The f-statistic values were positive when SNT49 (hpAsia2 strain from India) was set to H2. This means the gene flow from the H3 strains (hpRyukyu and Afghanistan-Nepal strains) to the Indian strain is stronger than the gene flow to H1 strains (PNG84A, hpSahul strain from Papua New Guinea, and East Asian strains). This implies that the gene flow was [H3] -> [India] -> [Oceania, East Asia].

In contrast, f-statistics values were all negative when the hpSahul strain was set to H2. This result suggests that gene flow from hpRyukyu to EA-Am was stronger than that from hpRyukyu to hpSahul, i.e., the gene flow skipped hpSahul. HpSahul is considered to have expanded via coastal line to reach Oceania (Moodley et al., 2009). If the ancestral hosts of hpRyukyu moved toward the south, there should be gene flow between hpRyukyu and hpSahul before hpRyukyu reached East Asia. The weaker gene flow from hpRyukyu to hpSahul than that from hpRyukyu to East Asia implies that the ancestral hosts of hpRyukyu might not take a southern course. Rather, hpRyukyu might go to the north circumventing the Himalayas.

The oldest Homo sapiens fossil in Okinawa, the Yamashita Cave Man, was estimated to be around 32,000 B.P. through radiocarbon dating (Kobayashi et al., 1971). The complete skeletons of 27,000 B.P. (Shiraho-Saonetabaru) (Shinoda and Adachi, 2017) and 2,2000 B.P. (Minatogawa) (Baba and Narasaki, 1991) were also discovered from Okinawa. This suggests that some Paleolithic human populations resided in Okinawa. The divergence dates of hpRyukyu and hspOkinawa correspond with these fossil records. However, human genome analyses indicate that the modern Okinawa population was more closely associated with the Japan main island than the Ainu people (Jinam et al., 2015; Kanzawa-Kiriyama et al., 2017).

One of the possible reasons for the discrepancy between human and H. pylori analyses is that ancient traces in the autosomal DNA are attenuated by admixture with new migrants. Actually, mitochondria DNA haplogroup M7a, which was found in 3 of 5 Paleolithic Shiraho-Saonetabal individuals (Shinoda and Adachi, 2017), is still observed in about 20% of the current Okinawa population.

Another possibility is that hpRyukyu in Okinawa was transmitted from the Paleolithic migrants to the later migrants without human admixture. Then, the old migrants went extinct and only the Paleolithic H. pylori remained in the new migrants. It is controversial whether the Paleolithic migrants contributed to the modern Japanese population, although at least the Paleolithic H. pylori survived up to now.

The divergence date of hspOkinawa and Ainu strains corresponded to the Jomon era. Both hspOkinawa and Ainu strains lack cagA (designated as cagA (−)). CagA (−) strains are often observed in Africa and Europe but are rare in Asia. As the farmer population expanded on the main island of Japan, the trace of Jomon people remained in the north (Hokkaido) and south (Okinawa) of the Japanese Archipelago.

However, hspOkinawa and Ainu strains belong to different sub-branches (Figures 1 and and2).2). In addition, hspOkinawa has similarity with the southern strains (hspMaori) in the STRUCTURE analysis (Figures S1 and S2). We assume that the hosts of hspOkinawa were in contact with people from Southeast Asia who might carry ancestral strains of hspMaori. From these observations, we assume that ancestral hspIndigenousAmericas from the north route and ancestral hspMaori from a southern route reached the east end of the Eurasian continent and hybridized to form hspOkinawa, so hspOkinawa has both characteristics (Figure 3B).

The scarce admixture between hpRyukyu and hspOkinawa suggests that they co-existed independently for a long time. This independence may reflect geographic isolation because the Okinawa area consists of many islands.

There were hpEurope strains that might be transmitted from Western people but its ratio was less than 5% of the strains obtained from Okinawa. On the contrary, the hpRyukyu and hspOkinawa strains accounted for 16% and 14%, respectively. H. pylori does not transmit easily between adults but mostly transmit from parents to children by close contacts while the immune system is not fully developed, therefore lineage expansion takes generations. Considering the substantial inflow of foreign population to Okinawa started only after 1945, it is unlikely that hpRyukyu and hspOkinawa strains derive from recent foreign sources.

Furthermore, strains that are close to hpRyukyu were found only from Afghanistan to Nepal, although we investigated a wide variety of Asian strains and public genome data of Western and African strains. Because current human flow from Afghanistan or Nepal to Okinawa is scarce, the similarity between Okinawa strains and Afghanistan or Nepal strains is likely due to common ancestry of the host human populations.