effects of natural selection, genetic drift, mutation and gene flow that can be used to develop suitable conservation methods and assist with future breeding programmes for plant species on the verge of extinction. Population genetics studies have played a significant role in the conservation of several species. For example, genetic diversity and inbreeding were significantly influenced by the size of the population of Chersophilus duponti, a threatened lark in the Alaudidae (Meffe and Carroll, 1997) and Hypericum cumulicola, a rare species of flowering plant found in small and isolated populations (Oakley, 2015).

As is the practice with several important medicinal plants, there is very little effort devoted to the conservation of C. sanguinolenta. There is an urgent need for establishing a conservation strategy for medicinal plant species in general and specifically for C. sanguinolenta. Currently, there is very limited knowledge of the population dynamics of C. sanguinolenta. As part of research efforts geared at conserving this species, our group has developed protocols for the cultivation of C. sanguinolenta (Jansen and Schmelzer, 2010), in addition, we have developed microsatellite loci for future genetic diversity studies (Amissah et al., 2016b). To address these knowledge gaps, this study seeks to: i) measure the genetic diversity of C. sanguinolenta across eight subpopulations from natural habitats within three regions of Ghana; and ii) describe the spatial structure of these subpopulations.

The large-scale use of artemisinin since its initial discovery in the early 1970s, has resulted in worldwide drug resistance prompting a switch in strategy to artemisinin-based combination therapy (ACT) in 2005 (Tu, 2011; WHO, 2020). The problem is further compounded by the increased resistance of malaria vectors to many insecticides currently available (Balkew et al., 2012; Massebo et al., 2013), which are cost-prohibitive on a large-scale and unsustainable in the long term. According to reports from WHO (WHO, 2015), artemisinin-based combination therapy (ACTs) had been adopted as a first-line treatment policy in several countries but may not be readily available in rural, remote and poverty-stricken communities of Western Africa, including Ghana. As a result, in Ghana, Mali, Zambia and Nigeria, traditional medicine approach is practiced with 60% of children who reported high fever resulting from malaria infection (Abdullahi, 2011).

People living in developing countries have limited access to modern medicine and sophisticated treatments (Reed and Frankham, 2003), thus often relying on medicinal plants that offer an alternative, cost-effective, and freely available traditional approach for malaria control (Turkson et al., 2019). In some areas, the traditional medicine approach is the only source of medical care available (Romero-Daza, 2002; Abdullahi, 2011). Based on African Health Monitor Report in 2003, there is one healer for every 500 individuals vs. one medical doctor for every 40,000 patients in Africa; for Ghana, there is one healer for every 200 individuals vs. one medical doctor for every 20,000 patients (Chatora, 2003; Abdullahi, 2011; WHO, 2014). These staggering numbers, combined with the fact that the majority of doctors reside in urban areas, stress the importance of traditional healers and indigenous plants in treatments of communities in rural Africa. The healers, also known as traditional doctors, approach the patients holistically, treating not only the given condition but restoring the social, emotional, and spiritual equilibrium of their patients based on community history and customs (Hillenbrand, 2006; Abdullahi, 2011).

Traditional African knowledge of indigenous plants have been utilized for healing and treating various ailments, including malaria, for centuries. Preservation and conservation of these nearly lost species is imperative, and documentation of alternative medicine applications and approaches used by traditional healers as an ancient and culture-bound method of healing for centuries should be prioritized (Abdullahi, 2011; Veeresham, 2012). These plants are largely understudied, and very limited financial and technological resources are available for research related to drug development in these countries (Reed and Frankham, 2003). Indigenous C. sanguinolenta contains not only antimalarial, anti-cancer, antihyperglycemic, antimicrobial, and antimycobacterial properties, but can be utilized to promote sustainable and climate-smart agro-ecosystems across West Africa. This, in turn, would allow sustainable commercialization of this plant as a cash crop across its native distribution, thus providing farmers with additional income while preventing destructive collection.

Despite overharvesting and near extinction of these indigenous plants, our findings provided evidence of high genetic diversity among C. sanguinolenta subpopulations across Ghana. In declining and isolated populations unable to respond to a variety of selection pressures, genetic variation can be reduced rapidly as a result of the limited number of individuals and inbreeding within the populations that are often driven by random genetic drift (Ellstrand and Elam, 1993; Hawley et al., 2006; Luan et al., 2006). This can further decrease species fitness, prospects for adaptive change, ultimately causing species extinction (Fischer and Matthies, 1998; Severns, 2003; Hawley et al., 2006; Hadziabdic, 2010). However, this is the first comprehensive study focusing on the population genetic and spatial structure of this medicinal plant, thus we can expect these individuals to be somewhat resistant to apparent population bottleneck and genetic drift effects, at least in the short term. Long term monitoring, better propagation systems, and selective breeding processes are necessary for better informed conservation efforts.

Our results indicated the presence of a weak population structure low genetic differentiation, and high gene flow among wild C. sanguinolenta subpopulations. Findings from this study can be attributed to the specialized wind dispersal mechanism of C. sanguinolenta seeds over long distances, as the main factor affecting variation in gene flow and population structure similarly reported in Cecropia obtusifolia (Epperson and Alvarez-Buylla, 1997), and some woody plant species (Hamrick et al., 1992), and thus providing long-term potential for the conservation of C. sanguinolenta. Gene flow is a directional transfer of genetic material across populations as a result of seed dispersal over a period of time and in some tree and shrub species, it is positively correlated with the direction of wind flows and rates (Kling and Ackerly, 2021). The signal of a weak population structure could be due to high gene flow in the species as reported by Waples (1998), since gene flow is known to be the predominant evolutionary force for the genetic structure (García-Verdugo et al., 2010). The unlimited gene flow and low magnitude of genetic differentiation observedcould be attributed to the relatively larger genetic neighbourhood and sampling size. According to Franceschinelli and Kesseli (1999), the relative magnitude of genetic differentiation is directly influenced by the size of the sampling area such that genetic differentiation increased with decreased sample plots in Helicteres brevispira.

In a large genetic neighbourhood, proposed by Wright (Wright, 1943; Wright, 1946), uniform selection pressures may limit differentiation among individuals. Alternatively, local differences and a relatively slow dispersal process of new alleles in these smaller geographical areas may increase differentiation between the groups (Wright, 1943; Wright, 1946). The presence of a contiguous genetic neighbourhood in C. sanguinolenta subpopulations in the present study is supported by STRUCTURE findings and indicated the presence of two distinct genetic clusters and extensive admixture as a result of gene flow. The high gene flow found in our study indicates that the effective population size of current C. sanguinolenta wild plants may be sufficient to offset the impact of population decline and the effect it has on genetic diversity. Similar to findings by Ratnaningrum et al. (2017), larger gene flow was found to be the main emphasis for natural genetical processes in Santalum album population.

The Mantel test revealed no correlation between Bruvo’s distance and geographic distance suggesting that genetic differentiation in the 8 subpopulations is not caused by geographic isolation due to distance. A possible explanation for the absence of the influence of isolation distance on genetic diversity could be due to high gene flow, sexual reproduction, and wind dispersal of seeds in C. sanguinolenta. The results of the Mantel test are similar to findings in Miscanthus, M. sinensis (Nie et al., 2014; Zhao et al., 2017).

These findings have practical implications for the conservation of C. sanguinolenta. First, the absence of geographic distance on genetic differentiation in C. sanguinolenta subpopulations, suggests that sampling from one geographic area could help cover a significant proportion of the genetic diversity in C. sanguinolenta. We recommend a protection strategy as follows: 1) protection of the plants in the Kwahu-Abene area, and 2) scaling-up the sampling to other C. sanguinolenta populations with high genetic diversity such as Hwehwee-Mempeasem, Hwehwee-Oboyan, Kwahu Pepease, and Asuafu-Sekyere West. In addition, ex situ conservation by encouraging the cultivation of C. sanguinolenta among smallholder farmers should be explored to ensure its availability in the needed quantities, thus contributing to a decrease in the harvest volumes from the wild, as a way of conserving the species in situ.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Conceptualization JNA, RNT, and DH; methodology JNA and SLB; validation JNA and SLB; formal analysis JNA and DH; investigation, JNA; resources JNA, DH, and RNT; data curation JNA and DH; writing—original draft preparation JNA and DH; writing—review and editing all authors; visualization JNA; supervision JNA; project administration JNA; funding acquisition JNA. All authors have read and agreed to the published version of the manuscript.

This research was funded by USDA NACA, grant number 58-6062-006, and the Volkswagen Foundation of Germany, grant number (85456). Funding from the Fulbright Scholar Fellowship supported DH’s visit to Ghana to draft portions of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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