Although a variety of non-LTR retrotransposons of the L1 superfamily have been found in plant genomes over recent decades, their diversity, distribution, and evolution have yet to be analyzed in depth. Here, we perform comprehensive comparative and evolutionary analyses of L1 retrotransposons from 29 genomes of land plants covering a wide range of taxa. We identify numerous L1 elements in these genomes and detect a striking diversity of their domain composition. We show that all known land plant L1 retrotransposons can be grouped into five major families based on their phylogenetic relationships and domain composition. Moreover, we trace the putative evolution timeline that created the current variants and reveal that evolutionary events included losses and acquisitions of diverse putative RNA-binding domains and the acquisition of an Archaea-like ribonuclease H (RNH) domain. We also show that the latter RNH domain is autonomously active in vitro and speculate that retrotransposons may play a role in the horizontal transfer of RNH between plants, Archaea, and bacteria. The acquisition of an Archaea-like RNH domain by plant L1 retrotransposons negates the hypothesis that RNH domains in non-LTR retrotransposons have a single origin and provides evidence that acquisition happened at least twice. Together, our data indicate that the evolution of the investigated retrotransposons can be mainly characterized by repeated events of domain rearrangements and identify modular evolution as a major trend in the evolution of plant L1 retrotransposons. E ver since the discovery of the first transposon by Barbara McClintock in 1948 in maize, plants have provided a prime model system to study transposition. Both DNA transposons and retrotransposons are abundant in plants (1) and play a major role in driving genetic diversity and evolution of these organisms. Interestingly, non-LTR retrotransposons, the class of retrotransposons that is most represented in mammals, are found less abundantly in plants (2-5). More recently, however, a variety of non-LTR retrotransposons has been discovered in plants, and they were classified to three non-LTR retrotransposon superfamilies: retrotransposable element (RTE), Dualen, and L1 (6-8).The L1 superfamily is perhaps the best-studied plant non-LTR retrotransposon group, members of which have been identified in most plants. Among other species, they occur in corn [Zea mays (Cin4)], thale cress [Arabidopsis thaliana (Ta11-1)], and the alga Chlorella vulgaris (Zepp) (9-11).In general, plant L1 retrotransposons carry two ORFs. The protein encoded by ORF1 (ORF1p) usually contains an RNA recognition motif (RRM) (12)(13)(14). Similarly, human L1 ORF1p, which presumably acts as an RNA chaperone (15), also contains an RRM that was shown to bind single-stranded nucleic acids in cooperation with the ORF1p C-terminal domain (14). The second ORF (ORF2) of plant L1 retrotransposons encodes a polyprotein (ORF2p) that exhibits apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) activities....