Introduction

An origins-of-life scenario depicts Darwinian evolution from self-replicating molecules, such as RNA, toward complex living systems1,2,3. How molecular replicators could develop complexity by continuously expanding information and functions is a central issue in prebiotic evolution4,5. An expected route for complexification is that novel RNA replicators successively emerged and co-replicated so that increased genetic information can be stored at a population level, before their assembly into a long genome4,5,6,7,8. Although several theoretical studies investigated the possibility of complexification9,10,11 and stable coexistence12,13,14,15,16 of molecular replicators, empirical demonstration has been challenging.

To date, diverse molecular replicators have been constructed based on biomaterials such as DNA, RNA, and peptides17,18,19,20,21. Although considerable efforts have been made to design interactions among these replicators22,23,24,25,26, spontaneous complexification was generally precluded due to their inability to undergo Darwinian evolution through continuous mutation accumulation and natural selection. The study by Ellinger et al.23 was an exception, but they used a predefined replicator network to initiate evolution, which was also limited to the short-term. Thus, it has remained an open question whether a single molecular replicator can evolve into a complex replicator network.

Previously, we constructed an RNA that replicates using an RNA replicase translated from itself27. During replication, mutations are introduced, and occasional recombination deletes replicase-encoded regions to generate a parasitic RNA that replicates by exploiting replicases derived from other RNAs (replicase-encoding “host” RNAs). The RNAs can undergo Darwinian evolution in a serial transfer experiment, and a previous attempt (120 rounds, 600 h) demonstrated the successive appearance of new host and parasitic RNA lineages showing defense and counter-defense properties28,29. However, these lineages dominated the population in turn and only transiently, possibly due to a short evolutionary timescale.

Here, we continued the serial transfer experiment up to 240 rounds (1200 h). Sequence analysis uncovered that two previously detected host RNA lineages became sustained and further diverged into multiple sublineages of host and parasitic RNAs. The population dynamics of each lineage gradually changed during the evolution, from dynamically fluctuating stages to quasi-stable coexistence, suggesting the appearance of co-replicative relationships among the lineages. Biochemical analyses supported the co-replication of dominant RNAs in the different lineages containing a cooperative RNA that replicates all other members, thus establishing a multiple replicator network.

Results

Long-term evolution of an RNA replicator

The RNA replication system (Fig. 1a) consists of a single-stranded RNA (host RNA) that encodes the catalytic subunit of Qβ replicase (an RNA-dependent RNA polymerase) and a reconstituted Escherichia coli translation system30. RNA replication occurs through the translation of the replicase subunit, which becomes active in association with elongation factors Tu and Ts (EF-Tu and EF-Ts) in the translation system. The introduction of mutations into the host RNA during replication generates host RNA variants with different features. Spontaneous RNA recombination deletes a part of the replicase gene to generate parasitic RNAs while retaining 5ʹ and 3ʹ terminal sequences for recognition by the replicase. When encapsulated in micro-sized water-in-oil droplets, host RNAs can sustainably replicate in the presence of parasitic RNAs28.