Phylogeny of Salix subgenus Salix s.l. (Salicaceae): delimitation, biogeography, and reticulate evolution

Abstract

Background: The taxonomy and systematics of Salix subgenus Salix s.l. is difficult. The reliability and evolutionary implications of two important morphological characters (number of stamens, and morphology of bud scales) used in subgeneric classification within Salix remain untested, and a disjunct Old-New World distribution pattern of a main clade of subgenus Salix s.l., revealed by a previous study, lacks a reasonable explanation. To study these questions, we conducted phylogenetic analyses based on 4,688 bp of sequence data from four plastid (rbcL, trnD-T, matK, and atpB-rbcL) and two nuclear markers (ETS and ITS) covering all subgenera of Salix, and all sections of subgenus Salix s.l.

Results: Subgenus Salix came out as para- or polyphyletic in both nrDNA and plastid trees. The plastid phylogeny successfully resolved relationships among the major clades of Salix, but resolution within subgenus Salix s.l. remained low. Nevertheless, three monophyletic groups were identifiable in subgenus Salix s.l.: the 'main clade' of subgenus Salix s.l., with New and Old World species being reciprocally monophyletic; the section Triandroides clade; and the subgenus Pleuradenia clade. While nrDNA regions showed higher resolution within subgenus Salix s.l., they failed to resolve subgeneric relationships. Extensive, statistically significant gene-tree incongruence was detected across nrDNA-plastid as well as nrDNA ETS-ITS phylogenies, suggesting reticulate evolution or hybridization within the group. The results were supported by network analyses. Ancestral-state reconstructions indicated that multiple stamens and free bud scales represent the plesiomorphic states within Salix, and that several significant shifts in stamen number and bud scale morphology have occurred.

Conclusions: Subgenus Salix s.l. is not monophyletic, and the evolutionary history of the subgenus has involved multiple reticulation events that may mainly be due to hybridization. The delimitation of subgenus Salix s.l. should be redefined by excluding section Triandrae and subgenus Pleuradenia from it. The evolutionary lability of bud-scale morphology and stamen number means that these characters are unreliable bases for classification. The disjunct Old-New World distribution of subgenus Salix s.l. appears to be linked to the profound climatic cooling during the Tertiary, which cut off gene exchange between New and Old World lineages.

Figure 1

Geographic distributions of the main lineages of Salix .

Figure 2

Maximum likelihood trees based on (A) nrDNA ETS, (B) ITS, and (C) combined plastid sequence datasets. ML/MP bootstrap support values above 50% are shown near branches. Dotted lines in (B) indicate branches that receive over 50% bootstrap support in the MP analysis, but which are not present in the ML topology. Bootstrap values of well-supported clades (BS ≥ 70%) are highlighted in bold.

Figure 3

Phylogenetic network of nrDNA ETS, ITS, and combined plastid ML trees with inferred reticulation events, drawn as (A) rectangular and (B) radial cladograms.

Figure 4

Relaxed molecular-clock chronogram based on combined plastid sequence data. Nodes used for calibration (A and B) are marked with black squares (see text for details). The main clades of Salix are numbered (1–8), and their estimated ages (with 95% HPD intervals in square brackets) are shown to the right of each node.

Figure 5

Ancestral state reconstruction of (B) bud scale morphology and (A) number of stamens, according to maximum likelihood optimization of states across the ML tree based on combined plastid datasets. Arrows indicate nodes with major character state shifts.