Phylogenetic distribution of phloem wedges in Malpighiaceae
Phloem wedges have evolved exclusively in lianescent lineages, with at least 10 independent origins in Malpighiaceae within eight clades (Fig. 2). This variant evolved once in the Bunchosia clade (Tristellateia), once in the Hiraea clade (Hiraea), once in the ancestral node of Tetrapteroids (Niedenzuella clade (Niedenzuella), + Christianella clade, + Carolus clade (Carolus), + Heteropterys clade (Heteropterys)), once in the Malpighia clade (Mascagnia) and six times within the species-rich Stigmaphyllon clade (Peixotoa, Diplopterys, some Banisteriopsis, some Stigmaphyllon, and Janusia).
The self-supporting was inferred to be the ancestral condition for Malpighiaceae, while the evolution of lianescent habit likely occurred twice in the family. Lianas evolved once in the Acridocarpus clade and once in the ancestral node of Bunchosia clade + Tetrapteroids + Stigmaphylloids, being the most common habit in the family. Many reversions back to the self-supporting habit occurred, two of them being noteworthy for leading to large neotropical genera within Malpighiaceae, Bunchosia, and Malpighia (Fig. 2).
The ancestral state estimation indicates that phloem wedges are a derived character in the family being present only in lianas. The Pagel 1994 test showed support for the dependent model of correlated evolution (p = 0.0000076), indicating the evolution of phloem wedges was contingent on the evolution of lianas. Except for Tristellateia (Bunchosia clade) and Hiraea (Hiraea clade), all other genera are in two large clades known as tetrapteroids and stimaphylloids (Fig. 2). Some of these lineages have members that reversed to the self-supporting habit (e.g., Heteropterys, Hiraea, Peixotoa), and the cambial variants are absent on them, giving further support to the results of our correlation analysis.
For members of Christianella clade, the genus Diplopterys, and some species of Banisteriopsis and Heteropterys, phloem wedges represent one of the stages of their development, which later progresses into fissured stems. In Tristellateia and Niedenzuella, interxylary phloem is formed right after phloem wedges formation. Exploring the formation of fissure stems in detail is beyond our goal here because their formation has been surveyed in previous works, which will be treated in the discussion.
Structure and development of phloem wedges
Phloem wedges external appearance and their position in the stems
Macro-morphologically, the presence of phloem wedges can be perceived from the outside in most species (Fig. 3). In these cases, the stems exhibit depressions where the phloem wedges develop, as we here illustrate in Heteropterys cordifolia (Fig. 3A, B) and Mascagnia sepium (Fig. 3C–E), where the depressions are so marked that their stems become non-cylindrical. In some lianas, such as Banisteriopsis caapi (popularly known as one of the main ingredients of the ritualistic Ayahuasca), the phloem wedges can merge with dividing non-lignified parenchyma to join the pith and produce fissured stems, with an architecture similar to that seen in Heteropterys cordifolia (Fig. 3B). In contrast, in some specimens of Tristellateia greveana, the stem remains with a round outline and shows no depressions, despite having deep phloem wedges. It is noticeable that the initial phloem wedges are located in between leaf insertions, with no phloem wedges under the leaves (Fig. 3D).
Two main ontogenies of phloem wedges are present in Malpighiaceae
Although we have detected 10 independent evolutions of phloem wedges in Malpighiaceae, we can group them under two main ontogenetic types (Fig. 4): (i) those which form phloem wedges with a single, continuous cambium (Fig. 4A); (ii) those whose variant cambia cease anticlinal divisions, disrupting the cambium continuity, leading to variant cambial portions, not in contact with the cambium of the regular portions (Fig. 4B). In the first type of ontogenetic trajectory, phloem wedges can be a stage in stem development that progresses to form fissured stems (Fig. 4A, ontogeny 1a) or included phloem (Fig. 4A, ontogeny 1b). In both ontogenies, the inclusion of portions of the phloem wedges into the xylem can occur, resulting in interxylary phloem (note the last stage in ontogeny depicted as Fig. 4A ontogeny 1b and Fig. 4B, ontogeny 2). Each of these two cases will be discussed in detail below.
Phloem wedges maintain a continuous cambium during their development (Ontogeny I)
All phloem wedges of Malpighiaceae have a continuous cambium in at least one stage of their development (Fig. 4). While in some species this configuration is kept as their final form (Fig. 5A–E), others progress to other stem anatomies (Fig. 5F–H). Anatomically, all species with phloem wedges start their secondary growth with a regular, continuous cambium producing tissues equally across its girth (Figs. 4, 6A). However, sooner or later, in certain cambial regions, the cambium switches its activity and starts producing less secondary xylem and more secondary phloem, initially forming shallow arcs or invaginations (Fig. 6B) and then progressing into phloem wedges (Fig. 6C, D). Because the amount of phloem produced does not always keep up with the amount of xylem produced by the regular cambium, the external depressions mentioned above are formed (as seen in Figs. 3, 6F, J).
It is common in most genera and species that we studied that the number of phloem wedges increases in time, such as seen in Mascagnia (Fig. 5B). This phenomenon is evidenced by the co-occurrence of wedges of different depths within the stem (Figs. 5B and 6D, F). In some species, the variant cambium of the wedges produces very little xylem, and the wedges can deeply furrow the xylem (Figs. 5B and 6D, E, H, K).
Regardless of the depth or number of the phloem wedges, the cambium in species with this configuration remains continuous. The cambium continuity is evidenced by the persistent production of phloem and xylem from the variant cambia, which will generate phloem and xylem with a slightly different orientation. This is more evident in the secondary xylem, where new tissue is inclined or even perpendicular to the other cells in the stem (Fig. 6G, H).
Another remarkable aspect of the variant cambia that generate phloem wedges is that the xylem produced is not similar to that of the interwedges. In Mascagnia and species of Stigmaphyllon, the variant cambium produces xylem with much more non-lignified parenchyma than the regular cambium of the interwedge (Fig. 6I). In Peixotoa the variant cambium produces xylem with fewer vessels and more fibers (Fig. 6J).
In thick stems of species of the Christianella clade, Carolus clade, Diplopterys, and some Banisteriopsis and Mascagnia, the non-lignified parenchyma, below the phloem wedges proliferates, inducing proliferation also of the pith in a continuum, anastomosing the deepest wedges in the stem, forming fissured stems. (Fig. 4A, ontogeny 1a, 6K). On the other hand, in Niedenzuella, the cambium is continuous throughout the plant ontogeny, but some portions of the phloem wedges get included by coalescence of the xylem on each side of the wedges. This inclusion form patches of interxylary phloem under the wedges (Fig. 7A, B), which maintain reminiscences of an active vascular cambium. The inclusion of phloem wedges is a result of mechanical pressure from both sides of the phloem wedge. The xylem encloses part of phloem wedges since wood production by the regular cambium is greater than that of the variant cambium. This differential production exerts pressure on both sides of the wedge including their innermost parts.
Phloem wedges with a discontinuous cambium form a stepwise pattern during their development (Ontogeny II)
This type of cambial variant was found exclusively in the paleotropical genus Tristellateia, from the Bunchosia clade (Fig. 4, ontogeny 2, Fig. 5G, H, Fig. 8). The secondary growth begins with the differentiation of a single and continuous cambium with regular activity (Fig. 8A), similarly to the previously discussed ontogeny. While the stem progresses with secondary growth, small phloem invaginations start to be formed by certain areas of the cambium which reduce the production of xylem and increase the production of phloem (Fig. 8B); these areas are alternate with leaf insertions. Later in development, the shallow phloem arcs acquire the shape of wedges (Fig. 8C, D). Although it can be perceived in much earlier stages (Fig. 8F), it is during this stage that cambium disruption is most evident (Fig. 8E). Disruptions are caused by a decrease or cessation of anticlinal divisions in the variant cambial region. Cambium continuity is lost, and the wedges become flanked by conspicuously wide rays, known as limiting rays (Fig. 8E).
Phloem wedges widen up towards the regular cambium region (Fig. 8E, G). The regular cambium near the original variant cambium switch from a regular to a variant activity, resulting in a stepwise pattern (Figs. 1C, D, 8E, G). The variant cambial portions get disconnected from the regular cambial portions as phloem wedges widen up. These variant cambial portions remain on the bottom of the wedges (Fig. 8E), being still active and producing both secondary xylem and phloem.
In some wedges, similarly to what is seen in Niedenzuella, the secondary xylem that flanks the external most part of the wedge, begins to exert pressure from both sides, eventually encapsulating portions of the phloem wedge within the wood, forming islands of interxylary phloem (Fig. 8H, I). It means that higher production of wood by the regular cambium generates pressure on both sides of the wedge and mechanically includes it. As portions of phloem wedges are included by adjacent wood, the limiting rays merge. This phenomenon evidences that the inclusion of phloem results from mechanical pressure that the adjacent wood exerts on both sides of the wedge. The interxylary phloem patches retain reminiscences of variant cambium. The wood produced by the variant cambial portions seems to be qualitatively equal to that produced by the regular cambial portions.