DISCUSSION
We aimed to identify the center of origin of the Indonesian nutmeg,Myristica fragrans . We applied a population genetic approach and used genetic data obtained from nuclear microsatellite genotyping and whole plastid genome sequencing. Our results suggest that M. fragrans originated from the South Moluccas, and colonized North Moluccas islands well before the management of the species by humans (Middle, Late Pleistocene). The species was thus present across the entire Moluccas at the start of species trading history.
We found two different intra-specific genetic clusters in the Moluccas, one in South Moluccas (Banda and Ambon) and the other in North Moluccas (Ternate, Tidore and Bacan). The South Moluccas cluster appeared to be ancestral as indicated by our ABC analyses conducted on both nuclear and cpDNA data sets. In these analyses, the best supported demographic scenario corresponded to an ancestral population of the species in the South Moluccas and a colonization of the North Moluccas 2159 to 8231 generations ago, followed by a demographic bottleneck in the South Moluccas 299 to 402 generations ago. Assuming a generation time of 50 years in M. fragrans , species migration from South to North would have thus occurred c. 411-108 ka ago (corresponding to the Middle, Late Pleistocene), and the demographic bottleneck would have occurred c. 20-15 ka ago (Late Pleistocene).
The first individuals that colonized North Moluccas were probably carrying the cpDNA haplotype Hap 3. The Hap 3 haplotype is frequent and widespread across the entire Moluccas region and was also inferred to be ancestral, being directly connected to the outgroup (M. argentea ) and had a central placement in the phylogenetic network. Most of the other haplotypes are endemic to a specific island or group of islands and appeared to have diverged from Hap 3. Also, South Moluccas and North Moluccas haplotypes tend to be in two different groups in the haplotype network, which is in line with the North/South genetic divide observed from nSSRs. Under this scenario, we would expect the presence of a phylogeographic signal, which is not the case. Recent human-driven dispersal of the species has potentially blurred this signal.
Species migration from South to North probably happened through animal or sea dispersal. Since the emergence of Moluccas islands in the Oligocene / Miocene and even during glacial maximas, no major land bridges have connected North and South Moluccas, even during the glacial maxima when the sea level was around 120 m lower than now (Faral et al., 2022; Hall, 1987; Hall & Wilson, 2000), which supports observed differences between the general flora of the North and South Moluccas (Rutgrink et al., 2018). The origin of the Asian genera Myristicahas been dated around the Late Miocene based on plastid data (Doyle et al., 2004; Frost et al., 2022; Sauquet et al., 2003), at a time when both North and South Moluccas were already present. The geological history of North and South Moluccas islands and the timing of colonization obtained in our analyses thus suggest that the dispersal ofM. fragrans was probably mediated by animals or ocean currents as opposed to rafting across tectonic terranes. Hornbills (Bucerotidae) are known to be involved in seed dispersal of Myristicaceae . These big birds potentially allow for long-dispersal events, although we cannot exclude that marine dispersal played a role in the demographic expansion of these species either. It has been demonstrated that seeds can be dispersed by marine currents over long geographic distances, andM. fragrans is not an exception (Bull, 2020). If some seeds kept their viability after marine dispersal, this might have also contributed to the dispersal of the species, as it has been shown for other tree species . It has also been suggested that the sea currents in the Moluccas during the Pleistocene flowed northwards (Barmawidjaja et al., 1993), allowing seed dispersal from South Moluccas to North Moluccas.
Since our demographic analyses suggest that M. fragrans colonized Northern Moluccas during the Pleistocene, this supports the presence of the species all across the Moluccas before the arrival of the Europeans. We found a number of historical accounts supporting this hypothesis
It seems that Europeans were in search of the mysterious historical place of cultivation of nutmeg, i.e. the place where nutmeg was cultivated before being imported to Europe along a complex network of different traders and nations. North Moluccas were known for the cultivation of cloves , and South Moluccas for the cultivation of nutmegs . Search for this ‘golden place’ and particular focus on the South Moluccas where the species was first grown in mass probably blurred their view that the species could be present elsewhere. Here, we can speculate that historical information on the distribution of the species was strongly controlled by the VOC. This idea is supported by the embargo set by the VOC on the publication of Rumphius ‘Herbarium amboinense’, a document that includes information about the trade and cultivation of the species (Friedrich, 2019; van der Werff & Raven, 2014).
Along the human-managed evolutionary history of M. fragrans , it is likely that the North Moluccas gene pool have remained unimpacted in contrast to the South Moluccas stock that have been strongly impacted. We observed higher levels of genetic diversity both for the SSR and cpDNA in the North Moluccas genetic cluster than for South Moluccas. The lowest level of genetic diversity observed in the South is probably due to the reduction in M. fragrans population size during the Dutch colonisation period in the 17th and 18th centuries. As a matter of fact, the Dutch secured a monopoly on the production (Robert & Trimen, 1880) and trade of nutmeg by adopting a strong control policy on the resource (extirpation of trees, destruction of a part of the production to keep prices high) (Wright, 1958). Such management practices have certainly negatively impacted the genetic diversity of the species. This reduction in population size in the South is actually supported by our demographic analyses, with a signal of a (relatively recent) bottleneck observed in Banda and a less-marked one in Ambon. In contrast, the demographic tests tend to support a constant population size in the North Moluccas islands. The dating of the population bottleneck is a bit older than the Holocene period, but dating recent bottleneck is almost impossible. Moreover, it is worth noting that it is extremely difficult to apply a time calibration in demographic analyses that use generation time as time unit for long-lived species such as trees. Here we have set an arbitrary generation time of 50 years which implies a detection of a bottleneck event before the Holocene period. Even though our analyses were based on only 15 nSSR, our results are roughly in agreement with the knowledge we have on the historical management of the species.
The observed pattern of higher genetic diversity in the North Moluccas cluster might also be the result of inter-specific gene flow. The potential hybridization of M. fragrans with otherMyristica species present in these islands is not known. According to de Wilde (2014), 20 Myristica species would be present in the Moluccas, which suggest that a number of Myristicasister species can be found in sympatry with M. fragrans . The radiation of Myristica species is probably quite recent, as demonstrated by the high rate of transferability of nSSR markers fromM. fragrans to sister species (Kusuma et al., 2020). A recent radiation implies that inter-specific gene flow may occur. It is worth noting that morphologically, M. fragrans individuals sampled in these islands are, to the best of our knowledge, similar to M. fragrans in South Moluccas. That would imply that if inter-specific gene flow occurred, it might have had a small effect on the phenotypic diversity of M. fragrans . In the absence of additional genetic data for other Myristica species present in the region, we cannot support or rule out this hypothesis that would deserve further investigation.
Perspectives about conservation and sustainable management and use .
Our results bring insights that will be important for conserving and managing the genetic diversity within M. fragrans . Genetic differentiation observed between North and South Moluccas in this study should be integrated into conservation strategies and management practices for this species. The genetic diversity of this species in Banda and Ambon has been particularly impacted by past human practices, with potential consequences on the adaptive potential of this species in these islands. To date, this lower level of genetic diversity does not seem to have caused any noticeable reduction in nutmeg production or susceptibility to pests and diseases, but such an impact may appear in the near future from indirect drivers of change, in particular climate change.
Banda and Ambon islands were, and still are the main sources of planting material introduced in other parts of Indonesia and across all major tropical regions in the world . Given the relatively low level of genetic diversity in these islands, low number of male trees kept on farms, and the fact that seeds used to produce planting material are usually taken from one mother tree only, it is likely that the genetic diversity introduced into other regions of the world is also quite low. In addition, we have no information about potential differences in quality between North and South Moluccas. The Banda island nutmeg ‘variety’ is the most well-known, but it is not known whether if the popularity of this stock is the legacy of historical cultivation being centered around the Banda islands, or if it was intentionally selected based on resilience to environmental factors (e.g., soil properties), or genetic differences, or the combination of the above. Testing differences in the quality of the nuts by conducting common garden experiments would be needed to inform strategies of genetic resource management for this species . If no significant differences exist, it would be interesting to consider the sourcing of additional material from North Moluccas in order to increase the level of genetic diversity and thus the potential resilience of introduced populations.
We cannot exclude that additional original genetic resources are present elsewhere. Here we have sampled only a representative part of the Moluccas islands. Indeed, this sampling regime was sufficient to infer the native range of the species, but it remains insufficient for conservation considerations. The presence of a third genetic cluster in Bacan island is interesting as it outlines an additional genetic structuring. We would need additional samples from the whole North Moluccas area (Halmahera, Sula, Obi, Misool in particular) to better interpret this result and get a more comprehensive view of the spatial genetic diversity of M. fragrans . Conducting an exhaustive sampling in a complex archipelago such as Indonesia is particularly complicated and time consuming (~1000 islands for the Moluccas only). This probably explains why local flora remains largely undocumented. Concerning M. fragrans , we cannot rule out that some additional unique resources exist somewhere else in the Moluccas, such as in the more Southern islands (Kai, Aru, Tanimbar, etc), but a much more intensive sampling is needed to get the answer.