The complete chloroplast genome of Fagus crenata (subgenus Fagus) and comparison with F. engleriana (subgenus Engleriana)

Introduction

The genus Fagus is a major tree of temperate forests of the northern hemisphere with two informal subgenera recognized (Shen, 1992): Engleriana with three species and Fagus with seven species (Oh, 2015; Renner et al., 2016). The genus has been the focus of intensive genetic studies over the last 30 years enabling insights into relationships of the extant species (Denk, Grimm & Hemleben, 2005), the impact of the interglacial-glacial cycles on extant genetic diversity (Fujii et al., 2002; Magri et al., 2006) and predictions of the impacts of ongoing climate change (Csilléry et al., 2014). However, despite the significance of the genus there remains a dearth of Next Generation Sequencing based-genetic resources for Fagus, including for the chloroplast genome, with the whole chloroplast genome of only a single species, the Chinese endemic F. engleriana of subgenus Engleriana (Yang et al., 2018), so far published.

This study reports the whole chloroplast genome of the Japanese endemic Fagus crenata, the first reported of subgenus Fagus. This species is a foundation tree of Japan’s cool temperate forest ecosystem and is distributed widely from the mountains of southern Kyushu (31.4° N 130.8° E) to southern Hokkaido (42.8° N 140.2° E). Phylogeographic studies based on Sanger sequencing of small portions of the chloroplast genome have revealed strong geographic structuring of chloroplast haplotypes (Fujii et al., 2002; Okaura & Harada, 2002) that, combined with fossil pollen data (Tsukada, 1982), suggests the species persisted in multiple coastal refugia and has occupied most of its current wide geographic range in the postglacial. Here we report the whole chloroplast genome sequence of F. crenata and compare it to the genome of F. engleriana (subgenus Engleriana). This data will be a useful genetic resource for investigating the phylogenetic relationship of Fagus and for developing chloroplast-based genetic markers, including both single nucleotide polymorphism- and microsatellite-based markers.

Materials and Methods

Results and Discussion

The assembled whole chloroplast genome of F. crenata has a total of 158,227 bp (Fig. 1: Genbank accession number MH171101) and consisted of an 87,557 bp large single copy region, a 18,928 bp small single copy region and two inverted repeats 25,871 bp in length. The genome contained 111 genes, including 76 protein-coding genes, 31 tRNA genes and 4 ribosomal RNA genes (see DatasetS1 for the genbank file of the chloroplast genome). The Gblocks alignment consisted of 143,882 bp of non-gapped sequence of which 11.48% of sites were variable (see DatasetS2 for Gblocks alignment). The resulting best ML tree had similar relationships to previous studies with Fagus as sister to all other Fagaceae (Manos & Steele, 1997) (Fig. 2). Fagus crenata and F. engleriana formed a strongly diverged clade consistent with previous evidence of the large divergence of Fagus from all other Fagaceae genera (Heenan & Smissen, 2013). The proportion of nucleotide sites that differed (p-distance) between F. crenata and F. engleriana was 0.0018 which was lower than any other pairwise differences observed including between five Quercus species which had values between 0.0035 and 0.0047 (average = 0.0042) (see DatasetS3 for a matrix of p-distances).

The two Fagus chloroplast genomes were relatively conserved (Fig. 3) with the IR region more conserved than both the large single copy (LSC) and small single copy (SSC) regions. We did not detect either inversions or translocations among the two genome sequences, and no rearrangement occurred in gene organization after verification (Fig. 4). There was high variation in nucleotide diversity values observed between the 101 regions of the two Fagus species with values ranging from 0.0003 (ycf2 gene) to 0.0781(ndhD-psaC) (Fig. 5). The six most variable regions were, in increasing order of variability, psbK-psbI, trnG-psbfM, rpl32, trnV, ndhI-ndh and ndhD-psaC of which four are located in the LSC region and two in the SSC region (Fig. 5). The nucleotide diversities of these variable regions between F. crenata and F. engleriana were higher than observed within some other studies of Fagaceae genera including East Asian (Yan et al., 2018) and Mediterranean oaks (Vitelli et al., 2017).

A total of 160 chloroplast microsatellites with a repeat unit length between 1 and 6 bp were identified based on the selection criteria in the two species of which mono- and tri-nucleotide repeat microsatellites were the most abundant with a frequency of 38.7% and 43.1%, respectively. This abundance of mono- and tri-repeats in the chloroplast is similar to a range of other angiosperms (Melotto-Passarin et al., 2011). Of these microsatellites, 46 displayed size variation between F. crenata and F. engleriana (see DatasetS4 for a table with details of all 46 variable chloroplast microsatellites). The majority (66.1%) of the variable chloroplast microsatellites were mono-nucleotide repeats while 20% of di-nucleotide repeats and both of the two hexa-nucleotide repeats were variable. On the other hand, zero of the tri-, tetra- and penta-nucleotide repeats showed size variation between the two species (Fig. 6). The length of variable versus non-variable chloroplast microsatellites was similar but with a greater length variation for variable microsatellites in both F. crenata and F. engleriana (Fig. 7).

Conclusion

Overall, the chloroplast genome of F. crenata will provide a useful genetic resource for future genetic studies into the foundation temperate tree genus Fagus. Specifically, the chloroplast genomes of both informal subgenera will provide useful references and sources of molecular markers to investigate phylogeographic patterns of the chloroplast within and between Fagus species. Some major questions are yet to be resolved in Fagus, including resolving taxonomic boundaries of western Eurasian Fagus populations which has remained a recalcitrant problem due to low marker resolution and high within-species genetic diversity (Denk et al., 2002) and the non-monophyly of the chloroplast of East Asian species as suggested by Sanger sequence-based data (Manos & Stanford, 2001; Okaura & Harada, 2002).

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