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Evidence of horizontal gene transfer between land plant plastids has surprising conservation implications

Lars Hedenäs1*, Petter Larsson2,3, Bodil Cronholm2, and Irene Bisang1

Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 1 Department of Botany, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden; 2 Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden; 3Centre for Palaeogenetics, Stockholm University, SE-106 91 Stockholm, Sweden

*For corresponding. E-mail: [email protected]

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 Background and Aims Horizontal Gene Transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions, between species or genomes. It is frequent in mitochondrial and nuclear genomes but has not been demonstrated between plastid genomes of different green land plant species.

 Methods We Sanger sequenced the nuclear Internal transcribed spacers 1&2 (ITS) Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 and the plastid rpl16 G2 intron (rpl16). In five individuals with foreign rpl16 we also

sequenced atpB-rbcL and trnLUAA-trnFGAA.

 Key Results We discovered 14 individuals of a moss species with typical nuclear ITS but foreign plastid rpl16, from a species of a distant lineage. None of the individuals with three plastid markers sequenced contained all foreign markers, demonstrating the transfer of plastid fragments rather than of the entire plastid genome, i.e., entire plastids were not transferred. The two lineages diverged 165-185 Myr BP. The extended time interval since lineage divergence suggests that the foreign rpl16 is more likely explained by HGT than by hybridisation or incomplete lineage sorting.

 Conclusions We provide the first conclusive evidence of interspecific plastid-to- plastid HGT among land plants. Two aspects are critical: it occurred at several localities during the massive colonization of recently disturbed open habitats that were created by large-scale liming as a freshwater biodiversity conservation measure. It also involved mosses whose unique life cycle includes spores that first develop a filamentous protonema phase. We hypothesize that gene transfer is facilitated when protonema filaments of different species intermix intimately when colonizing disturbed early succession habitats.

Key words: Biodiversity conservation, Bryum pseudotriquetrum, disturbed habitat, filamentousAccepted protonema, horizontal gene transfer, Manuscript moss, plastid fragments, Scorpidium cossonii, unique life history.

INTRODUCTION Horizontal Gene or genome fragment Transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions, between species or genomes. It is a well-known phenomenon between or to green land plant species (Bock, 2009; Renner and Bellot, 2012; Gao et al., 2014; Soucy et al., 2015; Dunning et al., 2019). It is commonly reported for mitochondrial genes, and possibly more widespread than Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 earlier believed also for nuclear genes (Dunning and Christin, 2020). However, HGT to plastid genomes in plants is rare (Straub et al., 2013). While it occurs from protobacteria or other sources to diatoms and red algae, it has not unambiguously been confirmed to occur between plastid genomes of land plants (Rice and Palmer, 2006; Janouškovec et al., 2013; Ruck et al., 2017). One case was reported between parasitic land plants, but the authors suggested that this was likely a transfer of entire plastids (Park et al., 2007). The limited evidence for HGT involving individual plastid genes of green land plants has led to the notion that plastid data yield more reliable phylogeny estimates than the labile mitochondria (Renner and Bellot, 2012; Gitzendanner et al., 2018). Conclusive evidence for HGT between plastids of different species is thus so far lacking. Nonetheless, should plastid HGT take place between green land plant species this will have major consequences not only for our understanding of plant interactions and evolution but potentially also regarding the reliability of plastid-based phylogenies. As we will show here, an entirely new aspect is that HGT has the potential to counter biodiversity conservation actions aimed at certain freshwater habitats.

In a recent study, described briefly in the Materials and Methods, we discovered the plastid rpl16 G2 intron (rpl16) from the moss Bryum pseudotriquetrum (Hedw.) G. Gaertn., B. Mey. & Scherb. in individual shoots of the moss Scorpidium cossonii (Schimp.) Hedenäs. Both species are common and widespread in base-rich wetlands. The atypical plants were present at two geographically separated sites that were recently disturbed through large- scale liming of wetlands (Svenson et al., 1995). Foreign rpl16 occurred in six shoots from three separateAccepted sampling spots, and one shoot, Manuscript respectively, from two different limed fens. The two moss genera Scorpidium and Bryum belong to lineages that are only distantly related (Liu et al., 2019), and diverged 165-185 Myr BP (Newton et al., 2007; Bell and Ignatov, 2019). Among closely related mosses, a few percent of the individual plants may have nuclear or plastid markers of other species than the morphologically defined species (Hedenäs, 2015b, 2017; Hedenäs et al., 2019). Typical divergence ages for such species’

lineages are up to a few tens of Myr BP (Laenen et al., 2014: their supplementary table S2), maybe up to 30-35 Myr BP. This is the time when the boreal zone, where many of the studied species have their main distribution areas, started to develop (Taggart and Cross, 2009). In such cases, hybridization or incomplete lineage sorting can explain foreign plastid genes. When lineages diverged much farther back in time, as for Scorpidium and Bryum,

such explanations are highly unlikely and suggests that recent HGT or transfer of entire Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 plastids is the most plausible explanation (Graham et al., 2012).

Here, we explored in detail whether our finds of Bryum rpl16 in Scorpidium cossonii were the result of true HGT, or if entire plastids were transferred between these species. We tested the two alternatives by sequencing additional plastid markers in S. cossonii shoots with Bryum rpl16: (i) If these additional markers also belonged to the Bryum genome, it implied the transfer of entire plastids. (ii) If these belonged to the Scorpidium genome, transfer of genome fragments only was substantiated. We further explored whether shoots of S. cossonii with noise in sequencing peaks and low-quality scores for rpl16 had mixed origins of rpl16. Finally, we studied the relationships among foreign Bryum rpl16 sequences from different S. cossonii shoots to evaluate whether potential HGT occurred once or several times. In the light of the outcome of our tests, we discuss which environmental factors and/or life history traits may facilitate HGT, and we highlight consequences for biodiversity conservation.

MATERIALS AND METHODS

We sampled the plants reported on in this paper for a study of the genetic variation in the wetland moss S. cossonii. We compared late succession habitats (natural rich fens; 150 shootsAccepted from 10 localities) and disturbed early Manuscript succession habitats (base-rich fens recently created through liming of originally acidic mires; 148 shoots from 10 localities) in mid- and northern Sweden, between 60.29-65.11° N and 12.40-20.68° E (Fig. 1a; Lönnell and Hylander, 2018; Hedenäs et al., unpublished).

We checked that no Bryum species were studied in the molecular laboratory of the Swedish Museum of Natural History during 15 years before the present study. We prepared

single shoots from the S. cossonii samples for DNA extractions under a dissecting microscope. We are therefore convinced that contamination from material in the laboratory is highly unlikely and cannot explain our results.

DNA extractions, PCRs and amplicon purification were done as earlier described in (Hedenäs, 2009a, b, 2015a). The purified PCR products were sequenced on an Applied Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 Biosystems 3730XL sequencer by Macrogen Europe B.V (Amsterdam, The Netherlands). Nucleotide sequence fragments were edited and assembled for each DNA region using PhyDE® 0.9971 (http://www.phyde.de/index.html; accessed 23 March 2020). We aligned the assembled sequences manually in PhyDE®. Regions of partially incomplete data in the beginning and end of the sequences were excluded from subsequent analyses. The generated sequences from the S. cossonii shoots were compared with each other and those of one sample of S. cossonii, specimen M104 in supplementary data table S1 from an earlier study (Hedenäs, 2009b). A BLAST at GenBank of the first encountered atypical rpl16 sequences from S. cossonii shoots aligned these closely with sequences from the Bryum pseudotriquetrum complex (Holyoak and Hedenäs, 2006). As a reference for Bryum pseudotriquetrum s.l., which includes, i.a., B. bimum (Schreb.) Turner (Holyoak and Hedenäs, 2006), we generated sequences from a B. bimum shoot, specimen P686 in Supplementary data Table S1) that was found intermixed in one of the S. cossonii samples of this study. We also included selected sequences from earlier studies (Pedersen et al., 2003; Holyoak and Hedenäs, 2006). Specimen information and GenBank accession numbers are provided in Supplementary data Table S1. Alignments for the individual molecular markers are included as Supplementary data Table S2. The sampled individual shoots were used up for DNA extraction. As specimen vouchers, we therefore deposited five S. cossonii samples from each site where we encountered shoots with foreign rpl16, each sample representing a differentAccepted spot at a site, in the herbarium of theManuscript Swedish Museum of Natural History (S). We checked that the nuclear Internal Transcribed Spacers 1 & 2 (ITS) in all sampled shoots belonged to S. cossonii. We indicate the individual shoots involved in each investigation step in Supplementary data Table S1 and Fig. 1. After the discovery of seven S. cossonii shoots with Bryum rpl16, we verified our results by running a new PCR and re- sequencing with the DNA extracts from five arbitrarily selected shoots among the seven atypical S. cossonii ones. We then generated sequences of two additional plastid markers

for the same five shoots to test whether plastid fragments or the entire plastids were transferred. We chose atpB-rbcL-spacer (atpB-rbcL), which is located relatively close to rpl16 on the plastid genome, and the more distantly located trnLUAA-trnFGAA spacer (trnL-trnF) (Stech and Quandt, 2010). Third, to explore the frequency of S. cossonii shoots with Bryum rpl16, we sampled twelve and two, respectively, additional S. cossonii shoots from the two

study sites where we first found potential rpl16 transfer. Finally, we ran DNA extracts from Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 shoots that had noise in sequencing peaks and low-quality scores in the earlier steps (∞ in Fig. 1) on long gels to attempt separating different variants of rpl16. This included such plants from two additional limed fen sites. Distinguished PCR bands were re-amplified directly from the gel, followed by Sanger sequencing.

We tested the difference in the frequencies of S. cossonii shoots with Bryum rpl16 between natural rich and limed fens by Pearson’s χ2 test. To evaluate whether Bryum rpl16 sequences in S. cossonii are of a single origin or were repeatedly transferred, we analysed the Bryum rpl16 relationships and differentiation in a haplotype context, using the program TCS (Clement et al., 2000).

RESULTS The re-sequencing of the DNA-extracts from five atypical S. cossonii shoots (Fig. 1b) confirmed the Bryum rpl16 identity (Fig. 1c, f).

All generated trnL-trnF sequences and three out of four atpB-rbcL sequences from S. cossonii shoots with atypical rpl16 belonged to the S. cossonii genome, whereas the fourth atpB-rbcL sequence belonged to Bryum (Fig. 1d). We were unable to generate the atpB-rbcL sequence for one shoot. This implies that only a portion of the plastid genome was transferred, but the size of the transferred genome portion differed between individual plants.Accepted In one individual, the transferred genome Manuscript portion included both rpl16 and atpB-rbcL.

The sampling of the additional 14 S. cossonii shoots, originating from both the original study sites (Fig. 1b, e), disclosed one additional S. cossonii plant with foreign Bryum rpl16 (Fig. 1e). Among eight shoots with noise in sequencing peaks and low-quality scores from steps b to e (∞ in Fig. 1) we recovered two shoots with Bryum rpl16, five shoots with

both S. cossonii and Bryum rpl16 (Fig. 1f) and were unable to generate the rpl16 sequence for one shoot.

None of the 150 shoots from natural rich fens displayed HGT, whereas 14 of the 162 (148 original +14 additional shoots) from limed fens harboured Bryum rpl16 (χ2 = 12.47; p = 0.0004). The haplotype network analysis showed that rpl16 differed strongly between sites Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 (Fig. 2). The differentiation between rpl16 from the site with the largest number of atypical S. cossonii shoots and rpl16 from the other three sites was eight mutational steps.

Our results verified inter-specific horizontal transfer of plastid genes in 14 investigated individual shoots of S. cossonii, from four geographically separated sites where we sampled 74 shoots in total. We confirmed Bryum rpl16 in nine S. cossonii shoots, mixed rpl16 in five S. cossonii shoots, and 60 shoots contained S. cossonii rpl16 (Fig. 1; the shoots specifically investigated here are listed in Supplementary data Table S1). Three sites harboured single S. cossonii shoots with Bryum rpl16, while the fourth site carried the remaining eleven atypical S. cossonii shoots.

DISCUSSION

We present the first unambiguous evidence for HGT between plastids of land plant species. We propose that a combination of environmental conditions during colonization and unique life history traits in our study plants are critical to explain our findings.

The sites where we disclosed interspecific HGT in plant plastids are environments that have recently undergone a major large-scale disturbance event, after which large open surfaces were created. The disturbance was caused by liming of originally acidic mires, which is regularly practised in Sweden since 1990 to conteract acidification (Svenson et al., 1995). This resulted in artificial fens with chemical conditions approaching those in natural rich fens,Accepted and vast bare surfaces exposed to colonizationManuscript by wind-dispersed spores and seeds of plants. All investigated shoots with foreign rpl16 (14) came from disturbed (limed) fens, while none of the shoots from natural rich fens indicated HGT. Previous extensive investigations of ITS and rpl16 in S. cossonii (>375 samples) and other mosses (1,812 samples) were conducted on plants from natural environments and failed to reveal HGT among distant lineages (Supplementary data Table S3), suggesting that HGT is rare in plants

from established, late-succession vegetation. Based on these two sets of observations, we suggest that the colonization phase in combination with unique features of the moss life cycle are key for interspecific plastid HGT to occur. Following spore germination, mosses form a (usually) filamentous and chlorophyllose protonema, from which the green ‘moss plants’ are produced. Early succession habitats, after disturbance, may be reached first by easily wind- dispersed spores, and developing protonemata filaments of different species can grow Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 intimately mixed during massive colonization events. Scorpidium cossonii and B. pseudotriquetrum frequently occur together in rich fens (Supplementary data Fig. S1), and we confirmed adult plants of both at all sites with S. cossonii shoots having foreign rpl16. In this study, we sequenced only ITS beyond the plastid markers, and future analyses need to explore whether other genomes may be involved in HGT under the conditions we describe above. However, the mitochondrial genome structure has remained evolutionary conservative across bryophytes lineages for 350 My (Liu et al., 2014).

We hypothesize that the protonema stage facilitates HGT according to the weak-link model (Husnik and McCutcheon, 2018). HGT from Bryum to S. cossonii plastids can potentially occur through various mechanisms that have been reported or suggested for plants, including grafting and plasmodesmata, or via vectors, such as pathogenic organisms, transgenic vectors like virus and bacteria, nematodes, insects, fungi, or transposable elements (Stegemann and Bock, 2009; Renner and Bellot, 2012; Gao et al., 2014; Soucy et al., 2015). However, the mechanism that is responsible for the plastid fragment transfer to S. cossonii remains to be established. The modified plastids, sometimes mixed with native plastids of the receiving species, will pass on to the developing moss plants. As outlined above, plastid HGT is presently unknown between moss species in natural habitats, which suggests that a mechanism exists that gradually eliminates foreign sequences in most cases. These are possibly inferior to the species-specific sequences. However, in situations when transferred genome fragments remain and integrate into the receiving genome, this could potentially lead to erroneousAccepted phylogenetic conclusions based onManuscript plastid data (Dunning and Christin, 2020). Moss plants rarely successfully establish in closed vegetation (Mishler and Newton, 1988; Cronberg, 1991; Bu et al., 2017) and large-scale disturbed environments are seldom in the focus of detailed genetic studies of bryophytes. This could explain the lack of previous evidence for HGT in mosses.

In this study, we demonstrate that plastid HGT can be recent and did not only occur in the early evolution of plants (Bock, 2009; Yue et al., 2012). A haplotype analysis of relationships among Bryum rpl16 from atypical S. cossonii shoots disclosed two distinct rpl16 haplotype groups separated by eight mutational changes (Fig. 2). This marked differentiation likely reflects two different species within the B. pseudotriquetrum complex

(Holyoak and Hedenäs, 2006), since the number of mutational differences per marker Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 between the two haplotype groups is similar to or larger than between other studied moss species (Draper and Hedenäs, 2009; Hedenäs, 2011; Stech et al., 2011; Carter, 2012). It strongly suggests that plastid HGT occurred at least twice. Dispersal of spores or plant fragments with atypical plastids can potentially account for the occurrence of similar haplotypes in three of the limed fens. However, considering that the localities are separated by 60-266 km from each other, dispersal cannot explain the genetically strongly different haplotype group at the fourth site. We believe that the observed HGT to S. cossonii occurred during the last few decades, i.e., the period after the major disturbance of the natural habitat. If the foreign rpl16 in S. cossonii reflected older HGT events, we would expect to have noted it also, at least occasionally, in the >375 S. cossonii samples from established natural vegetation.

To mitigate the effects of surface water acidification, active liming of lakes and mires has become a widespread practice since 1980 over large areas in Central and Northern Sweden (Svenson et al., 1995; Lönnell and Hylander, 2018). Such liming repeatedly results in large surfaces exposed to colonization by diaspores of plants that are adjusted to the created edaphic conditions. It is intriguing that a conservation measure intended to save or restore plant and animal diversity in streams and lakes could possibly have the unexpected effect of triggering processes at the genome level in a quantitatively important and widespread rich fen species. The massive colonization of new substrates by spores of differentAccepted species and subsequent intermixed Manuscriptprotonema growth seems to have enabled that plastid DNA was transferred between two, only distantly related species. In this study, we showed that HGT occurred at almost half of the limed study sites (four out of ten). Based on this frequency, a qualified estimate suggests that HGT has affected moss plants colonizing a significant proportion of the mires that have been subjected to liming or peat extraction. Two- to three-hundred km2 of wetlands (excluding lakes and streams) were limed in Sweden

during the last decades, and more than 700 km2 of Swedish and Finnish mires were mined for peat for energy production or soil improvement (Anonymous, 2003; Putkuri et al., 2013). This suggests that enormous surface areas have been strongly modified and opened-up for massive colonization, and thus for potential HGT between different establishing moss species and, maybe, between other species. The possible extent of such genetic

modification resulting from conservation measures turns our discovery into an issue of Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 significant concern to conservationists.

The two different rpl16 variants found in five shoots could occur together in the same plastid genomes or be present in different plastids. Given that numerous plastids occur per cell, mixtures of plastids with Bryum and S. cossonii rpl16 could potentially occur in additional shoots if plastids with rpl16 from one species constitutes a minor, undetected fraction. The proportion of shoots with pure Bryum rpl16 (or pure S. cossonii rpl16) could thus be lower, or even zero. Although this does not affect the qualitative evidence for HGT, it implies that our estimates of its frequency are conservative.

Concluding, we find evidence for plastid HGT in an early succession habitat, after a major disturbance event, exposed to colonization by plants with spore dispersal and a life cycle that includes a filamentous phase enabling intimate interspecific contact surfaces. The Scorpidium cossonii–Bryum system in recently disturbed (limed) fens provides an excellent experimental system. We propose further studies in other extensive and strongly disturbed habitats, including other genomes and plants with different life history traits. This study provides the first evidence of the repeated occurrence of HGT among plastids in land plants and adds HGT as an additional potentially negative effect of wetland liming, peat excavation, and similar measures that radically affect significant mire surfaces. We plan follow-up studies on the underlying mechanism for HGT between land plant plastids and suggest closer attention to this in future studies of the effects of wetland liming, peat excavation,Accepted and similar measures. Manuscript

ACKNOWLEDGEMENTS

We thank Niklas Lönnell for access to the studied material, and Johan Ehrlén, Kristoffer Hylander, and Helena Korpelainen for valuable and constructive comments on an earlier manuscript version.

FUNDING Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 This work was supported by Stiftelsen Anna och Gunnar Vidfelts fond för biologisk forskning [grant number 2018-010].

Accepted Manuscript

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Accepted Manuscript

Figure legends

Fig. 1. Graphic summary of study design and results. (a) Sampling design. We generated sequences of both ITS and rpl16 for 298 S. cossonii shoots from rich (late succession habitat; 150 shoots) and limed fens (disturbed early succession habitat; 148). (b) Five and one S. cossonii shoots from two separate limed fen sites, respectively, contained Bryum rpl16. Four

shoots from these and two additional limed fen sites yielded noise in sequencing peaks and Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 low-quality score curves (∞; M1144 yielded distinct Bryum motifs). (c) For five shoots, we run another PCR and re-sequenced rpl16, that yielded Bryum rpl16 again (M1144 again yielded distinct Bryum motifs). (d) For the same five shoots, we generated atpB-rbcL and trnL-trnF. The trnL-trnF sequences were all of S. cossonii type, implying that only plastid genome fragments were transferred. For atpB-rbcL the results were mixed; one shoot yielded a Bryum sequence. (e) We generated ITS and rpl16 sequences for 12 and 2 additional S. cossonii shoots from the two sites in b, respectively. Nine shoots contained S. cossonii-rpl16, one Bryum-rpl16, and four yielded noise in sequencing peaks and low-quality score curves. (f) For eight samples with noise in sequencing peaks and low-quality score curves, we attempted to separate different PCR products with gel electrophoresis, followed by reamplification and Sanger sequencing of each band. For two samples, we recovered Bryum rpl16, while five had rpl16 of both S. cossonii and Bryum and one shoot did not yield rpl16. Sample numbers correspond with those in Supplementary data Table S1.

Fig. 2. TCS haplotype network showing rpl16 relationships among 14 shoots of Scorpidium cossonii that had rpl16 from the Bryum pseudotriquetrum complex, for samples that yielded complete sequences. Circle sizes are proportional to the number of shoots with a certain haplotype. Circles connected by a line differ in a single mutational difference; black dots indicate ‘missing’ haplotypes. The Ångermanland site has the highest number of S. cossonii shoots with atypical rpl16 (12 shoots) and includes the B. bimum reference shoot (P686) in one haplotypeAccepted and the GenBank B. pseudotriquetrum Manuscript s.l. (GB1) specimen in another (Supplementary data Table S1). The group to the left comprises the haplotypes of the single S. cossonii shoots with foreign rpl16 from three sites.

Downloaded from https://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcab021/6145156 by guest on 08 March 2021 Figure 1

Accepted Manuscript

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Accepted Manuscript