Populus, a core genus of Salicaceae, plays a significant ecological role as a source of pioneer species in boreal forests. However, interspecific hybridization and high levels of morphological variation among poplars have resulted in great difficulty in classifying species for systematic and comparative evolutionary studies. Here, we present phylogenetic analyses of 24 newly sequenced Populus plastomes and 36 plastomes from GenBank, which represent seven genera of Salicaceae, in combination with a matrix of eighteen morphological characters of 40 Populus taxa to reconstruct highly supported relationships of genus Populus. Relationships among the 60 taxa of Salicaceae strongly supported two monophyletic genera: Populus and Salix. Chosenia was nested within the genus Salix, and five clades within Populus were divided. Clade I included the three taxa P. euphratica, P. pruinosa, and P. ilicifolia. Clade II contained thirteen taxa [P. adenopoda, P. alba, P. bolleana, P. davidiana, P. hopeiensis, P. nigra, P. qiongdaoensis, P. rotundifolia, P. rotundifolia var. duclouxiana, P. tremula, P. tremula × alba, P. tomentosa, and P. tomentosa (NC)]. Clade III included the ten taxa P. haoana, P. kangdingensis, P. lasiocarpa, P. pseudoglauca, P. qamdoensis, P. schneideri, P. simonii, P. szechuanica, P. szechuanica var. tibetica, and P. yunnanensis. Clade IV included P. cathayana, P. gonggaensis, P. koreana, P. laurifolia, P. trinervis, P. wilsonii, and P. xiangchengensis. The last clade comprised P. angustifolia, P. balsamifera, P. deltoides, P. deltoides × nigra, P. fremontii, P. mexicana, and P. trichocarpa. This phylogeny is also supported by morphological traits, including bark smoothness, bud size, petiole shape, leaf inflorescence, male anther length and male anther tip.

Species of the genus Populus, which is widely distributed in the northern hemisphere from subtropical to boreal forests, are among the most commercially exploited groups of forest trees. In this study, the complete chloroplast genomes of five Populus species (Populus cathayana, P. kangdingensis, P. pseudoglauca, P. schneideri, and P. xiangchengensis) were compared. The chloroplast genomes of the five Populus species are very similar. The total chloroplast genome sequence lengths for the five plastomes were 156,789, 156,523, 156,512, 156,513, and 156,465 bp, respectively. A total of 130 genes were identified in each genome, including 85 protein-coding genes, 37 tRNA genes and eight rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. The GC content was 36.7% for all plastomes. We analyzed nucleotide substitutions, small inversions, simple sequence repeats and long repeats in the chloroplast genomes and found nine divergence hotspots (ccsA+ccsA-ndhD, ndhC-trnV, psbZ-trnfM, trnG-atpA, trnL-ndhJ, trnR-trnN, ycf4-cemA, ycf1, and trnR-trnN), which could be useful molecular genetic markers for future population genetic and phylogenetic studies. We also observed that two genes (rpoC2 and rbcL) were subject to positive selection. Phylogenetic analysis based on whole cp genomes showed that P. schneideri had a close relationship with P. kangdingensis and P. pseudoglauca, while P. xiangchengensis was a sister to P. cathayana.

Methods for constructing trees using DNA sequences, known as molecular phylogenetics, have been applied to analyses of phylogenetic origin, evolutionary relatedness and taxonomic classification. Combining data sequenced in this study and downloaded from GenBank, we sampled 112 (chloroplast data) / 122 (ITS data) specimens belonging to 49 (chloroplast data) / 46 (ITS data) poplar species or hybrids from six (chloroplast data) / five sections (ITS data). Maximum parsimony and Bayesian inference were used to analyze phylogenetic relationships within the genus Populus based on eight chloroplast combinations and ITS regions. The results suggested that Bayesian inference might be more suitable for the phylogenetic reconstruction of Populus. All Populus species could be divided into two clades: clade 1, including subclades 1 and 2, and clade 2, including subclades 3 and 4. Species within clade 1, involving five sections except for Leuce, clustered coinciding with their two specific main geographical distribution areas: China (subclade 1) and North America (subclade 2). Clustering in subclade 3, section Leuce was confirmed to be of monophyletic origin and independent evolution. Its two subsections, namely Albidae and Trepidae, could be separated by chloroplast data but had frequent gene flow based on ITS data. Phylogeny analysis based on chloroplast data demonstrated once more that section Aigeiros was paraphyletic and further showed that the P. deltoides lineage is restricted in subclade 2 and that P. nigra lineage, located in subclade 3, originated from a hybrid of which an Albidae ancestor species was the material parent. Similarly, section Tacamahaca was found to be paraphyletic and had two lineages: a clade 1 lineage, such as P. cathayana, and a clade 2 lineage, such as P. simonii. Section Leucoides was paraphyletic and closely linked to section Tacamahaca. Their section boundaries were not conclusively delimitated by sequencing information.

Inverted cuttings of Populus yunnanensis remain alive by rooting from the original morphological apex and sprouting from the base, but the lateral branches exhibit less vigorous growth than those of the upright plant. In this study, we examined the changes in hormone contents, oxidase activities, and transcriptome profiles between upright and inverted cuttings of P. yunnanensis. The results showed that the indole-3-acetic acid (IAA) and gibberellic acid (GA3) contents were significantly lower in inverted cuttings than in upright cuttings only in the late growth period (September and October), while the abscisic acid (ABA) level was always similar between the two direction types. The biosynthesis of these hormones was surprisingly unrelated to the inversion of P. yunnanensis during the vegetative growth stage (July and August). Increased levels of peroxidases (PODs) encoded by 13 differentially expressed genes (DEGs) served as lignification promoters that protected plants against oxidative stress. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that most DEGs (107) were related to carbohydrate metabolism. Furthermore, altered activities of uridine diphosphate (UDP)-sugar pyrophosphorylase (USP, 15 DEGs) for nucleotide sugars, pectin methylesterase (PME, 7 DEGs) for pectin, and POD (13 DEGs) for lignin were important factors in the response of the trees to inversion, and these enzymes are all involved cell wall metabolism.