Trees https://doi.org/10.1007/s00468-021-02167-y ORIGINAL ARTICLE Comparative phylogeography of Juglans regia and J. mandshurica combining organellar and nuclear DNA markers to assess genetic diversity and introgression in regions of sympatry Meng Dang1 · Hui‑Juan Zhou2 · Keith E. Woeste3 · Ming Yue1,4 · Yi Zhang1 · Gui‑Fang Zhao1 · Shuo‑Xin Zhang2 · Peng Zhao1 Received: 23 February 2021 / Accepted: 24 June 2021 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Key message By comparing the phylogeography of Juglans regia and J. mandshurica, we found that two walnut spe- cies, even when sympatric, rarely introgress, suggesting that strong hybridization barriers exist between these species. Abstract The biogeographic investigation of temperate walnut (Juglans) trees is of great interest because of their ecological and economical importance. Our goal was to perform an in-depth investigation of the genetic and phylogeographic history of J. regia and J. mandshurica, two walnut species that are sympatric in parts of their ranges, including tests for gene fow and incomplete lineage sorting. We employed a 234 bp locus of mitochondrial DNA, a 1.8 Kbp locus of chloroplast DNA, 3 nuclear loci totaling 1740 bp in length, and 17 EST-SSRs. We sampled 559 individuals, 332 of J. regia and 227 of J. mand- shurica, from 69 locations. We found that J. regia and J. mandshurica, even when sympatric, rarely introgress, suggesting that strong barriers to hybridization exist between these species. Niche overlap analyses for the two species found that they occupy distinct ecological niches and that the sympatric populations may be the result of recent postglacial population expansion. Keywords Genetic diferentiation · Phylogeography · Organellar DNA · Nuclear DNA · Introgression · Juglans Introduction Phylogeography is the study of the geographical pattern of organisms from a phylogenetic perspective. Phylogeography correlates historical events such as gene fow, glaciation, Meng Dang and Hui-Juan Zhou contributed equally to this paper. and aridifcation to observed spatial‒genetic patterns in a species or group of species (Bai et al. 2010, 2014; Fan et al. Communicated by José I Hormaza . * Peng Zhao Shuo-Xin Zhang [email protected] [email protected] Meng Dang 1 Key Laboratory of Resource Biology and Biotechnology [email protected] in Western China, Ministry of Education, College of Life Hui-Juan Zhou Sciences, Northwest University, Xi’an 710069, Shaanxi, [email protected] China Keith E. Woeste 2 College of Forestry, Northwest A&F University, [email protected] Yangling 712100, Shaanxi, China Ming Yue 3 USDA Forest Service Hardwood Tree Improvement [email protected] and Regeneration Center (HTIRC), Department of Forestry and Natural Resources, Purdue University, 715 West State Yi Zhang Street, West Lafayette, Indiana 47907, USA [email protected] 4 Xi’an Botanical Garden of Shaanxi Province, Xi’an, Shaanxi, Gui-Fang Zhao People’s Republic of China [email protected] Vol.:(0123456789)1 3 Trees 2013; Avise 2000). Comparative phylogeography, when 2014, 2015). Juglans mandshurica is widely distributed in combined with a thorough consideration of ecological and China, ranging from the tropical South (< 22°N) and the life-history traits, can provide a better understanding of the Qinling Mountains to the Huai River line (~ 34°N) in hilly relative contribution of historical, contemporary, biotic, and mid-elevation areas (Bai et al. 2014; Dang et al. 2015). J. abiotic factors to the structure of genetic variation (Papa- regia and J. mandshurica difer in shell thickness, nut size, dopouloua et al. 2016). Recent studies have improved our color of stigma, number of fowers per cluster, and number understanding of the phylogeography of walnut (Juglans) of leafets per leaf (Lu et al. 1999a, b; Manning 1978). In species by analyzing the efects of geography, ecology, and China, local biogeographic history and climate have iso- a changing climate on walnut diversity and spatial genetic lated and led to the diversifcation of the J. regia and J. structure (Bai et al. 2010, 2014, 2015; Fan et al. 2013; mandshurica populations (Bai et al. 2014, 2015; Han et al. Wang et al. 2016; Han et al. 2016). The phylogeography of 2016; Pollegioni et al. 2015). J. mandshurica belongs to most temperate plant species in the northern hemisphere is the section Cardiocaryon along with J. hopeinesis (Ma strongly infuenced by repeated cycles of climatic suitability walnut), and J. ailantifolia (Japanese walnut) and, in some during the Quaternary period (Qiu et al. 2011). A species’ taxonomic delimitations, J. cinerea (American butternut) demographic, spatial, and genetic recovery from cold and (Zhao et al. 2018; Dang et al. 2019). The frst three species dry periods can be strongly infuenced by the location(s) are native to East Asia, but J. cinerea (butternut) is native and size of glacial refuges and what congeners may have to eastern North America (Stanford et al. 2000; Zhao et al. been present there. Refugia for walnut species are not fully 2018; Aradhya et al. 2007). The taxon J. cathayensis of Sect. resolved, but improved genetic data can reveal the history of Cardiocaryon is now considered invalid and all populations Asian walnut species (Chen et al. 2012). previously identifed as J. cathayensis are now J. mandshu- Chloroplast DNA (cpDNA) and mitochondrial DNA rica (Zhao et al. 2018; Dang et al. 2015, 2019). J. regia (mtDNA) are widely used in phylogeographic analyses to (Common walnut) belongs to the section Juglans, along with help reveal evolutionary history and lineage divergence J. sigillata (Iron walnut), a taxon native to southern China (Avise 1998, 2000; Casazza et al. 2016; Du et al. 2009; Gug- (Aradhya et al. 2007; Zhao et al. 2018; Feng et al. 2018a; ger et al. 2010). Both cytoplasmic organelles are in most Sun et al. 2019). Many species of Juglans can hybridize cases maternally inherited in angiosperms, so studies of the (Hoban et al. 2009; Zhao and Woeste 2011; Shu et al. 2016; spatial genetic structure can provide insight at deeper evo- Yuan et al. 2018; Mu et al. 2017). In North America, J. nigra lutionary time scales (Avise 1998, 2009; Liu et al. 2012; and J. cinerea are sympatric and reproductively isolated; Zhao et al. 2018; Sun et al. 2019). Data from nuclear DNA however, both species can hybridize with Asian Juglans and fragments [for example, barcodes such as the internal tran- appear in cultivated populations but not in the wild (Woeste scribed spacer (ITS)] are also commonly used for studies of and Michler 2011). Diferences in geographic range, climate, population genetics and speciation. Nuclear DNA sequences habitat, and the presence of natural geographical features can be used to reconstruct phylogenetic relationships among have (with certain exceptions) been inferred to be important related lineages (Dang et al. 2019), to identify hybrid indi- in maintaining genetic and spatial separation. To avoid the viduals (Zhao and Woeste 2011; Barton et al. 1993), and in interference of cultivated trees on gene fow or introgression, some cases to reveal patterns of sympatric speciation (Sun- we specifcally sampled locations that might be expected to nucks 2000; Van et al. 2008; Dang et al. 2019; Yuan et al. contain spontaneous wild hybrids. 2018). Nuclear DNA makers, such as SSRs, which are inher- We used mtDNA, cpDNA, nuclear DNA sequences, and ited biparentally, are also widely used in population genetic EST-SSRs to investigate the biogeography of J. regia and studies (Bai et al. 2015; Han et al. 2016; Yuan et al. 2018). J. mandshurica. Our goal was to understand J. regia and J. The combination of cytoplasmic (mitochondrial and chlo- mandshurica genetic diversity and introgression in regions roplast) DNA sequence data with nuclear DNA sequence of sympatry. We analyzed the composition of mtDNA, data provides a robust dataset for testing hypotheses related cpDNA, nuclear DNA sequences, and microsatellite length to phylogeography, population genetics, patterns of genetic variation within 69 populations of J. regia and J. mandshu- variation, and interspecifc gene fow. rica, 40 individuals from four other Juglans species, and 13 All Juglans species are monoecious, wind-pollinated, and individuals from other genera in the Juglandaceae (Carya deciduous trees (Bai et al. 2014, 2015; Zhao et al. 2018). cathayensis and Engelhardtia roxburghiana). Because J. Common walnut, or J. regia, and J. mandshurica are both regia and J. mandshurica cooccur in several sympatric temperate species that bear nutritious nuts and produce high- populations throughout China, we hypothesized that we quality timber, which makes them economically important would observe gene fow between these two walnut species (Bai et al. 2015; Han et al. 2016). Juglans regia has been in regions of sympatry. The objectives were to: (1) char- cultivated throughout temperate parts of China for millen- acterize mitochondrial and chloroplast haplotypes in Chi- nia, but its natural range there is uncertain (Pollegioni et al. nese Juglans and their distribution in two Chinese Juglans 1 3 Trees species; (2) determine if spatial genetic structure can shed markers (trnL-F and trnS-G), and three nuclear DNA loci light on how evolutionary history shaped the current geo- (ITS, 15R-8, and Jr5680) were polymorphic among indi- graphical distribution of these related, partially sympatric viduals of J. regia and J. mandshurica, so these were used species; (3) determine if nuclear and cytoplasmic DNA indi- for further analysis (Table S2). The internal transcribed cate similar or discordant evolutionary histories for J. regia spacer fragment ITS1 (primer Forward sequence: TCC GTA and J. mandshurica; and (4) determine if we could identify GGT GAA CCT GCG G, Reverse sequence: TCC TCC GCT evidence of gene fow between J. regia and J. mandshurica TAT TGA TAT GC) was used in this study (Table S2). In the and, if so, what the consequences might have been (parent- nuclear genome, the sequence variability at ITS (internal age of hybrid species).