The evolution of cambial variants in Nyctaginaceae represents an example of continuum morphology
Because evolution can be seen as the transformation of ontogenies, many authors have argued that plant morphology is better understood under more dynamic and process thinking than the typological view of classical morphologists (e.g., Wilhelm Troll, Donald Kaplan) [63, 64]. One of the alternative worldviews is based on processes and continuum morphology which has been built upon the works of several botanists from the 20th century such as Agnes Arber  and Rolf Sattler [3, 66, 67]. This approach implies that the static view of plants having structures (such as organs or the different variants here) with clear cut boundaries seems no longer sufficient to explain plant morpho-anatomical diversity . In contrast, the process and continuum morphology recognize plants as combinations of developmental processes or as dynamic continua, based on the acceptance of the partial homology concept [65, 67], which reiterates the blurry boundaries (mixed identities, fuzzy morphology) between what used to be considered as separate structural categories. In other words, plant organs are no longer seen as structural categories, but they are likely to intergrade to a considerable degree [65, 64, 67]. This means that diversity is more likely to result from quantitative rather than from qualitative differences in development, which leads to the identification of several intermediate forms between two categories . Although these concepts are not new, they have recently inspired many developmental evolutionary biologists to look at morphology and anatomy in a more holistic way [63, 64]. The process thinking and the continuum approach may be as well the better way to look at the evolution of successive cambia from the interxylary phloem in Nyctaginaceae.
Although successive cambia and interxylary phloem are recognized in the literature as two types of cambial variants, their occurrence in Nyctaginaceae indicates that in some cases there is a blurry boundary delimitation between these two patterns – which are represented in two levels. First, plants with ontogeny 1 that are characterized with interxylary phloem may present phloem strands immersed within the xylem with different arrangements (i.e., phloem islands, patches or bands). These arrangements result from different extensions of the coalescent cambium which is formed in continuity with the single cambium and encloses the phloem strands. Given that in plants with bands the stem initiates forming phloem islands followed by patches and then bands, the development of this ontogeny itself indicate the existence of a continuum between these different stem macromorphologies. Second, the ontogenies characterized with successive cambia shows that the independent cambia arise and form usually long tangential bands of vascular tissues similar to the phloem strands in bands of some species with interxylary phloem (the type of cambial variant present in the ancestor of the family). In this sense, in the context of continuum morphology, we can propose that the successive cambia arise from interxylary phloem, by topological change of the new cambia differentiation migrating from secondary phloem parenchyma to pericycle (heterotopy), as a homologue of the coalescent cambium, but with a larger extension, so large that they start constituting completely independent additional cambia. In addition, the diverse topologies observed in plants with successive cambia which has received numerous attempts of subcategorization is another evidence of these blurry categories (Additional file 7: Table S4).
The fuzzy and continuum worldview has been used to distinct morphological systems especially in organ identity [68–71]. Here, for the first time, the diversity of cambial variants is interpreted under the concept of continuum morphology. These observations for Nyctaginaceae enrich our understanding of these complex vascular morphologies, as it gives us the notion of how ontogenies changed across evolutionary time producing intermediate forms that at some point can be distinguished as discrete categories.
The origins and developments determining the cambial variants in Nyctaginaceae
Different developmental pathways accounts for the disparate secondary vascular architectures observed in Nyctaginaceae. The recognition of interxylary phloem along with the occurrence of successive cambia is based primarily on their differences in development, i.e., single vs. multiple cambia respectively , but these developmental pathways result in architectonically similar anatomies and to a certain level can be considered to integrate into intermediate forms in Nyctaginaceae.
Here we identified that to the four ontogenies, all events share an origin to their secondary growth internally to the pericyclic fibres. These findings contradict that the cambial variants in Nyctaginaceae are formed from a meristem arising in the cortex as previously suggested [29, 39–41, 72]. Instead, all cambial variants originated from procambial-derived cells (i.e., either pericycle or arching cambia induced in phloem axial parenchyma), corroborating previous findings [37, 38]. Therefore, multiple origins (e.g., primary phloem, secondary phloem, cortex - [32, 73–76]) and developmental trajectories, as we discuss below, can lead to the formation of successive cambia, unlike the idea of a universal phenomenon for the formation of this cambial variant across different plant groups [39, 72, 77]. Indeed, successive cambia can be established from different stem regions, such as primary phloem , secondary phloem , pericycle [32, 70], or cortex . Nevertheless, except for the cortex and secondary phloem, in all other cases the origin of the new cambium can be considered developmentally linked with a pre-existing vascular meristem because both pericycle and primary phloem are produced by the procambium. A similar developmental parallel can be established for the origin of interxylary phloem and successive cambia in Nyctaginaceae, because both the cambium giving rise to phloem strands within the secondary xylem (interxylary phloem) and the meristematic zone producing a de novo cambium (successive cambia) may be traced back to the procambium at some point in stem development. Therefore, successive cambia and interxylary are evolutionarily and developmentally linked in Nyctaginaceae.
Although the cambial variants in Nyctaginaceae present similar origins at the cell lineage level (i.e., procambium-derived cells), the eustele types and subsequent events in their development are diverse, leading to four distinct ontogenies. In a previous work we showed that the origin and development of interxylary phloem in Nyctaginaceae is similar to that shown in other groups, except for the fact that the single cambium is originated from the continuous cylindrical procambium (CCP), which is part of the polycyclic eustele, that also includes the medullary bundles . Although ontogenies 2, 3 and 4 are characterized as successive cambia at maturity, they are built upon three different step-wise developmental pathways. The developmental steps in the formation of successive cambia in Reichenbachia (ontogeny 3) is the same as described in most families with this cambial variant, i.e., a new cambium is formed de novo (mostly but not always) from the pericycle in stems with regular eustele; this is the case of species both in the gymnosperms (e.g., Gnetum, Cycas – ) and several families of angiosperms (e.g., Menispermaceae ; Convolvulaceae ; Sapindaceae ). On the other hand, successive cambia, as described in ontogenies 3 and 4, differ from the taxa mentioned above because they either do not produce a regular cambium, forming an extra-fascicular cambium (ontogeny 2), or because the stem begins with a polycyclic eustele and the first cambium is derived from the CCP instead of a regular cambium (ontogeny 4). It is important to highlight that the appearance of the extra-fascicular cambium, which is independent from the primary vasculature, corroborates the potential of perivascular tissues (i.e., the pericycle) to produce new meristems, as it is observed in species with successive cambia following ontogeny 3 or 4. In addition, all developmental pathways in Nyctaginaceae include a de novo formation of new cambia, either entirely autonomously formed in the pericycle generating successive cambia or as short to long coalescing, arching cambia connected to the original single cambium generating interxylary phloem.
Besides the presence of distinct developmental pathways, Nyctaginaceae stands out for having all extant lineages characterized by some type of variant anatomy (see discussion on ‘evolution of development’ below). Regular vascular growth with eustele and a regular cambium forming xylem centripetally and phloem centrifugally, as observed in most eudicots, is observed only during the initial developmental stages of Reichenbachia (ontogeny 1), which later develops successive cambia. Species with ontogeny 4 also have a short period of secondary vascular tissues being produced by a bifacial cambium in the central cylinder, but it is developed from a polycyclic eustele .
Our observations on the origin and development of vascular meristems in Nyctaginaceae, as presented here and in previous studies [26, 35, 38], challenge a number of interpretations raised in previous investigations: (1) Cambial variants in Nyctaginaceae arise in the cortex [29, 39, 72] – we showed that the origin of cambial variants is from the pericycle (which is procambium-derived) . (2) Successive cambia are the only type of cambial variant in Nyctaginaceae [29, 39, 58, 68] – two types of cambial variants occur in Nyctaginaceae, interxylary phloem and successive cambia . (3) The existence of an ‘extra-fascicular cambium’ forming vascular bundles  – the extra-fascicular cambium produces secondary vascular tissues, observed exclusively in ontogeny 2 [this study]. (4) The presence of an unidirectional cambium forming both secondary xylem and secondary phloem to the inside  – instead of an unidirectional cambium we found that a single (bidirectional) cambium acts in the formation of interxylary phloem . (5) The presence of a “Primary Thickening Meristem” similar to the monocots [81–83] – the presence of medullary bundles was probably the main reason for this interpretation, and we have demonstrated that these vascular structures arise from a continuous cylindrical procambium, different from what is observed in the monocots [26, 38].
In addition, because the vessel elements and sieve-tube elements are usually restricted to short tangential areas resembling discrete “vascular bundles”, authors have used different terminologies to describe the secondary vascular tissues in stems of Nyctaginaceae, such as “secondary medullary bundles” , “vascular strands” , “vascular bundles” [28, 86] or “secondary bundles” , but in the secondary body this is a misnomer.
The evolutionary history of the diverse cambial variants in Nyctaginaceae
The ancestral state reconstruction presented here demonstrates that the ancestor of Nyctaginaceae already had cambial variant, with interxylary phloem (ontogeny 1) reconstructed as the most likely character state for the ancestral node of the family. This observation is remarkable because it indicates that Nyctaginaceae is one of the few examples where the cambial variants are present in all members and is shown to be shared with the other members of the phytolaccoid clade, being therefore plesiomorphic for Nyctaginaceae. In most families with cambial variants they appear only in one group, mostly in clades containing lianas or descending from lianas (e.g., Bignoniaceae, Convolvulaceae) [11, 12, 31, 87]. Contrary to expectations, interxylary phloem (ontogeny 1) is also the most common type of cambial variant in the family, occurring in five out of seven tribes, inclusive in those most studied genera such as Bougainvillea, Boerhavia, Mirabilis and Pisonia, which used to be classified as having successive cambia (reviewed by ).
The ontogenies are not evolutionary labile since each of them appeared only once, except for ontogeny 4 that evolved twice. The evolution of ontogenies 2 and 3 in members of Leucastereae is interesting because the tribe has other morphological (e.g., type of trichomes, pollen and fruit - [42, 76, 77] and vascular anatomical characters (e.g., type of stele, ) that are exclusive or unusual if compared to other lineages of the family. Curiously, from all stem ontogenies of Nyctaginaceae, Reichenbachia (ontogeny 3) is the only taxon following the commonly described development for successive cambia, i.e., regular cylinder + successive cambia [29, 39, 72].
Ontogeny 4 is the only type with more than one evolution with two transitions in tribe Nyctagineae. The evolution of successive cambia in Allionia and Okenia is noteworthy for the fact that they are both small herbs with limited secondary growth in the regular cylinder, but the successive cambia still develop in some way. However, this might not be surprising since the presence of cambial variants in other herbs (annuals or perennials) is reported in many sister-related families across the Caryophyllales [72, 90]. This observation suggests that if given time to grow, most herbaceous plants in this lineage can form new additional rings of successive cambia or as presented here, forming new phloem islands/patches/bands as in species with interxylary phloem. Nevertheless, the occurrence and diversity of variant anatomies in Nyctaginaceae seem to be not contingent on specific habits, since both cambial variants occurs in species with all the range of growth forms present in the family.
The development and evolution of distinct patterns of cambial variants in Nyctaginaceae is remarkable because successive cambia has been reiterated as the only cambial variant in the family [29, 41, 72, 90]. Although we observed multiple ontogenetic pathways resulting in distinct cambial variants in Nyctaginaceae, similar patterns may also be present in other caryophyllalean families . Comparably to other traits that seem to represent apomorphic tendencies (e.g., floral morphology [91–93]), the presence of cambial variants likely share developmental and genetic programmes (deep homology) triggering the recurrent evolution of this morphological feature in multiple Caryophyllales lineages.
Evolution of development: how different ontogenies generate similar stem macromorphologies
Because ontogeny is a linear process and given that primary and secondary vascular tissue may have intrinsic developmental relations, the investigation of the diversity and evolution of vascular anatomies in Nyctaginaceae needs to include the products of procambium, cambium and cambial variants to thoroughly comprehend the anatomical and developmental shifts in stem ontogeny. Here the integration between ontogeny and phylogeny showed that adult stems with distinct cambial variants evolved from different eustele types. Therefore, the organization of the primary vascular system is not a prerequisite for the evolution of patterns of secondary growth in Nyctaginaceae, achieved through distinct ontogenetic pathways. This scenario diverges from the evolution of secondary growth patterns in stems of Bignoniaceae  or Paullinia from the Sapindaceae  where cambial variants trace back to stems with regular growth in previous stem developmental stages.
For the evolution of interxylary phloem in the ancestor of Nyctaginaceae, several developmental modifications were needed in stem development of the putative ancestor of inner nodes of the phylogeny of the Caryophyllales, which likely had a regular anatomy (Fig. 15). Considering that some of the anatomical changes include: i) the decrease in the formation of xylem derivatives at specific locations, ii) the increased phloem production at the same location and iii) the development of the coalescent cambium. Similarly, considering that the ancestor of Nyctaginaceae had interxylary phloem (ontogeny 1), the appearance of each new ontogeny requires a set of transformations in the developmental pathway of its ancestor. First, in the case of ontogeny 2, the main steps include the loss of the regular cambium and the appearance of the extra-fascicular cambium, which is probably regulated by the same mechanism leading to the evolution of the new cambium from the pericycle in ontogeny 3, that result in the formation of successive cambia. The difference from ontogeny 3 to ontogeny 2 is that the later regained the regular cambium and form regular xylem and phloem for some period. For the evolution of ontogeny 4, the evolution of new cambia from the pericycle as in ontogeny 2 and 3 is also observed, but this time the primary vascular system is characterized by the polycyclic eustele, while the main cambium produces regular tissue for a shorter period of time compared to ontogeny 3. Surprisingly, at maturity, the stem macro-anatomy of all ontogenies in Nyctaginaceae are alike, especially the ones with successive cambia (e.g., Reichenbachia – ontogeny 3) or interxylary phloem forming bands (e.g., Bougainvillea, Colignonia – ontogeny 1). Species with interxylary phloem forming only phloem islands are more easily distinguished in stem topology, although the development follow virtually the same steps in species with patches/bands, except for the length of the coalescent cambium.
Evolutionary mechanisms are hard to be interpreted for the evolution of secondary vascular patterns in Nyctaginaceae due to multiple and complex developmental transitions (Fig. 15). Here, three processes are inferred to generate the stem diversity found in the family (Fig. 15): homeosis, heterochrony, and heterotopy. (1) The formation of interxylary phloem (ontogeny 1) in relation to a putative ancestor with regular anatomy seems likely to represent a case of homeosis, since the unusual activity of the cambium leads to the presence of phloem strands in the place of secondary xylem. Similar cases of homeosis in woody plants has been hypothesized for example in species with parenchymatized xylem, that is, in cases where non-lignified parenchyma occur where fibres, vessels and lignified axial parenchyma would be present (e.g., lianas, succulents) [10, 11]. (2) In the evolution of ontogeny 2 from ontogeny 1, the extra-fascicular cambium appeared, suggesting a case of heterotopy since the first vascular cambium arises in a different position from that present in the ancestor. In addition, at the structural level the development of ontogeny 2 is based on the earlier onset of formation of the cambial variant by suppressing one of the ontogenetic stages (i.e., formation of a cambium from the primary vascular system), therefore, it may also illustrate for the first time a case of predisplacement, a form of peramorphosis (heterochrony). In wood anatomy, most cases of heterochrony suggest the occurrence of prolonged juvenile characteristics into adult forms (paedomorphosis) [10, 94, 95], and a case of peramorphosis (hypermorphosis - evolution by developmental additions) is also suggested for the origin of successive cambia in Paullinia, Sapindaceae . Moreover, because ontogeny 2 evolved from ontogeny 1, this transition also requires modifications in the primary vascular system which indicates developmental changes that are regulated by an independent developmental module . Thus, modularity may also be a source for anatomical diversity in this group. The evolution of ontogeny 3 from ontogeny 2 implicates in the appearance of a regular cambium. This transition indicates that a partial regression to the state of the ancestor of the family occurred in this lineage, if we consider that the regular cambium occurs in the same position of the single cambium generating interxylary phloem. Similarly, the evolution of ontogeny 4 requires a reversion from the cambium with unusual activity to the regular cambium, and then a new cambium is formed constituting the successive cambia, which suggests an additional developmental event. Whether these interpretations would hold under a genetic developmental approach is something yet to be explored.
In the challenge to understand developmental changes during evolution, one can notice that evolutionary changes such as heterochrony and heterotopy are likely to involve fewer processes than other changes such as homeosis or evolutionary novelty . Regardless, these combinations of developmental processes as observed in Nyctaginaceae may be under complex gene regulation, given that multiple cellular and tissue processes are involved in the formation of each cambial variant  and that morphological fuzziness results from overlapping developmental programs . For example, it is likely that the loss-of-function related to receptor-like kinases (PHLOEM INTERCALATED WITH XYLEM - PXY), which positively regulates the WUSCHEL-RELATED HOMEOBOX4 (WOX4), and III HD ZIPs genes (e.g., PtrHB4) implicated in regulating the rate of cambial cell division and development of interfascicular cambium [98–102] could be involved in the development of interxylary phloem macromorphologies. In contrast, the superexpression of other class III HD ZIPs orthologous to Populus (e.g., popREVOLUTA) may result in formation of ectopic cambia [102, 103] which may be similar to the formation of de novo cambium in patterns of cambial variants with multiple cambia, as in cases of successive cambia. In addition, it is likely that the formation of the primary vascular system function as a module independent from the establishment of secondary growth since different secondary architectures can evolve from distinct pre-vascular conditions in the primary stem.
Sheathing axial parenchyma vs. conjunctive tissue: origin, classification, and functional significance.
The term conjunctive tissue has been applied predominantly in the context of cambial variants, particularly for successive cambia [18, 22, 29, 39, 68], but also in cases of interxylary phloem (=included phloem, ). In the context of cambial variants, conjunctive tissue is described as the parenchymatous or fibrous tissue between vascular increments (rings) derived from the meristematic zones that produce the new cambium in the successive cambia system. This interpretation has been maintained for cases of successive cambia in Nyctaginaceae . However, to describe the parenchymatous tissue bordering the conducting cells of phloem strands in species with interxylary phloem a different name (i.e., sheathing axial parenchyma) has been applied because this tissue originates from the phloem axial parenchyma formed by the main cambium , while conjunctive tissue is formed from remaining cells of the meristematic zone that originates the variant cambium [38, 61, 67]. As indicated in relation to the origin of the cambial variants, the sheathing axial parenchyma and conjunctive tissue as products of these two systems are also regarded as developmentally linked.
The spatial distribution of conjunctive tissue and sheathing axial parenchyma is one of the main aspects resulting in the diversity of stem macromophologies in Nyctaginaceae. Given their structural and organization similarities, these tissues are likely to develop the same functions indicated to wood axial parenchyma (e.g., storage, involvement in mechanical strength, defence against pathogens and in hydraulic maintenance - [87, 88, 89]). In addition, the abundant parenchyma present in some plants with cambial variants may also represent adaptive advantages for increased flexibility, mechanical strength and injury repair as suggested to climbing plants [20–22, 68]. Other functions have also been attributed to species with successive cambia (e.g., salt sequestration, xylem and phloem three-dimensional network – ). Curiously, successive cambia and interxylary phloem, are two types of cambial variants that can be now observed in species with distinct habits, from herbs to lianas and large trees [32,68,90,this study]. In any case, the capacity of producing multiple cambia or secondary phloem within the secondary xylem, in such intricate organization, might represent a beneficial physiological alternative to the typical regular growth of woody plants [29, 39, 86], given the multiple evolution of these cambial variants across angiosperms in both scandent and self-supporting plants. Experimental work is still needed to substantiate these hypotheses.
The impact of transitions in habits, habitats and cambial variants in the diversification of Nyctaginaceae
The diversification of Nyctaginaceae was most probably in the Middle Eocene (~48 Ma), when most of the extant angiosperm families were already established forming the contemporaneous tropical biomes . Other estimates for the split between Nyctaginaceae and close-related families assumes an interval lying between 13 and 33 Ma , as inferred for the divergence from Aizoaceae + Phytolaccaceae (e.g., 26 Myr, ).
Speciation/diversification rate increased in Nyctaginaceae 28.62 Ma, at the time of emergence of a group comprising the Bougainvilleeae and Pisonieae (‘‘B&P’’) clade + the Nyctagineae (“NAX”) clade , and has been maintained since then. There is not an apparent unique characteristic for this group that could explain its increase in diversification. However, different hypotheses have been pointed out for the high number of species in each tribe individually. For instance, a remarkable radiation of genera from the NAX clade occurred in deserts of North America, and they are associated with multiple evolutions of cleistogamy and edaphic endemism to grow on gypsum soils (Douglas & Manos, 2007); the B&P clade stands out by having most of the neotropical and large, woody species of the family, which include both the Guapira/Neea/Pisonia trees, as well as the shrubby-scandent or tree species of Bougainvillea . In addition, the evolution of fleshy anthocarps (the fruits of Nyctaginaceae) in the Guapira/Neea lineage and the likely appearance of endozoochory, seems to be one of the possible explanations for the rapid radiation of taxa of this lineage [42, 43].
Commicarpus has experienced a high turnover of species, where many species have been generated but also went extinct, as observed by the rise in both speciation and extinction rate that ultimately involve an increase in the diversification rate. This genus is one of several lineages of Caryophyllales where a diversification rate shift has been detected, indicating a very recent and rapid radiation . In some other caryophyllalean lineages, genome duplications (polyploidy species) were associated with diversification shifts, which was not identified in Commicarpus in that study sampling. Within the NAX clade, Commicarpus stands out for having few American species and being mostly diverse in Africa, with several species showing restricted distributions (endemics) in tropical regions, some of them also growing on gypsum or limestone [42, 112, 113].
Our results also suggest that there was no increase in diversification in the lineages containing lianas (e.g., Colignonia), therefore, contradicting previous hypotheses [33, 34]. This observation is noteworthy because it indicates for the first time with an explicit analysis that higher speciation rates correlated to the evolution of lianas seem not to hold, at least when the whole group has a cambial variant, as it is the case of Nyctaginaceae. On the other hand, we found out that there is a transition from regular to polycyclic eustele in the clade comprising the tribes Boldoeae+Colignonieae+Pisonieae+Bougainvillea+Nyctagineae, which is probably the main vascular character associated to an increase in diversification rate (Fig. 14). However, diversification rate shift results obtained with BAMM (Fig. 14) show that a slight increase in diversification rate occurred in a clade comprising Pisonieae+Bougainvillea+Nyctagineae, a less inclusive group compared to Boldoeae+Colignonieae+Pisonieae+Bougainvillea+Nyctagineae observed in the HiSSE results, thus possibly the shift to a polycyclic eustele (medullary bundles) type was a precursor, or a background variable, for diversification rate shift instead of a trigger . This may suggest that other factors can be involved in the diversification rate shifts within Nyctaginaceae, such as extrinsic variables like habitat occupation.