Journal of Systematics JSE and Evolution doi: 10.1111/jse.12479 Research Article Plastomes of Betulaceae and phylogenetic implications † † Xiao-Yue Yang1 , Ze-Fu Wang2 , Wen-Chun Luo1, Xin-Yi Guo2, Cai-Hua Zhang1, Jian-Quan Liu1,2, and Guang-Peng Ren1* 1State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, Lanzhou University, Lanzhou 730000, China 2Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610000, China † These authors contributed equally to this work. *Author for correspondence. E-mail: [email protected] Received 4 July 2018; Accepted 16 November 2018; Article first published online31 xx December Month 2019 2018 Abstract Betulaceae is a well-defined family of Fagales, including six living genera and more than 160 modern species. Species of the family have high ecological and economic value for the abundant production of wood. However, phylogenetic relationships within Betulaceae have remained partly unresolved, likely due to the lack of a sufficient number of informative sites used in previous studies. Here, we re-investigate the Betulaceae phylogeny with whole chloroplast genomes from 24 species (17 newly assembled), representing all genera of the family. All the 24 plastomes are relatively conserved with four regions, and each genome is 158–161 kb long, with 111 genes. The six genera are all monophyletic in the plastome tree, whereas Ostrya Scop. is nested in the Carpinus clade in the internal transcribed spacer tree. Further incongruencies are also detected within some genera between species. Incomplete lineage sorting and/or hybrid introgression during the diversification of the family could account for such incongruencies. Our dating analysis, based on four fossils, suggests that the most recent common ancestors of the extant genera date back to the mid- to late Miocene, and confirms that Betulaceae started to diversify in the upper Cretaceous/early Paleocene. Our results highlight the significance of using more informative sites in resolving phylogenetic relationships. Plastome data and increased taxon sampling will help to better understand the evolutionary history of Betulaceae in the future. Key words: Betulaceae, chloroplast genome, divergence time, fossil calibration, phylogenomics. 1 Introduction easier to sequence the entire plastome, phylogenetic studies using plastomes in plants are still relatively rare. Here, we Accurate and comprehensive phylogenetic reconstructions present a phylogenetic study based on plastomes of 33 can help to understand the origin of life, biotic diversification Fagales species, representing all the six genera of Betulaceae, and evolutionary history (Janko et al., 2003; Fry & Wuster,€ and related taxa. 2004; Pramual & Nanork, 2012; Wen et al., 2016). However, due Betulaceae, a morphologically well-defined family of to the complex evolutionary histories (e.g., hybrid introgres- Fagales, consists of six living genera and more than 160 sion, incomplete lineage sorting, polyploidization, and natural modern species (Christenhusz & Byng, 2016). Most species of selection) of related groups, phylogenetic studies often the family are distributed in the Northern Hemisphere, and a encounter low resolution and inconsistencies of phylogenetic few occur in Africa or South America (Kubitzki, 2004). As trees relationships between gene trees, especially if the number of or shrubs, species of Betulaceae have high ecological and informative sites of sampled fragments (e.g., a few plastid or economic value for the production of wood (Abbe, 1974; nuclear DNA markers) is low. This phenomenon is commonly Normand & Bousquet, 1989; Furlow, 1990; Salojarvi€ et al., found in numerous groups (e.g., Zhang et al., 2011; Xu et al., 2017). The six genera of Betulaceae are divided into two 2012; Ren et al., 2015; Mallet et al., 2016; Zeng et al., 2017). The subfamilies: Coryloideae (Corylus L., Ostryopsis Decne., whole chloroplast (cp) genome (plastome), containing more Carpinus L. and Ostrya Scop.) and Betuloideae (Alnus Mill. informative sites, has been recently suggested to give higher and Betula L.) (Takhtajan, 1980; Heywood, 1993; Thome, 2010). resolution of phylogenetic relationships compared with Although the phylogenetic relationships among or within the traditional DNA markers (Erst & Vaulin, 2014; Hu et al., genera of Betulaceae have been extensively examined (Chen, 2016c; Zeng et al., 2017). Furthermore, well-resolved cpDNA 1994; Kato et al., 1999; Forest & Bruneau, 2000; Whitcher & phylogenies could greatly facilitate clarification of reticulate Wen, 2001; Chen & Li, 2004; Jarvinen et al., 2004; Li et al., 2007; evolution during species diversification (Jansen et al., 2007; Xu Erdogan & Mehlenbacher, 2009; Lu et al., 2014), phylogenetic et al., 2012). Despite the fact that high-throughput sequencing relationships among genera have remained ambiguous in methods have recently become cost-effective and it is now some clades. In most studies, Ostryopsis was placed between ©XXX 2018 2019 Institute | Volume of Botany, 9999 |Chinese Issue 9999 Academy | 1– 11of Sciences © 2019 InstituteSeptember of Botany, 2019 | Volume Chinese 57 Academy | Issue 5 of | 508–518 Sciences 2Phylogenetics Yang of et Betulaceae al. 509 Corylus and Carpinus–Ostrya clades, whereas several studies the mapped reads for the subsequent analyses. We then found Ostryopsis as a sister to Corylus (Yoo & Wen, 2002; constructed a joint pipeline to assemble the plastomes using Larson-Johnson, 2016). The most recent phylogenetic study of Velvet version 1.2.04 (Zerbino & Birney, 2008), NOVOPlasty this family, by Grimm & Renner (2013), using 46 species and version 2.5.9 (Dierckxsens et al., 2017) and Platanus version 9300 nucleotides of nuclear and plastid DNA, supported the 1.2.1 (Kajitani et al., 2014). All of the resulting contigs were two proposed subfamilies and found Ostryopsis as sister to aligned to the reference Juglans regia L. plastome sequence Ostrya/Carpinus, with Carpinus being nested in Ostrya. To our (NC_028617) (Hu et al., 2016d), using Bwa version 0.7.12 (Li, knowledge, all of the former phylogenetic studies were 2014) and Samtools (Li et al., 2009), respectively. Geneious undertaken with only a few DNA fragments and/or a small version 9.1.8 (Kearse et al., 2012) was used to manually check number of species (Bousquet et al., 1992; Chen et al., 1999; the assembled contigs and to further process them into a Yoo & Wen, 2002; Li, 2008; Ma et al., 2015). At present, an consensus linear sequence. After filling the gaps with increasing number of plastomes have been published, shared GapCloser (Luo et al., 2012), we obtained the complete and widely used in phylogenetic analysis, and several plastome sequences. Finally, we used Plann version 1.1.2 plastomes of Betulaceae have been published (Hu et al., (Huang & Cronk, 2015) to annotate plastomes and Sequin 2016a, 2016b, 2017; Salojarvi€ et al., 2017). Therefore, it is version 15.10 (http://www.ncbi.nlm.nih.gov/Sequin/) to map possible for us to undertake a comprehensive phylogenetic the predicted genes to the reference annotation. The visual analysis of Betulaceae at a genomic-level scale. Moreover, images of the annotations were generated by OGDRAW phylogenetic trees combined with the relatively good fossil version 1.1 (Lohse et al., 2013) (http://ogdraw.mpimp-golm. record for the family (Table S1; Forest et al., 2005; Grimm & mpg.de/). The 17 newly assembled plastomes were deposited Renner, 2013) could provide good means for molecular dating in GenBank under the accession numbers KY312849 and analyses. In this study, we took advantage of the Betulaceae MG386363-MG386378 (Table S2). plastomes now available in GenBank and newly assembled 17 additional plastomes of Betulaceae, representing all the six 2.3 Sequence variation within Betulaceae genera, to reconstruct a comprehensive phylogenetic tree of this We first estimated the variation level of Betulaceae species family. Specifically, we aimed to address the following questions: using mVISTA (Frazer et al., 2004) (http://genome.lbl.gov/ (i) Do the plastome datasets increase phylogenetic resolution in vista/mvista/submit.shtml) with Betula pendula Roth as comparison to traditional DNA markers? (ii) Do plastome data reference. We then extracted the coding sequences for and nuclear internal transcribed spacer (ITS) data yield protein-coding genes (PCGs) of the 24 species from the topologically congruent trees? and (iii) Do plastome datasets GenBank format files using a custom Perl script and assessed facilitate our understanding of divergence in Betulaceae? their variation. After stop codons were removed, PRANK (Loytynoja€ & Goldman, 2008) was used to carry out the multiple alignment for each gene in each genus, and the whole family. Finally, we estimated the variation ratios for the coding 2 Material and Methods regions with a custom Perl script. 2.1 Taxon sampling and data description We used a total of 33 plastomes including 24 species of 2.4 Sequence alignment Betulaceae that represented all the six genera of the family For the multiple alignment of 33 plastomes, MAFFT version and nine outgroup species chosen from Fagales (Table S2). We 7.221 (Katoh & Standley, 2013) was used under the default downloaded the plastomes of seven Betulaceae species and settings. Ambiguous alignment regions were trimmed by nine outgroups from GenBank, and the other 17 species of Gblocks 0.91b (Castresana, 2000). We also took the multiple Betulaceae were newly