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Journal of Systematics JSE and Evolution doi: 10.1111/jse.12515 Research Article

Exploring the plastid genome disparity of liverworts

† † Ying Yu1,2 , Hong‐Mei Liu3 , Jun‐Bo Yang4, Wen‐Zhang Ma2, Silvia Pressel5,Yu‐Huan Wu1, and Harald Schneider6*

1College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China 2Key Laboratory for Diversity and Biogeography of East Asia, Kunming Institute of , Chinese Academy of Sciences, Kunming 650201, China 3Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, China 4Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China 5Department of Life Sciences, Natural History Museum, London SW7 5BD, UK 6Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, China † These authors contributed equally to this study. *Author for correspondence. E‐mail: [email protected] Received 14 February 2019; Accepted 16 May 2019; Article first published online 22 May 2019

Abstract Sequencing the plastid genomes of land provides crucial improvements to our understanding of the plastome evolution of land plants. Although the number of available complete plastid genome sequences has rapidly increased in the recent years, only a few sequences have been yet released for the three lineages, namely , liverworts, and . Here, we explore the disparity of the plastome structure of liverworts by increasing the number of sequenced liverwort plastomes from five to 18. The expanded sampling included representatives of all major lineages of liverworts including the Haplomitrium. The disparity of the liverwort genomes was compared with other 2386 land plant plastomes with emphasis on genome size and GC‐ content. We found evidence for structural conservatism of the plastid genomes in liverworts and a trend towards reduced plastome sequence length in liverworts and derived mosses compared to other land plants, including hornworts and basal lineages of mosses. Furthermore, Aneura and Haplomitrium were distinct from other liverworts by an increased GC content, with the one found in Haplomitrium only second to the . The results suggest the hypothesis that liverworts and other land plants inherited and conserved the plastome structure of their most recent algal ancestors. Key words: bryophyte, chloroplast, evolutionary conservatism, genome evolution, land plant evolution.

1 Introduction has been given to the three lineages of (28 spp.). Sparsity of sampling is remarkable given the fact that the Reconstructing the evolution of key features of the plastid plastid genome of the liverwort Marchantia polymorpha was genome (plastome) of land plants is a crucial task to improve among the first three plastid genomes sequenced together our understanding of the adaptation of plants to terrestrial with the eudicot Nicotiana tabacum L. and the monocot Oryza environments. Research aiming to elucidate the evolutionary sativa L. (Ohyama et al., 1986). history of the plastid genome has been enabled by the rapid Until 2018, the available plastid genomes of bryophytes increase of sequenced whole plastid genomes that have consisted of two hornworts [Anthoceros angustus Steph. become available in recent years as a consequence of improved (NC004543), Nothoceros aenigmaticus J.C. Villarreal & K.D. sequencing technology (Gao et al., 2010; Daniel et al., 2016; MacFarland (NC020259)], five liverworts [Aneura mirabilis Wang et al., 2018). Thus, the number of sequenced whole (Malmb.) Wickett & Goffinet (NC010359), Aneura pinguis (L.) plastid genomes made available has accelerated rapidly in Dumort. (NC035617), Apopellia endiviifolia (Dicks.) Nebel & D. ‐ recent years, enabling large scale phylogenetic studies, in- Quandt (NC019628), Marchantia polymorpha L. (NC001319), and cluding all branches of the land plant phylogeny (Gitzendanner Ptilidium pulcherrimum (Weber) Vain. (NC015402)], and six et al., 2018) and/or several hundred species of a single lineage, mosses [Nyholmiella obtusifolia (Brid.) Holmen & E. Warncke such as the grasses (Saarela et al., 2018). In 2018, approximately (NC026979), Orthotrichum rogeri Brid. (NC026212), Physcomitrella 2313 species had at least one plastid genome sequenced patens (Hedw.) Bruch & Schimp. (NC00587), Sanionia uncinata (Table 1). However, the majority of species with at least one (Hedw.) Loeske (NC025668), Syntrichia ruralis (Hedw.) F. Weber plastid genome sequenced belongs to seed plants (2213 spp.) & D. Mohr (NC012052), and Tetraphis pellucida Hedw. fl especially owering plants (2120 spp.), and other vascular (NC024291)]. As a consequence, the evolutionary history of plants, such as (60 spp.), whereas relatively little attention plastid genomes during the colonization of land by plants is still

July 2019 | Volume 57 | Issue 4 | 382–394 © 2019 Institute of Botany, Chinese Academy of Sciences Liverwort plastome diversity 383

Table 1 Summary of the taxonomic coverage of plastid genomes available in February 2018 plus newly generated liverwort sequences Lineage Orders SaOrd PrSaOrd (%) Families SaFam PrSaFam (%) Species SaSpp PrSaSpp (%) Hornworts 5 2 40.0 5 2 40.0 250 2 0.80 Liverworts 15 7 46.7 87 16 18.4 7236 18 0.25 Mosses 29 7 24.1 109 7 6.4 12 000 8 0.07 3 3 100.0 3 3 100.0 1338 6 0.45 Ferns 11 11 100.0 49 25 51.0 10 578 61 0.58 Acrogymnosperms 8 8 100.0 11 11 100.0 1080 102 9.44 Angiosperms 64 55 85.9 418 251 60.0 295 383 2195 0.74 All land plants 135 93 68.9 682 315 46.2 327 865 2392 0.73 Families, number of families currently recognized per lineage; Lineage, major lineages of land plants; , number of orders currently recognized for each of the major lineages; PrSaFam, proportion of families sampled; PrSaOrd, proportion of orders sampled; SaFam, number of families with at least one plastome sequence sampled; PrSaSpp, proportion of species sampled; SaOrd, number of orders with at least one plastome sequence sampled; SaSpp, number of species with at least one plastome sequence sampled; Species, number of species currently recognized for each of the major lineages. poorly understood. The few available bryophyte plastomes conservatism of the plastid genome structure but a trend suggest evolutionary conservatism of the genome structures, towards reduced sequence‐length compared to plastomes especially among liverworts (Wickett et al., 2008; Forrest et al., of vascular plants (Mower & Vickrey, 2018). This hypothesis 2011; Wicke et al., 2011; Grosche et al., 2012; Myszczyński et al., assumes that plastomes of land plants differ in their 2017), although some gene deletions were found in the non‐ evolutionary fate from those of algae, with the latter photosynthetic liverwort A. mirabilis (Wickett et al., 2008; showing a remarkable variation of the plastid genome Myszczyński et al., 2017). Thus, liverworts may diverge from structure (Smith, 2017; Turmel & Lemieux, 2018). Second, vascular plants and even mosses by the absence of evidence genome size and GC‐content might vary among liverwort supporting major structural reorganization as a major process in genomes as a consequence of the length variation of the plastid genome evolution (Wicke et al., 2011; Mower & introns, deletions of small regions, and frequency of RNA Vickrey, 2018). In contrast, the frequency of RNA editing varies editing. Besides focusing on liverworts, the study also aims among the studied liverwort plastid genomes (Grosche et al., to improve our understanding of evolution of the plastid 2012). The recorded variation in putative RNA editing sites among genome in the other two bryophyte lineages, namely the mitochondrial genome sequences of liverworts ranges from zero mosses and hornworts (Puttick et al., 2018; Sousa et al., in M. polymorpha, only a few edited nucleotides in Lejeunea 2018), by enabling identification of biases required to be cavifolia (Ehrh.) Lindb., to up to 20% editing of cytidine in considered in the set‐up of the models in phylogenetic Haplomitrium mnioides (Lindb.) R.M. Schust. (Schallenberg‐ analyses (Nishiyama et al., 2004; Cox et al., 2014; Cox, 2018; Rüdinger et al., 2012; Schallenberg‐Rüdinger & Knoop, 2016; Puttick et al., 2018; Sousa et al., 2018). Ichinose & Sugita, 2017). This pattern might also be found in the plastome of liverworts. Some studies increased the phylogenetic sampling by extracting protein sequences from transcriptome data (Gitzendanner et al., 2018) but they are still not sufficient to 2 Material and Methods reconstruct the evolution of the plastome structure. 2.1 Sampling strategy This study aims to contribute new data to reconstruct The study aimed to increase not only the taxonomic the evolutionary history of the liverwort plastid genome coverage but also to improve the sampling of the and elucidate the fate of the plastid genome in one of the phylogenetic diversity of liverworts by including at least major lineages of land plants. Liverworts represent a more one species of each major lineage of liverworts (see than 400‐million‐year separate history of adaptation to Table S1). To achieve the aims, we obtained plastid genomes terrestrial environments (M orris et al., 2018). Therefore, of 13 previously non‐sampled species of liverworts, and understanding the evolution of the liverwort plastome is increased the number of available genomes to 18, corre- crucial, although current phylogenetic studies rejected the sponding to approximately 0.25% of the extant liverwort hypothesis of liverworts as sister to all land plants (Puttick species diversity (Table 1). The dataset comprised represen- et al., 2018; Renzaglia et al., 2018; Sousa et al., 2018). tatives of all three major lineages of liverworts, namely Despite increasing evidence supporting liverworts as sister (1 sp.), (1 sp.), and to mosses and together forming the Setaphyta clade, (16 spp.). The sampling comprises hypotheses such as the liverwort basal proposal could be representatives of seven out of 14 orders currently recog- still considered as alternatives (Cox, 2018). Throughout the nized (Söderström et al., 2016). Special attention was given study we consider the Setaphyta clade (liverworts–mosses to the sampling from the species‐rich leafy liverwort lineages, sister relationships) as the most likely true topology but Jungermanniales and Porellales. Both lineages were repre- also consider proposed alternative hypotheses. Based on sented by six species, enabling us to explore the existence of the existing evidence, we aim to test the following distinct trends linked to the diversification in epiphytic predictions. First, released plastid genomes suggest the environments (Feldberg et al., 2014). www.jse.ac.cn J. Syst. Evol. 57(4): 382–394, 2019 384 Yu et al.

2.2 Genome sequencing, assembly, and comparative mosses (8 spp.), hornworts (2 spp.), lycophytes (6 spp.), analyses ferns (60 spp.), acrogymnosperms (102 spp.), and angios- Total genomic DNA was extracted from 10–50 mg dry perms (2195 spp.). In addition to 2391 land plant species, the tissue using a modified CTAB protocol (Yang dataset included 19 streptophytic algae and 50 chlorophytic et al., 2014). A genome library was constructed using Illumina algae. The proportion of taxa sampled per main lineage of Nextera XT DNA library preparation based on ca. 500‐bp long land plants was correlated with the estimated species DNA fragments obtained by shearing the obtained genomic diversity (r2 = 0.956, P = 0.011). The sampling size exceeds DNA following the manufacturer’s manual (Illumina, San all previous studies in number of taxa included (Wicke et al., Diego, CA, USA). By generating 90‐bp long paired‐end 2011; Zheng et al., 2017). Instead of focusing on individual sequences using an Illumina HiSeq 2000 at BGI‐Shenzhen genes, we emphasized two major parameters, plastid (Guangdong, China), approximately 200 Mb of sequences genome length and GC‐content. The evolution of these two was generated for each sample. These sequences were values was reconstructed by plotting these two values onto a assembled using CLC Genomic workbench version 10.1.1 consensus phylogeny of land plants using the linear cost (https://www.qiagenbioinformatics.com) involving quality model implemented in Mesquite 3.4 (Maddison & Maddison, control of the raw sequences with the NGS QC Tool Kit 2018). The impact of the different positions of hornworts was (http://nipgr.res.in/ngsqctoolkit.html) with cut‐off values for considered by exploring the estimates using three alternative read length and PHRED quality scores set as recommended positions, namely hornworts sister to all land plants, in Yang et al. (2014). Annotation was made with the support hornworts sister to vascular plants, and hornworts sister to of DOGMA (Wyman et al., 2004) and Geneious (Kearse et al., a liverworts–mosses clade (Puttick et al., 2018; Renzaglia 2012). The previously released liverwort plastomes, Aneura et al., 2018; Sousa et al., 2018). These alternative positions mirabilis (NC010359), Aneura pinguis (NC035617), Apopellia seem to have little effect on the estimated ancestral values endiviifolia (NC019628), Marchantia polymorpha (NC001319), for the major land plant lineages. The phylogenetic relation- and Ptilidium pulcherrimum (NC015402) were used as ships among liverworts were reconstructed according to the reference genomes during the assembly of the plastomes most recent phylogenetic hypothesis (Heinrichs et al., 2005; for newly generated accessions. The newly generated plastid Feldberg et al., 2014; Laenen et al., 2014) with the genome sequences were deposited in GenBank (http://www. Haplomitriopsida represented by Haplomitrium as a sister ncbi/nlm.nih.gov; see Table S1). to the remaining liverworts and complex thalloid liverworts To enable a reliable comparison of all available plastomes Marchantiopsida, represented by Marchantia, as the sister to of liverworts, we extracted the following information: Jungermanniospida. The latter includes the leafy liverwort genome size (kb), GC‐content, the length of the main orders Jungermanniales (Bazzania, Calypogeia, Heteroscyphus, structural features, namely the large single copy (LSC), the Plagiochila, Scapania, and Schistochilia), Ptilidiales (Ptilidium), small single copy (SSC), and the length of the two inverted and Porellales (Cheilolejeunea, Cololejeunea, Frullania, Jubula, repeats (IR), and the number of genes, proteins, rRNAs, Porella, and Radula), and the simple thalloid Metzgeriales tRNAs, and pseudogenes. The number and total length of (Aneura) and Pelliales (Apopellia) (see Table S1). introns were estimated without and with the separation into introns located in genes and those located in tRNAs. The identity of pseudogenes and lost genes were identified. Using the assembled genomes we identified putative lost 3 Results genes and determined gene arrangements and their loca- The 18 liverwort plastid genomes showed a genome 1.2‐fold tions. Finally, we determined the number of putative RNA range variation with a minimum size of 108 007 kb found in editing sites per genome (Table S1). Correlations among the non‐photosynthetic Aneura mirabilis and a maximum size these parameters were explored by using Pearson’s correla- of 128 728 kb found in the terrestrial Haplomitrium blumei tion coefficient reporting r2‐values and permutated P‐values. (Nees) R.M. Schust. (see Table S1). The plastome of the latter To enable visual comparison, the plastid genomes were species also shows the highest GC‐content, with 44.4%, drawn in linear shape using OrganellarGenomeDRAW (Lohse whereas the lowest GC‐content (28.9%) was found in the et al., 2013). plastid genome of the complex thalloid Marchantia poly- morpha. In total, the 18 liverwort plastid genomes showed a 2.3 Comparing liverwort plastid genomes in the context of 1.54‐fold variation in GC‐content. All liverwort plastid land plant phylogeny genomes showed a highly conserved genome structure To compare the observed variation among the plastid including the LSC, IRA, SSC, and IRB (Fig. 1). Only a few genomes of liverworts, we compiled a dataset based on all plastids showed evidence for genome rearrangements, plastid DNA genomes available in GenBank at the end of namely the plastid genome of H. blumei having a rearranged February 2018 (https://www.ncbi.nlm.nih.gov/genome/ SSC as well as a duplicated nadF, and the plastid genome of organelle/). We extracted the information reported in the the non‐photosynthetic A. mirabilis, which has a modified LSC summary reports, namely genome size, GC‐content, number as a consequence of the deletion of six genes (see Table S1). of proteins, number of rRNAs, number of tRNAs, total Gene deletion was also found in the plastid genome of five number of genes, and number of pseudogenes. In cases with other liverworts, namely one gene in A. pinguis and H. blumei, more than one sequence available per species, we estimated two genes in Ptilidium pulcherrimum and Cheilolejeunea mean values. These data were used to characterize the xanthocarpa (Lehm. & Lindb.) Malcombe, and six genes in variation of genome size and GC‐content for each of the Cololejeunea lanciloba Steph. (Table S1). The increased major lineages of land plants, namely liverworts (18 spp.), number of lost genes in the last species was likely the

J. Syst. Evol. 57(4): 382–394, 2019 www.jse.ac.cn Liverwort plastome diversity 385

Fig. 1 Continued.

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Table 2 Correlations between main characteristics of liver- mirabilis was contributed by the loss of six genes, the wort plastid genomes reduction in Cololejeunea was contributed mainly by the deletion of a region ranging from nadF to ccsA. A reduction Statistics r2 P‐values (perm) of the plastome length was also found in the closest sampled GS/GC 0.0631 0.2964 relatives, the epiphyllous Cheilolejeunea xanthocarpa (117 138 GS/LSC 0.8333 0.0001 kb). The two Lejeuneaceae species shared the deletion of GS/IR 0.577 0.0014 cysA and cysT but also a reduction of the total length of GS/SSC 0.6142 0.0035 introns that are located in genes (Table S1). Length reduction GS/LGI 0.8262 0.0001 of introns was also found in A. pinguis and in Radula japonica GS/LtRNAI 0.0866 0.2184 Gotsche ex Steph. Among the plastid genomes of liverworts GS/RNAES 0.0252 0.2583 observed, H. blumei had the largest total length of introns GC/LSC 0.297 0.0247 with 7412 bp, whereas the smallest total length of introns GC/IR 0.0244 0.5263 was found with 6723 bp in Scapania ciliata Sande Lac. (Table GC/SSC 0.0306 0.4993 S1). In addition to Haplomitrium, a total intron length of more GC/LGI 0.1152 0.1735 than 7000 bp was found in the six species of Porellales. GC/LtRNAI 0.0537 0.3382 The majority of land plants had plastid genomes with a GC/RNAES 0.0453 0.3348 length of 134 000 kb and 196 000 kb (GSL in Figs. 2, S1), but LGI/LtRNAI 0.1692 0.0936 liverworts and the majority of mosses showed plastid LGI/RNAES 0.0593 0.1466 genomes with a length below 134 000 kb. However, the LtRNAI/RNAES 0.0198 0.5315 plastid genome length was within the range of the majority LSC/RNAES 0.0341 0.3209 of land plants in hornworts and the two basal lineages of SSC/RNAES 0.0063 0.6945 mosses represented by and Sphagnum (Fig. 2). These IR/RNAES 0.0130 0.4548 results suggested either independent genome size reduc- Correlations were tested between two of the following tions in liverworts and derived mosses or a small genome parameters: length of the plastid genome (GS), GC‐content size in the common ancestor of the two lineages. The latter (GC), length of long single copy section (LSC), length of the scenario includes the proposal of independent expansions of two inverted repeats (IR), length of small single copy section the genome size in some early diverging bryophyte lineages. (SSC), total length of the introns nested in proteins (LGI), In angiosperms, small plastid genomes (<130 000 bp) were total length of introns nested in tRNAs (LtRNAI), and found exclusively in parasitic species, whereas slightly estimated number of putative RNA editing sites (RNAES). reduced genome sizes were the consequence of genome The r2 and permutated (perm) P‐values are reported for rearrangements (Fig. 3). Among land plants, only angios- each. R2‐values >0.5 and P‐values <0.005 are considered perms contained species with a plastid genome size above significant. 175 000 bp. In ferns and acrogymnosperms opposite evolutionary trends exist (Fig. 3). A trend towards expanded consequence of two deletion events, one effecting cysA‐cysT plastome size was observed in ferns, whereas a trend and the other one effecting the region ranging from nadF to towards reduced plastomes was found in . Strepto- ccsA. In total, the number of protein coding genes (mean, phytic algae showed a wide range of genome size variation 80.7; minimum, 64; maximum, 89), rRNA (always 8), and (Fig. 4). tRNA (mean, 36; minimum, 35; maximum, 38) showed only a Liverworts showed a rather notable variation in the GC‐ limited variation as a consequence of genome structural content of the plastome (Fig. 2, GCC). The majority of conservatism. In contrast, the number of pseudogenes and liverworts showed relatively low GC‐content (28.8%–35.9%), putative RNA editing sites were more variable (Table S1). but Aneura (40.0%–40.6%) and Haplomitrium (44.4%) showed Genome size was correlated with the size of the LSC (P = much higher GC‐content (Table S1). Differentiation of the GC‐ 0.0001), IR (P = 0.0014), and SSC (P = 0.0035) (Table 2). The content was also observed in mosses, in which Takakia length of the plastome was correlated with the length of (37.1%) and Sphagnum (36.9%) showed higher content than introns nested in genes (P = 0.0001), but not with the length other mosses (28.4%–29.4%). The two species with of introns nested in tRNAs (P = 0.2184). The variation of GC‐ sequenced plastomes differed in the GC‐content (32.9% and content was not correlated with genome size (P > 0.01), 35.0%) and plastome length (153 206 bp and 161 162 bp). The number of RNA editing sites, or any other parameters tested plastid genome of Haplomitrium was only second in the GC‐ (Table 2). Comparing the genome size among liverworts, it content to the champion of the GC‐content among land was notable that small genomes with a length approximately plants, the lycophyte genus Selaginella (GCC = 50%–51%). 108 000 kb were not only found in the non‐photosynthetic A. Another remarkable trend was observed in leptosporangiate mirabilis (108 007 kb), but also in the epiphyllous liverwort C. ferns that show a trend towards an increase of the GC‐ lanciloba (108 673 kb). Whereas the size reduction in A. content during their diversification (Fig. 5). The GC‐content of

Fig. 1. Synteny of plastid chromosomes of 18 liverworts organized according to their phylogenetic relationships. The plastid genomes are shown in linearized form illustrating the relative gene synteny. Solid line connects the end of the long single copy, whereas the colored bars represent proteins, rRNA, and tRNA regions. Names of the regions are displayed above and below the graphics. The sequence of the regions is highly conserved across the phylogeny of liverworts. The depicted phylogeny was obtained by pruning a liverwort phylogeny obtained using complete chloroplast genome sequences (Yu et al., unpublished data).

J. Syst. Evol. 57(4): 382–394, 2019 www.jse.ac.cn Liverwort plastome diversity 387

Fig. 2. Evolutionary scenarios of the genome size (GSL) and GC‐content (GCC) evolution in liverworts based on a consensus phylogeny. The results are based on the analyses of a dataset including the plastid genomes of 18 liverworts (LI),8mosses(MO),2 hornworts (HO), 6 lycophytes (LY), 60 ferns (FE), and seed plants (SP) including 93 acrogymnosperms and 2120 angiosperms. Among liverworts, the following major lineages are marked: Ha = Haplomitriopsida, Ju = Jungermaniopsida, and Ma = Marchantiospida. The dataset has an outgroup rooting with 17 streptophytic algae (Charales, Chlorokybophyta, Coleeochaetales, Desmidiales, Klebsormidiophyta, Mes- ostimgatophyceae, and Zygnematales) and 50 chlorophytic algae (). Branches colored in green show values within the range found in the majority of land plants namely for GSL with a range from 134 to 196 kb and a GCC range from 34.0% to 39.7%. Blue colored branches had estimated values below the majority range value (<134 kb and <34%, respectively), whereas red colored branches had estimated values above themajorityrangevalue(>196 kb and >39.7%, respectively). Instead of individual species, the lineages are annotated according to orders for the three lineages of bryophytes or higher taxonomic tanks for other land plants. Inferred decrease/increase events are marked by blue “<” or red “>”, respectively. some plastomes was similar to the one recovered in the million years of independent phylogenetic history of liver- Haplomitrium plastome. Similar to plastid genome size, the worts. Both GC‐content and genome size varied as predicted streptophytic algae showed a notable variation (Fig. 6). as a consequence of changes effecting the length of introns, deletions of short plastid genome regions, and the frequency of RNA editing. 4 Discussion 4.1 Taxon sampling quality By generating new complete chloroplast genomes of The study increased the sampling of liverwort plastid liverworts and the interpretation of these new evidence in genomes from five to 18 species. This increased the species the most comprehensive sampled framework available, we coverage to 0.25% of liverwort species having one plastome were able to explore several hypothetical frameworks sequenced, but this is still below the average of plastid outlined above. We found no evidence for major reorganiza- genomes available for land plant species of 0.73% (Table 1). tion of the plastid genome structure during the ca. 500 Together with its sister lineage the mosses (0.07%), www.jse.ac.cn J. Syst. Evol. 57(4): 382–394, 2019 388 Yu et al.

Fig. 3. Phylogenetic distribution of plastid DNA sequence length (GSL) of the dataset. Branches colored in green show values within the range found in the majority of land plants namely for GSL with a range from 134 to 196 kb and a GCC range from 34.0% to 39.7%. Blue colored branches had estimated values below the majority range value (<134 kb and <34%, respectively), whereas red colored branches had estimated values above the majority range value (>196 kb and >39.7%, respectively). Blue colored branches occur preferences in four lineages, namely chlorophytic algae, streptophytic algae, mosses, and liverworts but are also found among seed plants. A notable small plastid genome size is also shown by horsetails (). Reduction of genome size was found in and some legumes as a consequence of genome rearrangements or in parasitic angiosperm clades as the consequence of gene loss. Red colored branches are also found in chlorophytic algae, streptophytic algae, and liverworts, but characterize mainly the lineage and . A few angiosperm clades also show high GC content values. liverworts rank lowest in the coverage per species be better covered in all three criteria with acrogymnos- (Table 1). The phylogenetic coverage was assessed by perms having the best coverage in each of the three criteria determining the number of families and orders represented (Table 1). Lycophytes have similar excellent coverage in the in our sampling. Our sampling covered 18.4% of the order and family coverage criteria but the reliability of the liverwort families and 46.7% of the liverworts orders. Both observed trend is constrained by the limited taxon values are below the average of land plant families (46.2%) sampling. Ferns show a declining trend in the taxon and orders (68.9%) covered (Table 1). Again, mosses and coverage from order to family to species (Table 1). With liverworts are among the lineage with the lowest coverage the recent publication of the first plastome sequence of a but are joined by hornworts in the low coverage of their filmy fern (Kuo et al., 2018), ferns have remarkably good deep node diversity (= order level). Vascular plants tend to coverage, with all orders having at least one complete

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Fig. 4. Distribution of the plastid DNA sequence length variation based on the dataset assembled. The x‐axis shows plastid DNA length ranging from 10 kb to 300 kb separated into step classes of 5 kb. The y‐axis shows the number of sequences belonging to length classes separated for the main lineages of land plants plus the streptophytic algae. plastid genome available. The relatively good coverage of plant plastomes, attention should not only be given to the angiosperms in all three criteria is notable given the number three lineages of bryophytes but also to selected lineages of species required to be covered. Angiosperm orders of angiosperms. Efforts focusing on the phylogenomics of lacking at least one accessible plastome, namely Berber- economically important lineages, such as the grape genus idopsidales (4 spp.), Boraginales (3095 spp.), Bruneales (79 Vitis (Wen et al., 2018) or the birch family Betulaceae (Yang spp.), Crossomatales (66 spp.), Escallonicales (130 spp.), et al., 2019), will enable us to fill some of the crucial gaps in Mettentiusales (55 spp.), Paracryphyalles (36 spp.), and our current understanding of the evolution of plastid Picramniales (49 spp.), all belong to the eudicot lineages genomes. asterids and rosids. With the exception of Boraginales they tend to be rather species‐poor and often have rather small distribution ranges. However, some orders have notably 4.2 Evolutionary trends in liverwort plastomes compared to poor coverage, such as Oxalidales with only one sampled other land plants out of 1815 species, and Fabales with 98 sampled species The newly generated liverwort plastid genomes sup- representing only the Fabaceae and data are missing for the ported the hypothesis of a remarkable conservatism of remaining three families including Polygalaceae. Thus, to the plastid genome structure among land plants plus their achieve an excellent coverage of the phylogeny of land closest relatives, the streptophytic algae (Wicke et al., 2011; Daniel et al., 2016; Zheng et al., 2017; Mower & www.jse.ac.cn J. Syst. Evol. 57(4): 382–394, 2019 390 Yu et al.

Fig. 5. Phylogenetic distribution of the GC‐content (GCC) of the dataset. Branches colored in green show values within the range found in the majority of land plants, namely for a plastid DNA sequence length with a range from 134 to 196 kb and a GCC range from 34.0% to 39.7%. Blue colored branches had estimated values below the majority range value (<134 kb and <34%, respectively), whereas red colored branches had estimated values above the majority range value (>196 kb and >39.7%, respectively). Blue colored branches occur in four lineages, namely chlorophytic algae, streptophytic algae, mosses, and liverworts but are also found in a few derived clades of angiosperms. Red colored branches are also found in chlorophytic algae and streptophytic algae but are rather exclusive among land plants. Increased GCC characterizes ferns, especially leptosporangiate ferns, the heterosporous lycophyte lineage Selaginellales, most cycads, and among liverworts, the Haplomitriaceae and Aneuraceae. A few angiosperm clades also show high GCC values.

Vickrey, 2018). This conservatism is most obvious in the genomes smaller than 120 000 bp are contributed mostly widespread occurrence of the four‐partition structure of by non‐photosynthetic angiosperms that show deletion plastidgenomes:LCS,IRA,SSC,andIRB.Aconsequence of several genes during their diversification (Wicke et al., of the conserved structure is arguably the limited 2011, 2016). A similar pattern is found in acrogymnos- variation of the plastome length and GC‐content among perms. Some acrogymnosperms, such as Ginkgo and most land plants (Figs. 3–6). For example, the majority of cycads, possess plastid genomes of a similar size as the angiosperms (ca. 70%) share the four‐partitioned DNA majority fraction of angiosperms, whereas most conifers structure, with plastomes of a length between 150 000 bp show smaller sized plastomes. The variation of the and 165 000 bp, whereas a second fraction (17.2%) is plastome size in gymnosperms is arguably the conse- contributed by species with rearrangements of the quence of expansion of the IR and/or the reduction of the genome structure having genome sizes between length of non‐coding DNA regions (Wu & Chaw, 2014; 130 000 bp and 145 000 bp (mostly Poaceae). Plastid Chaw et al., 2018; Mower & Vickrey, 2018).

J. Syst. Evol. 57(4): 382–394, 2019 www.jse.ac.cn Liverwort plastome diversity 391

Fig. 6. Frequency distribution of the GC‐content (GCC) based on the dataset assembled. The x‐axis shows GCC ranging from 20% to 51% seated in step classes of 1%; the y‐axis shows the number of sequences belonging to GCC classes. The number of complete plastome sequences belonging to each GCC is given for each lineage of land plants and the streptophytic algae. Lineages with extremely high GCC values include the leptosporangiate (Lepto.) ferns, and the heterosporous lycophyte Selaginellaceae among vascular plants, and Haplomitrium and Aneura among liverworts genera. It is also worth noting that the two genera Takakia (Tak) and Sphagnum (Sph) show GCC comparable to other land plants, whereas the remaining mosses show highly reduced GCC. Hornworts and liverworts show notable variation in their GCC.

Liverworts and the majority of mosses are distinct from genomes. Furthermore, streptophytic algae show a remark- non‐parasitic vascular plants by their smaller plastome size. able variation of their plastid genome size, ranging from The reduced sequence length in liverworts is not the approximately 90 000 bp up to nearly 210 000 bp (Fig. 3). consequence of genome rearrangements or gene loss but Among land plants, only angiosperms possess plastid likely the consequence of reduced length of introns. The genomes exceeding the length of 170 000 bp, but they are trend might have evolved independently from mosses rare (1.4% of all angiosperm species) and, as far as known, because the two taxa representing basal moss lineages, restricted to the magnoliid family Annonaceae and the rosid namely the genera Takakia and Sphagnum, possess larger family Geraniaceae (Fig. 3); the latter include the largest genomes. Thus, the trends towards smaller genomes are genomes as a consequence of genome rearrangements likely independent in the two Setophyta lineages (Fig. 2). (Ruhlman & Jansen, 2018). Frequent genome rearrange- However, this hypothesis needs further study by increasing ments could cause substantial length difference among the sampling of mosses. It is worth noting that the hornwort closely related species, as recently shown for the plastid plastid genomes are similar in their size to the majority genome of Passiflora (Rabah et al., 2018). The observed fraction of angiosperms, but again this needs to be variation in the length of plastomes is arguably partly confirmed by expanding the sampling of hornwort plastid contributed by changes in gene content (Zheng et al., 2017) www.jse.ac.cn J. Syst. Evol. 57(4): 382–394, 2019 392 Yu et al. and partly by the length of non‐coding DNA. Similar trends 5 Conclusions were detected in the evolution of the plastid genome GC‐ content (Fig. 5). The majority of land plants shows a GC‐ The increase of the sampled liverwort plastid genomes fi content ranging from 34% to 39.7% (Fig. 6). Lower GC‐content con rmed the prediction that the plastid genome structure is was found in some angiosperms, hornworts, and mosses. highly conserved across the phylogeny of land plants. During fi However, the GC‐content is within the range of the majority their diversi cation, both liverworts and mosses might have of land plants in the two most basal lineages of mosses, experienced a reduction of the genome length as a ‐ namely Takakia (37.1%) and Sphagnum (36.9%). Two liver- consequence of constraints on the length of non coding worts, Aneura (40.2%–40.62%) and Haplomitrium (44.4%) DNA regions. Currently we are lacking a reliable hypothesis showed GC‐content above the values found in the majority to explain the observed genome size reduction. Future of land plants. Notably, Haplomitrium has one of the highest studies could focus in particular on the reduction of the GC‐contents observed until now. Similar high GC‐contents plastome length in adaptation to epiphyllic habitats because were found in leptosporangiate ferns, which tend to taxa with this life strategy show evidence of enhanced accumulate high GC‐content, and the heterosporous lyco- reduction of the plastid genome length. Another intriguing phyte genus Selaginella (Figs. 5,6). The latter has the highest question is whether the conservatism observed in plastid GC‐content found so far in land plants (50%–51%). genomes of liverworts coincides with the conservatism of Ś Compared to genome size and structure, the GC‐content the mitochondrial genomes ( lipiko et al., 2017) and perhaps changes are arguably the most prominent (Fig. 6). This also the nuclear genome. Furthermore, the evolution of the pattern might be caused by the additive effects of intron plastid genome structure of liverworts in arguably the fl length (see above), small structural deletions, and amino acid majority of land plants does not re ect recent changes in fi composition bias (see Cox, 2018). Here, we argue that this the diversi cation rates of these plants (Laenen et al., 2014). pattern needs further investigation using a denser taxon Given the relative stability of the plastid genome structure sampling and analyses focusing specifically on the GC‐ across land plants, the evolutionary rate of the plastid fi content of the genes and pseudogenes in the plastid genomes may be uncoupled to species diversi cation rate genomes. during the phylogenetic history of liverworts as well as other Similar to genome size, the streptophytic algae showed a land plants. notable variation of the GC‐content, ranging from 27.64% in Coleochaete to 42.2% in Mesotaenium (Fig. 5). These trends need to be taken into account in studies aiming to Acknowledgements reconstruct the relationships of the sister lineages of land We thank colleagues at KIB, NHM, and XTBG for sharing plants because biases in the base‐pair composition need to materials and comments. The authors acknowledge the be considered in the models of sequence evolution applied financial support by the Chinese National Natural Science (Cox et al., 2014). Foundation to YY (No. 31500182) and YHW (No. 41571049), The newly generated complete chloroplast genomes the Large‐scale Scientific Facilities of the Chinese Academy of provided further support to the hypotheses of frequent Sciences to JY (No. 2017‐LSF‐GBOWS‐02), and NERC (“Origin pseudogenization of the plastid‐encoded sulfate transport of Land Plants: Rocks, Genomes, and Geochemical Cycles”) gene cysA in liverworts (Wickett et al., 2008, 2011). In total, to HS. 72% of the assembled liverwort plastid genomes showed pseudogenization of this gene. For the first time, the pseudogene cysT was found in Haplomitrium, which adds References to the previous report of the pseudogene in Treubia (Wickett et al., 2011). Thus, the gene might be pseudogenized in all Chaw SM, Wu CS, Sudianto E. 2018. Evolution of plastid – Haplomirtriopsida. The new data also confirmed the putative genomes. Advances in Botanical Research 85: 195 222. fi xation of the pseudogenization in Porellales (see Wickett Civáň P, Foster PG, Embley MT, Seneca A, Cox CJ. 2014. Analyses of et al., 2011) and the repeated loss of the gene function in charophyte chloroplast genomes help characterize the ancestral Jungermanniales. chloroplast genome of land plants. Genome Biology and Evolution So far, no convincing evidence exists that the plas- 6: 897–911. tomes of land plants are structurally rearranged com- Cox CJ. 2018. Land plant : A review with pared to their closest living relatives the Zygnematophy- comments on evaluating incongruence among phylogenies. ň ceae (Civá et al., 2014). Thus, the colonization of land did Critical Reviews in Plant Sciences 37: 1–15. not trigger major changes in the composition of plas- ň fl tomes during the initial radiation of land plants. These Cox CJ, Li B, Foster PG, Embley MT, Civá P. 2014. Con icting results suggest that the genome structure and gene phylogenies for early land plants are caused by composition biases among synonymous substitutions. Systematic Biology 63: content have been highly conserved during the evolution 272–279. of land plants, whereas plastome evolution differs substantially in terrestrial environments compared to Daniel H, Lin CS, Yu M, Chang WJ. 2016. Chloroplast genomes: the plastome evolution in aquatic (Smith, Diversity, evolution, and applications in genetic engineering. 2017). 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gene content, gene order, gene function. Plant Molecular Biology Supplementary Material 76: 273–297. The following supplementary material is available online Wickett NJ, Forrest LL, Budke JM, Shaw B, Goffinet B. 2011. Frequent for this article at http://onlinelibrary.wiley.com/doi/10.1111/ pseudogeneization and loss of the plastid‐encoded sulfate‐ jse.12515/suppinfo: transport gene cysA throughout the evolution of liverworts. Table S1. Summary of the plastid genomes of 18 liverworts. – American Journal of Botany 98: 1263 1275. Information provided from left to right for rows 2 to 19: fi Wickett NJ, Zhang Y, Hansen SK, Roper JM, Kuehl JV, Plock SA, Wolf Genbank Accession Numbers of ve previously released PG, dePamphilis CW, Boore JL, Goffinet B. 2008. Functional gene liverwort plastid genomes and accession numbers of the 13 losses occur with minimal size reduction in the plastid genome newly generated plastid genome sequences. Classification of parasitic liverwort Aneura mirabilis. Molecular Biology and of these liverworts was given as lineage, order, family, and Evolution 25: 393–401. taxon (genus & species) following Söderström et al. Wu CS, Chaw SM. 2014. Highly rearranged and size‐variable (2016). The following columns provide the basic informa- chloroplast genomes in conifers II clade (cupressophytes): tion about each of the plastid genome sequences namely: Evolution towards shorter intergenic spacers. Plant Biotech- plastid genome size in kb, GC‐content, length of the large nology Journal 12: 344–353. single component (LSC) in kb, the length of the inverted repeat (IR), length of the small single component (SSC), Wyman SK, Jansen RK, Moore JL. 2004. Automatic annotation of number of genes (Genes), number of proteins (Proteins), organelles genomes with DOGMA. Bioinformatics 20: 3252–3255. number of rRNAs (rRNA), Number of tRNA (tRNA), total Yang JB, Li DZ, Li HT. 2014. Highly effective sequencing whole number of Introns (Introns), number of introns located in chloroplast genomes of angiosperms by nine novel universal protein coding genes (NGI), total length of introns located – primer pairs. Molecular Ecology Resources 14: 1024 1031. in protein coding genes (LGI) in kb, number of introns Yang XY, Wang ZF, Luo WC, Guo XY, Zhang CH, Liu JQ, Ren GP. 2019. located in tRNAs (NItRNA), total length of introns located Plastomes of Betulaceae and phylogenetic implications. Journal in tRNAs (LtRNAI), annotated pseudogenes (cysA, cysT are of Systematics and Evolution https://doi.org/10.1111/jse.12479. highlighted, annotated loss of genes, annotated genome arrangements (GA), number of RNA editing sides Zheng XM, Wang JR, Feng L, Liu S, Pang HB, Qi L, Li J, Sun Y, Qiao WH, Zhang LF, Cheng YL, Yang QW. 2017. Inferring the (NRNAES). Raws 20 to 23 provide the following summary evolutionary mechanisms of the chloroplast genome size by statistics as applicable: mean value, minimum value, ‐ comparing whole‐chloroplast genome sequences in seed plants. maximum value, x fold value. * Values based on Grosche Scientific Reports 7: 1555. et al. (2012).

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