Comparison Genomes of Juglandaceae
Both genome size and GC content among Juglandaceae plastomes were consistently more than the median genome size and GC content for land plant plastomes (Table 1). The nucleotide variability (Pi) across all 27 plastomes of Juglandaceae included in this study was 0.00791 (Fig. 2; Fig. S1). Coding regions with the highest variation included matK, atpI, rpoC2, rps14, aacD, psaI, ycf4, cemA, rpl33, infA, rpl22, rps19, ndhF, rpl32, ndhD, ndhI, and ycf1. Non-coding regions that were most variable were matK-rps16, petN-psbD, ndhC-trnV-UAC, rbcL-psaI, psbE-petL, and rpl14-ycf1. These regions of maximum variability will no doubt prove the most informative for phylogenetic studies in the Juglandaceae [6, 12]. Previous studies have identified rpl22, rps19 and ycf1 genes as the most variable genes in the Juglandaceae plastomes based on high indel density [12]. It was surprising, however, that the LSC region also contained variation, including 2,577 bp differences among Juglandaceae plastomes, while SSC had 237 bp and IR had 296 bp differences among plastomes (Table 1).
Backbone Relationship of Juglandaceae
The phylogeny of family Juglandaceae has been inferred based on microsporogenesis, morphology [22–23], fossils [24, 78], molecular markers [26, 35], and combined data (morphology, fossils, and molecular data) [16]. Several recent studies of phylogeny in the Juglandaceae have included data from plastomes [12, 35–38, 56, 79–80]. The previously recognized subfamilies (Engelhardioideae and Juglandoideae), tribes (Platycaryeae and Juglandeae) and subtribes (Caryinae and Juglandinae) were all strongly supported [26, 30, 35–38]. Our phylogenetic analyses indicated that the Juglandaceae is subdivided into three major clades corresponding to the three subfamilies Rhoipteleoideae, Engelhardioideae, and Juglandoideae [16, 26–27, 29, 34–35, 81] (Fig. 1). The evidence for these three subfamilies can be found from morphology, fossil, and molecular data [16], fruits [43], and flower development [40]. The subfamily Engelhardioideae includes Engelhardia, Oreomunnea, and Alfaroa [22] (Fig. 1). Our results supported the separation of Alfaropsis [16] as a separate genus within Engelhardioideae (Fig. S4). The Rhoipteleoideae (Rhoiptelea chiliantha) was a basal, monophyletic branch, which indicated that winged (dry) fruit was an ancestral character for the Juglandaceae (Fig. 3, Fig. 4). The fruits of Myricaceae, the closest relative of the Juglandaceae, are small and fleshy, of a type common among Cretaceous flora [34–38]. The subfamily Rhoipteleoideae has only one species (Rhoiptelea chiliantha), which is a threatened and endemic in China [35–38, 47]
The subfamily Juglandoideae includes the commercially important nut-producing trees commonly called walnuts and butternuts (Juglans), pecan and hickory (Carya) [15, 26, 29] (Fig. 1). The Persian walnut, Juglans regia, is one of the major nut crops of the world. Walnuts and hickories are also valuable timber trees [65]. Our plastid phylogenomic analyses fully resolved relationships among the major clades and genera of Juglandoideae (Fig. 3). Within subfamily Juglandoideae, four tribes are recognized (Juglandeae, Cyclocaryae, Platycaryae, and Hicorieae). Our results strongly supported the previously published merger of the genera Annamocarya and Carya into the genus Carya [26, 43]. Five genera, with their subgenera and sections were identified previously [22, 24, 26], i.e., Carya (here including Annamocarya), Platycarya, Cyclocarya, Pterocarya, and Juglans. These five genera resolved in our analysis with 100 % support (Fig. 3). The phylogenetic relationships of the genera of the Juglandaceae reveal that Carya retains more primitive characters than Platycarya based on chloroplast DNA variation and morphology [81].
In previous studies, it was suggested the genus Cyclocarya is sister to genus Platycarya [16] based on fossil, chloroplast DNA fragments, and morphological data. Our data also confirm this relationship (Fig. 5). Alternatively, it was suggested by Xiang et al. (2014) that Platycarya is sister to Juglans based on five chloroplast markers [30], that Carya and Platycarya are sister groups [30]. Others considered Cyclocarya and Juglans to be sister groups [28]. Using criteria based on fruit morphology, however, Carya and Juglans are sister groups [34], this relationship was not confirmed by our DNA-based analysis (Fig. 5), and Cyclocarya and Pterocarya are sister groups [this relationship was supported in our data (Fig. 3, Fig. 5) [34, 43]. Previously, Smith and Doyle (1995) [81], based on chloroplast DNA and morphological data, concluded that Platycarya evolved earlier than Carya; our results based on nuclear resequencing (Fig. 4) supported this conclusion. Our results based on sequencing the entire chloroplasts, however, indicated that the differentiation of Carya preceded Platycarya (Fig. 3, Fig. 5; Fig. S4), as suggested by Zhang et al. (2013), although their differentiation, about 57 Mya, was roughly simultaneous.
The Phylogenetic Relationships within genus of Juglandaceae
Our analyses fully resolved some previously unresolved intergeneric relationships and added additional evidence supporting some of the recently altered generic circumscriptions based on analyses with much more appropriate representation at the species level. The species C. sinensis (Chinese Hickory, beaked walnut, or beaked hickory) was resolved into Carya (Annamocarya sinensis) [82]. The generic circumscription of Annamocarya (also C. sinensis) has frequently been altered, and many genera have been segregated from or merged with Carya [26, 79, 83].
The previously unresolved intrageneric relationships of Pterocarya were also resolved with high support. P. stenoptera var. zhijiangensis and P. hupehensis were clustered together (Fig. 3). These two species are sympatric and P. stenoptera var. zhijiangensis may be a subspecies of P. hupehensis (Fig. 3, Fig. 5, but see Fig. 4). The taxonomy of sub-species P. stenoptera var. zhijiangensis and P. macroptera var. insignis conflicted with the previous study of Wu and Raven (1999) [82]. We consider these taxa subspecies based on our data (Fig. 3; Fig. 4, but see Fig. 5), however we did not complete a detailed phylogeny of Pterocarya because our sample pool was too small.
Our phylogenomic analyses resolved genus Juglans into three well sections (Cardiocaryon, Dioscaryon, and Rhysocaryon) with high support (Fig. 3; Fig. 4). Earlier phylogenies [22, 24] based on limited molecular data sometimes included a fourth section (Trachycaryon) containing only the North American species J. cinerea. The separation of Trachycaryon as distinct from section Cardiocaryon was inconsistent with morphology [21–23] and nuclear markers [84–85], but congruent with fossil data [24] and the results of other analyses based on plastid sequences [12, 15]. In our phylogenetic analysis of nuclear genome SNPs, American butternut (J. cinerea) has high support (100%) as sister to Section Cardiocaryon (Asian butternut, J. cathayensis, J. mandshurica, and J. ailantifolia) (Fig. 4).
Based on sequence data from 16 mtCDS and 61 chloroplast protein-coding genes, our results supported the unification of J. mandshurica, J. ailantifolia, and J. cathayensis within sect. Cardiocaryon (Fig. 3; Fig. S4), consistent with a previous conclusion based on genotyping by sequencing data [22, 86]. We also confirmed that the Ma walnut (J. hopeiensis) arose from the resent hybridization of J. regia and J. mandshurica based on both matrilineal and biparental inheritance data (Fig. 3; Fig. 4) [12, 86]. The placement of J. cinerea into Rhysocaryon (black walnuts) based on plastome sequence was clear (Fig. 3), however, it belongs to Cardiocaryon (Asian butternuts) based on nuclear sequences (Fig. 4), and its morphology is consistent with Cardiocaryon [12, 15]. In addition, J. cinerea can hybridize with members of Cardiocaryon and even Dioscaryon, but not with Rhysocaryon [87]. All other North American Rhysocaryon freely hybridize. The discordance between the J. cinerea nuclear genome and its plastome is almost certainly the result of a chloroplast capture [15, 31]. It is notable that the chloroplast of J. cinerea is not an ancient one (ancestral to the Rhysocaryon) but is instead most like J. nigra (Fig. 5). Our results indicated that the capture of a Rhysocaryon chloroplast by J. cinerea capture was relatively recent (Fig. 5). Hybridization and chloroplast capture between Rhysocaryon and Cardiocaryon apparently played a major role in the diversification of Juglans, as it did in other plant families [88–90].
Dating of the Origin and Historical Diversification of Juglandaceae
Stem ages in the Juglandaceae are controversial [13, 16, 28–29, 91]. Most previous studies estimated a stem age of Juglandaceae about 84 Mya in the Cretaceous [24, 28], however, the divergence times for some genera remain uncertain [28–29, 91], as only a few studies have examined the divergence times among the major genera and within the species of the family [16, 29, 91]. The lack of a robust phylogenetic framework and time tree has hindered development of a full understanding of the diversification of Juglandaceae.
The crown ages of Betulaceae, Myricaceae, and Casuarinaceae were 74.0 Mya (66.9–80.3), 90.4 Mya (85.0-94.6), and 82.8 Mya (74.7–88.6), respectively [30]. The crown age of Juglandaceae varied among previous studies, 78 Mya by Manos et al. (2007) [16], 71 Mya by Larson-Johnson (2016) [34], 85.5 Mya by Sauquet et al. (2012) [28] and 79.9 Mya by Xiang et al. (2014). Our results indicated the stem age of Juglandaceae to be during the late Cretaceous (78.58 Mya with 95% HPD: 76.58–80.50 Mya). The major diversification of the family is recorded in the pollen and megafossil record of the early Tertiary (~ 65Mya) at the K-T boundary [24]. The three subfamilies diverged during the Late Cretaceous to Early Palaeocene (60.65–68.64 Mya) (Fig. 5). Our estimates of divergence times among subfamilies and major genera were from 50.93 to 61.98 Mya in warm and dry habitats during the Middle Palaeocene to the Early Eocene (Fig. 5), which is largely consistent with the estimates of Xiang et al. (2014) and Larson-Johnson (2016) [34]. We estimated the divergence time of Juglans and Pterocarya to have been ~ 47 Mya (Fig. 5; Manos et al. 2007, ~ 55 Mya) [16], and ~ 56 Mya between Pterocarya and Cyclocarya (Fig. 5; Manos et al. 2007, ~ 59 Mya) [16], however, both Xiang et al. (2014) and Larson-Johnson (2016) estimated a divergence time between Juglans and Pterocarya of ~ 24 Mya [30, 34], and ~ 18 Mya between Pterocarya and Cyclocarya [34]. During the end of the Eocene, Cyclocarya and Platycarya became extinct in North America but survived in Eurasia [24]. Our results indicated Carya emerged as an animal-dispersed genus about 58 Mya, considerably earlier than the estimate (44 Mya) of Larson-Johnson (2016) [34], although we agree that the overwhelming majority of winged and wingless fruited genera diverged or diversified during the Paleogene, probably reflecting adaptation to changing regeneration regimes[92].
During the early Tertiary to the Neogene there was likely extensive migration and exchange among North Atlantic, North America, western Europe, and Asia [24]. Interestingly, most species within the extant genera diversified between 18.54 and 8.52 Mya in warm and dry environments of the Early Miocene (Fig. 5), a period of especially rapid speciation within Juglans and Pterocarya. Some closely related species pairs within Juglans appear to have diverged relatively recently, under the influence of climate change during the Quaternary glacial period (Fig. 5; Bai et al. 2017). For example, J. regia and J. sigillata, J. mandshurica and J. hopeiensis, and Carya hunanesis and C. kweichwensis (Fig. 5). Overall, the Juglandaceae reflect a complex evolutionary history and diversification affected by changes in geography, distinctive distributions, climate changes, coevolution with animals. Biotic interactions (e.g., pathogens) no doubt also had a role in driving species abundance and distribution [93], but biotic interactions of that type are difficult to detect from current data [35–39].