DOI: 10.5061/dryad.12jm63xtk
title: Contact zone of two different chloroplast lineages and genetic guidelines for seed transfer in Quercus serrata and Q. crispula

description: Within their natural distribution ranges, plant species exhibit genetic
 structure which has been created by global climate change and natural
 selection over long periods. To conserve local forests with different
 genetic structures, genetic guidelines for seed and seedling transfer in
 individual species are therefore necessary. Genetic guidelines have been
 published for 43 Japanese tree species using population genetic data;
 however, for practical use, more detailed genetic borders between
 important genetic lineages should be clarified to inform seed collection
 and planting. Thus, we investigated in detail the genetic borders between
 two important Japanese oak species, Quercus serrata and Q. crispula, in
 the Chubu region of Japan using chloroplast and nuclear DNA markers, and
 we discuss the factors that influenced border creation using the results
 of species distribution modelling (SDM). Two distinct cpDNA haplotypes
 were found for each species (northern and southern haplotype) within the
 Chubu region of Japan but the difference in nuclear DNA between northern
 and southern haplotype populations was very small both in Q. serrata and
 Q. crispula. The results of SDM showed that during the LGM Q. serrata was
 distributed mostly along the coastline but Q. crispula was distributed not
 only along the coast but also in mountainous areas further inland. The
 cpDNA genetic borders of these two oak species are complex and seem to
 have been influenced by topography and their distribution during the LGM.
 We propose and discuss genetic guidelines for these two oak species based
 on the results of this study.
description: <em>Chloroplast DNA analysis</em><br> DNA was
 extracted using a DNeasy Plant mini kit (Qiagen, Germany). To amplify the
 3’to_rps2 fragment, which distinguishes between the northern and southern
 type of cpDNA, the following forward and reverse primers were used:
 5’-GTCATATATTTGATCCCGCC-3’ and 5’-AACCGGAACTAGTCGGATG-3’ respectively (San
 Jose-Maldia et al. 2017). The PCR conditions and sequencing procedure were
 as in San Jose-Maldia et al.(2017). The northern and southern haplotypes
 for the sequenced fragment are shared between both species. The PCR
 products were purified using Exo-SAP ITTM (GE Healthcare Limited) and
 sequenced in both directions using a BigDye Terminator Sequencing Kit (PE
 Biosystems). The sequencing products were then purified by ethanol
 precipitation and analyzed in a 3100 Genetic Analyzer(Applied Biosystems).
 Sequence data were assembled and manually edited using Sequencher 10.4.1
 (Gene Codes Corporation). <em>MIG-seq
 analysis</em><br> Multiplexed inter-simple sequence repeat
 (ISSR) genotyping by sequencing (MIG-seq; Suyama and Matsuki 2015) was
 conducted to genotype selected samples. We used 36 and 36 samples of Q.
 serrata, and 59 and 37 samples of Q. crispula, for the northern and
 southern haplotypes, respectively. These samples were selected from meshes
 in which the two cpDNA haplotypes did not coexist in each species. The
 technical implementation for MIG-seq analysis followed the protocol by
 Suyama & Matsuki (2015). Based on the protocol, we have
 constructed a MIG-seq library with two polymerase chain reactions(PCR) and
 conducted the sequencing. The first PCR is conducted to amplify ISSR
 regions from genomic DNA with a MIG-seq primer set. The first PCR product
 is used as the template for the second PCR. The latter is performed to add
 the complementary sequences that serve as an index for subsequent analyses
 to the primary PCR products. Fragments in the size range 300-800 bp were
 isolated using the magnetic bead method and quantified using Real-time
 PCR. The DNA library was sequenced using an Illumina MiSeq Sequencer
 platform.<br> The obtained sequence data were filtered using
 FASTX_toolkit to remove low-quality and adapter sequences. The
 quality-filtered reads were used for SNP detection with the “ustack”,
 “cstack” and “sstack” modules in Stacks v. 2.2, software that identifies
 loci (Catchen et al. 2011). SNPs were called using the “population”
 module. Minimum number of populations in which a locus must be present (p)
 and minimum percentage of individuals in a population required to have a
 locus (r) were set to 1 and 0.8, respectively. Thus we retained only those
 loci that were present in ≥ 80% of individuals.
 <em>Data analysis of MIG-seq output</em><br>
 The observed heterozygosity (Ho), expected heterozygosity (He), nucleotide
 diversity (π), inbreeding coefficient (FIS) and genetic differentiation
 (FST) were estimated using Populations in Stacks (Catchen et al.2013).
 Allelic richness (AR; El Mousadik & Petit 1996) was estimated
 using FSTAT 2.9.4 (Goudet 2003). We also carried out STRUCTURE analysis
 (version 2.3; Pritchard et al. 2000) which is Bayesian cluster analyses to
 clarify the genetic structure of the two cpDNA groups in the Chubu region.
 We ran ten replications with each K from 1 to 10 with burn-in length of
 100,000 and following 100,000 Markov Chain Monte Carlo (MCMC) iterations.
 To determine the most appropriate number of K, we plotted both of the log
 probability(L(K)) and applied the ΔK method of Evanno et al. (2005) using
 STRUCTURE HARVESTER (Earl and VonHoldt 2012). Then the output results from
 STRUCTURE and STRUCTURE HARVESTER were aligned using CLUMPP (Jakobsson
 & Rosenberg 2007) and displayed using DISTRUCT (Rosenberg
 2004).