MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants

Abstract

The evolutionary origin of the angiosperms (flowering plants sensu stricto) is still enigmatic. Answers to the question of angiosperm origins are intimately connected to the identification of their sister group among extinct and extant taxa. Most phylogenetic analyses based on morphological data agree that among the groups of extant seed plants, the gnetophytes are the sister group of the angiosperms. According to this view, angiosperms and gnetophytes are the only extant members of a clade called "anthophytes" to emphasize their shared possession of flower-like reproductive structures. However, most phylogeny reconstructions based on molecular data so far did not support an anthophyte clade, but also could not clarify the case because support for alternative groupings has been weak or controversial. We have isolated 13 different homologs of MADS-type floral homeotic genes from the gnetophyte Gnetum gnemon. Five of these genes fall into monophyletic gene clades also comprising putatively orthologous genes from flowering plants and conifers, among them orthologs of floral homeotic B and C function genes. Within these clades the Gnetum genes always form distinct subclades together with the respective conifer genes, to the exclusion of the angiosperm genes. This provides strong molecular evidence for a sister-group relationship between gnetophytes and conifers, which is in contradiction to widely accepted interpretations of morphological data for almost a century. Our phylogeny reconstructions and the outcome of expression studies suggest that complex features such as flower-like reproductive structures and double-fertilization arose independently in gnetophytes and angiosperms.

Figure 1

Phylogenetic tree showing the relationships between a subset of MIKC-type MADS-domain proteins known. The tree was constructed by using the “MIK-domain” sequences and the neighbor-joining algorithm. Genus names of species from which the respective genes were isolated are given in parentheses after the protein names. Gnetum proteins are indicated by inverted boxes, and genes from non-gnetalean gymnosperms are indicated by shaded boxes. Proteins from ferns are highlighted by open boxes. Proteins that are not boxed have been derived from angiosperm gene sequences. The numbers next to some nodes give bootstrap percentages, which are shown only for relevant nodes and those defining gene subfamilies. Subfamilies, which generally represent monophyletic gene clades (17, 23), are labeled by brackets at the right margin.

Figure 2

Northern blot analysis of GGM gene expression. The names and subfamily memberships of the respective genes are indicated at the right. At the left, the apparent length of the major band is indicated in kb. RNA sources were young leaves and male or female cones from two individual G. gnemon trees as indicated.

Figure 3

Expression patterns of GGM2 and GGM3 as determined by in situ hybridization. In A–E, digoxigenin-labeled antisense probes were used, which detect GGM2 (B and E) or GGM3 (A, C, and D) transcripts, respectively. Using sense probes of GGM3 as control did not result in visible signals (F and G). All sections are longitudinal ones. (A, B, and F) Sections of a node of a male strobilus at an early developmental stage. (C) Section through a sterile female reproductive unit of a male strobilus at a relatively late developmental stage. (D) Section through male reproductive units at a relatively late developmental stage. (E) Section of a node of a male strobilus at a relatively late developmental stage. (G) Section through male reproductive units at a relatively late developmental stage. (Bar = 100 μm.) a, antherophore; f, female reproductive unit (sterile); m, male reproductive unit; n, nucellus; oe, outer envelope.