Secondary Phloem Diversity

Annals of Botany 116: 333–358, 2015
doi:10.1093/aob/mcv106, available online at www.aob.oxfordjournals.org
Secondary phloem diversity and evolution in Bignonieae (Bignoniaceae)

Marcelo R. Pace1,*, Suzana Alcantara3, Lúcia G. Lohmann2 and Veronica Angyalossy1
1
Laboratório de Anatomia Vegetal, Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do
Matão, 277, Cidade Universitária, CEP 05508-090, São Paulo, SP, Brazil, 2Laboratório de Sistemática Vegetal, Departamento
de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, Cidade Universitária, CEP 05508-090,
São Paulo, SP, Brazil and 3Laboratório de Sistemática de Plantas Vasculares, Departamento de Botânica, Centro de Ciências
Downloaded from https://academic.oup.com/aob/article/116/3/333/170093 by guest on 13 September 2021

Biológicas, Universidade Federal de Santa Catarina, CEP 88040-970, Florianópolis, SC, Brazil
* For correspondence. E-mail [email protected] or [email protected]
Received: 13 April 2015 Returned for revision: 19 May 2015 Accepted: 29 May 2015
Background and Aims Phloem evolution has been explored in the literature across very broad scales, either for
vascular plants as a whole or for major plant groups, such as the monocotyledons or the former dicotyledons.
However, it has never been examined in a way that would elucidate evolutionary shifts leading to the diversification
of phloem in single lineages. Therefore, the present study explores in detail the patterns of phloem evolution in the
tribe Bignonieae (Bignoniaceae). This group represents a particularly good model for phloem studies since it is
known to have a very conspicuous and diverse phloem.
Methods A total of 19 phloem characters were coded in 56 species from all 21 genera currently recognized in the
tribe Bignonieae, accounting for phloem wedge growth and for all the anatomical cell diversity encountered in the
phloem. Phloem evolution was explored by reconstructing ancestral character states using maximum-likelihood as-
sumptions with a time-calibrated molecular phylogeny for the group. Directionality and the effect of phylogenetic
transformations in the current variation of quantitative traits and evolutionary correlations of selected discrete
phloem traits were also tested under a maximum-likelihood approach.
Key Results Individual phloem features are quite diverse in the tribe, but generally conserved within smaller
clades. Contrasting phloem patterns were found when comparing major groups, with certain lineages having the
phloem marked by a background of phloem fibres where all other cells are embedded, tangentially arranged sieve
tubes and sieve-tubecentric parenchyma. In contrast, other lineages exhibited a scarcely fibrous phloem, regularly
stratified phloem, sieve tube elements in radial or diffuse arrangement, and diffuse parenchyma. We found signals
of directional evolution in fibre abundance and number of sieve areas, which increased in the ‘Fridericia and allies
extended clade’ and decreased in the ‘Multiples of four extended clade’, resulting in no signal of directionality
when the whole Bignonieae was considered. In contrast, no indication of directional evolution was found for the ax-
ial parenchyma, either in single clades within Bignonieae or in the entire tribe. Positive correlation was found be-
tween sieve element length and both sieve plate type and the presence of a storied structure. Correlated evolution
was also found between fibre abundance and several traits, such as sieve tube arrangement, sieve plate type, paren-
chyma arrangement, ray lignification and number of companion cells.
Conclusions The secondary phloem of Bignonieae is extremely diverse, with sister lineages exhibiting
distinct phloem anatomies derived from contrasting patterns of evolution in fibre abundance. Fibre abundance in
the tribe has diversified in correlation with sieve tube arrangement, sieve tube morphology, number of companion
cells and parenchyma type. The results challenge long-standing hypotheses regarding general trends in cell abun-
dance and morphological cell evolution within the phloem, and demonstrate the need to expand studies in phloem
anatomy both at a narrow taxonomic scale and at a broad one, such as to families and orders.
Key words: Phloem diversity, Bignonieae, Bignoniaceae, cambial variant, diversification, ancestral character state
reconstructions, directional evolution, lianas, sieve elements, parenchyma, sclerenchyma, fibres, correlated
evolution.
INTRODUCTION these cell types, their relative abundance in the tissue and their
spatial organization are all extremely diverse, although they
Phloem is a vascular tissue of vital importance to plants since it may be conserved within plant families, subfamilies and tribes
carries all the photosynthates produced by the leaves and sig- (Roth, 1981). Thus, these characteristics have been useful in de-
nalling molecules, such as mRNAs, to all plant parts. Formed scriptions and used as a tool to identify large plant groups
by more than one cell type, the secondary phloem is considered (Solereder, 1908; Zahur, 1959; Esau, 1969; Roth, 1981; Costa
a complex tissue composed of conducting cells (sieve et al., 1997). Despite all this diversity, few studies have ad-
elements), parenchyma (axial and radial) and generally scleren- dressed when and how all these different phloem cell types and
chyma (fibres and/or sclereids). The general morphology of their spatial organization evolved.
C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.
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334 Pace et al. — Secondary phloem diversity and evolution in Bignonieae
Most studies on phloem diversity have been descriptive

(Chattaway, 1953; Parameswaran and Liese, 1970; Roth, 1981; Interwedge
Angyalossy-Alfonso and Richter, 1991), and the only studies VP RP
RP
addressing phloem evolution were done on a large scale, con-
sidering either vascular plants as a whole (Hemenway, 1911,
1913; Zahur, 1959; Chavan et al., 1983, 2000) or major plant
groups, such as the monocotyledons (Cheadle and Whitford, Wedge
1941; Cheadle, 1948) or the former dicotyledons (Den Outer VP
1983, 1993). In these works, the authors mainly focused on
characteristics of the sieve elements and their correlation with
the relative abundance and morphology of other cell types, i.e.
parenchyma and sclerenchyma. Based on the observation of nu-

merous angiosperm plant families and the notion that phloem
evolution should parallel xylem evolution, general ‘trends of
phloem evolution’ have been proposed. Evolutionary trends
proposed for sieve elements include a decrease in length, an in-
crease in diameter and a gradual modification in the sieve plates
from compound to simple sieve plates, generating more effi- FIG. 1. Transverse section of Tynanthus cognatus. Wedges of variant phloem
cient conducting cells over time (Hemenway, 1913; Zahur, (VP) and interwedges with the regular phloem (RP).
1959; Esau, 1969; Den Outer, 1993). For parenchyma and scle-
renchyma, a gradually increasing trend in abundance of paren-
cambial variant results in the formation of two distinct types of
chyma, but a decrease in abundance of fibres, has been
secondary phloem in the same stem: a regular phloem and a
suggested, the latter being replaced by sclereids (Zahur, 1959).
variant phloem (Fig. 1) (Solereder, 1908; Dobbins, 1971; Pace
Such hypotheses were mainly based on empirical correlations
et al., 2011). The two phloem types were shown to have several
observed in sieve element morphology and sclerenchyma and
anatomical differences, including sieve tube element width,
parenchyma type and abundance, suggesting that sieve ele-
abundance of parenchyma, distribution of fibres and ray width
ments, parenchyma and sclerenchyma evolved together in the
(Pace et al., 2011). Given these differences, it has been sug-
phloem (Zahur, 1959). However, no further explicit hypotheses
gested that both phloem types in the tribe have evolved towards
have been proposed to add functional and/or adaptive explana-
a division of labour, with the regular phloem assuming the
tions to these general evolutionary trends observed. However,
function of storage and the variant phloem assuming the func-
these hypotheses have been challenged by authors such as
tion of photosynthate conduction (Pace et al., 2011), a phenom-
MacDaniels (1918), who suggested that the morphologies of
enon also reported in other plants in which a regular and variant
sieve tubes and the organization of other cells in the phloem
phloem coexist (Carlquist, 2013).
would be more related to physiological (including plant matu-
Here, we conducted a detailed anatomical study of the vari-
rity) and environmental aspects than phylogeny, but without
ant phloem of Bignonieae and investigated its pattern of evolu-
providing explicit explanations on this matter. Despite these
tion within the tribe using a time-calibrated phylogeny as
very broad analyses, to the best of our knowledge no studies
framework for the analyses (Lohmann et al., 2013). We focus
have investigated phloem evolution on a narrower scale, in a
on (1) exploring the dynamics of phloem wedge growth; (2) ex-
way that would elucidate evolutionary shifts leading to the di-
ploring phloem diversity in Bignonieae by delimiting phloem
versification of phloem in single lineages. Studies on a nar-
characters and character states according to their anatomical
rower scale would allow delimitation of the diversity present in
variation; (3) exploring the distribution of characters across the
lineages and specifically address directionality in the evolution
phylogeny, their ancestral character states and evolutionary
of phloem characters and whether correlated evolution has oc-
transitions within the tribe; (4) testing for directionality in the
curred between these characters. Therefore, the present study
evolution of four continuous traits, including sieve element
explored in detail the patterns of phloem evolution in the tribe
width, number of sieve areas per sieve plate and fibre and pa-
Bignonieae (Bignoniaceae). The family Bignoniaceae repre-
renchyma abundance; and (5) testing for evolutionary correla-
sents a particularly good model for phloem studies since it is
tion between specific pairs of characters involving sieve
known to have a very conspicuous (Roth, 1981) and diverse
element dimensions and fibre abundance.
phloem (Pace et al., 2011).
The tribe Bignonieae (Bignoniaceae, Lamiales) is a mono-
phyletic group (Spangler and Olmstead, 1999; Lohmann, 2006;
Olmstead et al., 2009) and contains approximately half the spe- MATERIALS AND METHODS
cies of the family, representing the most species-rich group of
Taxon sampling and anatomical procedure
lianas in the Neotropics (393 species in Bignonieae; Gentry,
1991; Lohmann, 2006). This group is particularly interesting We sampled the stems of 56 species representing all 21 genera
anatomically for having a cambial variant characterized by the currently recognized in Bignonieae (Lohmann and Taylor,
formation of four or multiples of four phloem wedges that fur- 2014) and accounting for all the known anatomical diversity
row the xylem (Schenck, 1893; Dobbins, 1971; Pace et al., recorded for the tribe (Schenck, 1893; Dos Santos, 1995).
2009; Pace and Angyalossy, 2013) (Fig. 1), a synapomorphy of Because the phylogenetic position of the monotypic
the tribe (Lohmann, 2006; Lohmann and Taylor, 2014). This Callichlamys is uncertain within Bignonieae (Lohmann, 2006;
Pace et al. — Secondary phloem diversity and evolution in Bignonieae 335
Lohmann et al., 2013), we only analysed the genus anatomi- IAWA Committee list (2016) for most terms and Pace et al.
cally, and data for it were not incorporated in the phylogenetic (2009) for phloem wedges and interwedges. Phloem wedges
analyses. Here we only consider the variant phloem of are characterized as the regions produced by the activity of a
Bignonieae since the regular interwedge phloem has been variant cambium that produces less secondary xylem and more
explored elsewhere (Pace et al., 2011). As the phloem is an secondary phloem. Interwedges are characterized as the regions
extremely fragile tissue, most samples were collected in their between phloem wedges, in which the cambium maintains reg-
natural habitat or from living collections (botanical gardens), ular activity (Pace et al., 2009) (Fig. 1). As a result of variant
with just a few specimens coming from wood collections (see cambial activity of the cambia within the phloem wedges, a
Appendix). All specimens had their transverse section surfaces variant phloem is formed (Fig. 1), while regular phloem is
clear-cut to allow better penetration of the fixative, and they located in the interwedges (Fig. 1). Furthermore, we use the
were immediately fixed in FAA 70 according to Berlyn and term ‘sieve-tubecentric axial parenchyma’ to refer to a sheath
Miksche (1976) or Karnovsky (1965) while in the field. After a of phloem present around the sieve tubes whenever the variant

few days under vacuum, samples were transferred to a conserv- phloem was characterized by a large amount of fibres forming
ing solution of 50–70 % ethanol. Collected samples were a background tissue in which all other cell types are embedded
standardized to have the same diameter (2 cm). This diameter is (IAWA Committee, 2016).
known to represent adult lianas already established in the can-
opy (Gerwing et al., 2006), with fully developed stems (Pace
et al., 2009), and it is the most abundant diameter found for lia-
Phylogenetic comparative analyses
nas growing in their natural habitats and already in their repro-
ductive stage (Schnitzer et al., 2006). To evaluate the patterns of phloem evolution, we performed
Before sectioning, all samples were softened in ethylenedi- statistical phylogenetic comparative analyses using a well-sup-
amine for up to 4 d (Carlquist, 1982), rinsed in distilled water ported time-calibrated molecular phylogeny of the Bignonieae
and gradually embedded in 1500 polyethylene glycol (Rupp, (Lohmann et al., 2013). This phylogenetic framework was
1964). Samples were subsequently sectioned with a sliding reconstructed based on a combined molecular dataset that
microtome with the aid of an anti-tearing resin made of included chloroplast (ndhF) and nuclear gene (PepC) sequences
expanded polystyrene (foam) dissolved in butyl acetate (Barbosa (Lohmann, 2006). Since a few species sampled in this study
et al., 2010). Sections were double-stained in astra blue and were not sampled in that phylogeny, we built a tree including
safranin (Bukatsch, 1972), or sometimes with tannic acid, fol- the sampled species along with all the non-sampled species by
lowed by ferric chloride and resorcin blue (lacmoid) whenever collapsing them into the least inclusive node that represented
we wanted to better visualize the sieve plates (Cheadle et al., genera or an existent subclade within the genera, in the most
1953). Sections were then mounted in Canada balsam to make recent generic classification of the group (Lohmann and Taylor,
permanent slides. Furthermore, some samples were cut into 2014). In addition, we pruned the species that were not sampled
cubes of 3 mm with phloem, cambium and xylem and embedded in our anatomical study so that the final tree only contained the
R
in HistoresinV (Leica Microsystems). In this case, sections were sampled taxa to which the relationships were known. This
made in a rotary microtome and stained in 005 % toluidine resulted in a totally resolved tree, which was then used for all
blue in glacial acetic buffer at pH 47 (O’Brien, 1964). the analyses performed here. We follow the clade names estab-
lished in Lohmann et al. (2013): ‘Fridericia and allies clade’,
which includes the genera Cuspidaria, Fridericia, Lundia,
Phloem description, character coding and terminology
Tynanthus and Xylophragma, and ‘Multiples of four clade’,
To properly analyse the development of phloem wedges and which includes the genera Amphilophium, Anemopaegma,
the variant phloem in Bignonieae, we performed detailed Bignonia, Mansoa and Pyrostegia. However, sometimes we
descriptions of their anatomy and development based on use both clades with additions: the ‘Fridericia and allies clade’,
sequential sections of collected stems. The variation in anatomi- together with their sister clade formed by Pachyptera,
cal traits among the analysed species was recorded as qualita- Pleonotoma and Manaosella, was termed ‘Fridericia and allies
tive or quantitative traits, depending on their mode of variation. extended clade’, and the ‘Multiples of four’ þ Dolichandra was
Sieve tube element length, sieve tube area, sieve tube diameter, termed ‘Multiples of four extended clade’.
percentage of phloem parenchyma and fibres in the phloem Characters were unordered in all analyses. Ancestral state
were calculated with ImageJ software, version 139 d (http:// reconstructions of discrete characters were performed in order
rsb.info.nih.gov/ij/), adopting the same procedures as described to identify major state transitions along the phylogeny, using
in Pace et al. (2011). The percentages were calculated using a maximum-likelihood assumptions as implemented in Mesquite
grid of 0.05 mm2 (50 000 mm2) to calculate the total area occu- 275 (Maddison and Maddison, 2009). Continuous characters
pied by each cell type, and at least four replications were made were optimized by parsimony, as implemented in Mesquite
for each specimen analysed, and the average of all measure- 275 (Maddison and Maddison, 2009).
ments was used to represent each species. The number of sieve We tested for directional evolution in the following traits: (1)
areas was calculated in at least 20 sieve tube elements by man- sieve tube diameters; (2) number of sieve areas per sieve plates;
ually counting the number of sieve areas in radial longitudinal (3) percentage of fibres; (4) percentage of axial parenchyma;
sections of stems. and (5) percentage of axial parenchyma. All analyses were per-
The anatomical terminology for phloem adopted here follows formed using the same phylogeny as that described above for
that proposed by Trockenbrodt (1990), Angyalossy-Alfonso the tribe Bignonieae (Lohmann et al., 2013), as well as major
and Richter (1991), Richter et al. (1996), Evert (2006) and the subclades within the tribe. This procedure was done in order to
test whether individual clades within the phylogeny had a dif- phloem wedges (hereafter termed ‘limiting rays’, a term
ferent evolutionary pattern when compared with the entire tribe. coined by Schenck (1893)).
The analyses used the maximum-likelihood (ML) Pagel’s
(1999) test of models of evolution. This model approach is
Phloem wedges types. When phloem wedges are first formed,
based on ML estimation for two alternative models of evolu-
they typically have straight lateral margins (Fig. 2A), and in
tion, i.e. model A, which corresponds to the standard constant-
some species it seems that the wedges remain straight through-
variance (also called Brownian motion) random-walk model,
out stem development (Fig. 2C). However, in many species the
and model B, which corresponds to the directional random-
formation of lateral steps at both sides of the phloem wedges is
walk model, equivalent to model A, but with one additional
common (Fig. 2B). These steps represent parts of a regular
parameter accounting for the directional rate of evolution of a
cambium on the sides of the phloem wedges that switch from
trait. Model B detects any directional evolutionary signal.
regular to variant activity. According to the pattern these steps
Likelihood ratio tests (LRTs) were performed to evaluate the

acquire, they may be symmetrically formed early in stem devel-
best-fit model. In addition to the test for the best fit of model A
opment, with each side of the phloem wedge having nearly the
(undirectional) or B (directional evolution), we also evaluate
same pattern of steps (Fig. 2D), or they can form lateral steps at
the effect of the tree-scaling parameters in the ML estimation in
different times, resulting in an asymmetrical pattern on both
both model A and model B. These parameters are denoted j, k
sides of the wedge, with one side having more steps than the
and d and allow the respective tests of tempo (gradual versus
other (Fig. 2E). Straight margins kept throughout stem develop-
punctuational evolution), phylogenetic associations and trait
ment are encountered solely in Tanaecium (Fig. 2C).
evolution (whether the phylogeny correctly predicts the cova-
Symmetrical phloem wedge steps are encountered in
riance patterns of the traits) and mode (early versus late evolu-
Cuspidaria, Tynanthus (these are sister groups; Fig. 2D) and
tion; Pagel, 1999). We performed these analyses as
Manaosella. All other genera display asymmetrical phloem
implemented in BayesTraits V2 in accordance with the specifi-
wedge steps (Fig. 2E).
cations in the companion manual.
Tests for correlated evolution between traits were performed
for the following characters: length and type of sieve plates and Formation of limiting rays The four to multiples of four equidis-
features related to increase in fibre abundance, such as sieve tant regions of the phloem wedges are known not to grow in
plate type, arrangement of sieve tubes, fibre lignification and girth, since they lack anticlinal divisions (shown in Pace et al.,
companion cell number. Since some of these features relate to 2009). Therefore, while the stem thickens in Bignonieae, multi-
continuous characters (e.g. sieve tube length), they were trans- seriate rays are formed on the sides of the phloem wedges in
formed into binary characters to allow the correlation analyses, order to accommodate secondary growth (Fig. 2C, arrows). The
and the characters and character states are presented in Table 3. limiting rays appear as a single structure, but they are actually
Correlations were performed in Mesquite 275 (Maddison and mixed in origin. The lateral outermost portion of the limiting
Maddison, 2009), using Pagel’s 1994 test of correlated charac- rays is produced by the regular cambium at the sides of the
ter evolution, which uses an ML approach, and the LRT to eval- phloem wedges (ray xylematic portion), while their innermost
uate models of correlated and independent evolution of any part is produced by the variant cambium from inside the
paired character states, using 1000 simulations (Midford and phloem wedge (ray phloematic portion). The limiting rays are
Maddison, 2006). adjacent to the entire variant phloem and leave a conspicuous
mark in the secondary xylem (Fig. 2C). Three groups can be
distinguished, depending on the lignification status: (1) limiting
RESULTS ray cells only lignified next to the xylem and non-lignified in
its phloematic portion (Fig. 3A); (2) limiting ray cells lignified
This study compared the secondary phloem of all 21 genera next to both xylem and phloem, but with a row of non-lignified
currently recognized in Bignonieae (Bignoniaceae) within a cells between the two parts (Fig. 3B); and (3) non-lignified lim-
phylogenetic framework. We first explored the outline and iting ray cells (Fig. 3C).
development of phloem wedges, then the character and charac- After the lateral steps are formed, some limiting rays
ter states delimited for the secondary phloem in detail accompa- start to be derived exclusively from the variant cambium
nied by analyses of ancestral character state reconstructions and (Fig. 2E). In the taxa in which the ray xylematic portion is
analyses on directional evolution, and separately a test of corre- lignified and the ray phloematic portion is non-lignified, the
lated evolution between phloem traits. All the quantitative and limiting rays will be exclusively non-lignified (except some-
qualitative data are summarized in Table 1, while the results of times when crossing the fibre bands), since now all the lim-
directionality analyses of continuous phloem traits are summar- iting rays from the cambium outward will be exclusively
ized in Table 2 and those of correlation analyses are summar- phloematic (Fig. 2E).
ized in Table 3. Ancestral character state reconstructions unambiguously
indicated that the ancestral condition of the tribe was the pres-
ence of limiting rays only lignified next to the xylem (Fig. 3D).
In addition, a single origin of non-lignified limiting rays was
Growth dynamics of phloem wedges
observed in Dolichandra (Fig. 3D), and at least three independ-
Here we explored two variable characters related to ent origins of double-faced lignification were observed, once in
phloem wedges: their general outline and the diversity in Adenocalymma salmoneum, once in the ‘Fridericia and allies
the lignification status of the wide rays that border the clade’ (except for Lundia, with reversal to rays lignified only to
A B

E
C D
FIG. 2. Growth dynamics of phloem wedges. (A, B) Transverse sections of Stizophyllum riparium. (A) At the early stage of development, the phloem wedges have
straight margins and no lateral steps. (B) At later stages of development, lateral steps are formed on the margins of phloem wedges (arrowheads), while the stem
thickens. (C) Transverse section of Tanaecium pyramidatum: phloem wedges not forming lateral steps. Note the conspicuous multiseriate limiting rays on both sides
of the phloem wedges (arrows). (D) Transverse section of Tynanthus cognatus: phloem wedges with early and regular formation of lateral steps. (E) Transverse sec-
tion of Fridericia samydoides: phloem wedges with irregular formation of steps on each side of the phloem wedge. Scale bars ¼ 3 mm.
the xylem face) and once in the clade formed by Martinella and Conducting phloem Sieve elements on the conducting
Stizophyllum (Fig. 3D). phloem are generally grouped in multiples (Fig. 4A), but are
sometimes solitary. The sieve elements are usually >500 mm in
length, as seen in tangential section (Fig. 4B), and P-protein is
frequently found as a slime plug at the sieve plates (Fig. 4B).
Phloem diversity: characters, ancestral state reconstructions Inside the wedges, rays are always uniseriate to biseriate and
and tests of directional evolution shorter than 1 mm in tangential section (Fig. 4B), except when
Variant secondary phloem is located in phloem wedges (Fig. these are limiting rays. When a ray borders a group of sieve
1) and exhibits remarkable differences among genera. elements, their companion cells are usually adjacent to the ray
Nevertheless, here we first treat the features that are common to cells (Fig. 4C). Fibres in the fibre bands are more thick-walled
all genera and then discuss the variable characters, their ances- than xylem fibres (Fig. 4A, arrows) and frequently exhibit a
tral character state reconstructions and the results of directional polylamellate structure. Furthermore, fibres possess a distinc-
evolution. tive appearance, being square to rectangular in shape, as seen in
X VC

X
VC VC
VC
A B C
D Fridericia and allies
Adenocalymma tanaecicarpum
Anemopaegma chamberlaynii
Amphilophium magnoliifolium
Adenocalymma neoflavidum
Adenocalymma salmoneum
Adenocalymma divaricatum
Adenocalymma peregrinum
Adenocalymma bracteatum
Amphilophium paniculatum
Adenocalymma flaviflorum
Amphilophium bracteatum
Amphilophium crucigerum
Adenocalymma comosum
Xylophragma myrianthum
Amphilophium elongatum
Adenocalymma nodosum
Anemopaegma robustum
Dolichandra quadrivalvis
Tanaecium pyramidatum
Dolichandra unguiculata
Pleonotoma stichadenia
Pleonotoma tetraquetra
Dolichandra unguis cati
Perianthomega vellozoi
Mansoa onohualcoides
Bignonia campanulata
Fridericia samydoides
Tanaecium bilabiatum
Stizophyllum riparium
Anemopaegma laeve
Manaosella cordifolia
Cuspidaria convoluta
Pleonotoma albiflora
Bignonia corymbosa
Neojobertia mirabilis
Fridericia platyphylla
Neojobertia sp. nov.

Tynanthus cognatus
Bignonia capreolata
Fridericia conjugata
Lundia corymbifera
Pyrostegia venusta
Bignonia magnifica
Cuspidaria pulchra
Tynanthus elegans
Fridericia speciosa
Martinella obovata
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
To the xylem face

To both faces, with interior non-lignified
Do not lignify
FIG. 3. Limiting ray lignification (variant portion). (A) Transverse section of Pyrostegia venusta. Limiting rays lignify only next to the xylem (white arrow), with its
phloem portion non-lignified (black arrow). (B) Transverse section of Tynanthus elegans. Limiting rays are lignified next to the xylem and phloem (black arrows),
with a row of cells remaining non-lignified in the middle portion (white arrow). (C) Transverse section of Dolichandra unguiculata. Limiting rays do not lignify,
even in the xylem (arrows). (D) Ancestral character state reconstruction of limiting ray lignification. VC, variant cambium; X, xylem Scale bars ¼ 200 mm.
transverse section (Fig. 4A). Radial sieve elements are rare, but expansion of the phloem parenchyma cells. Ray dilatation is
were found in many species and may be widespread in the rarely seen, but when present it occurs in the outermost part of
tribe. the wedge (Fig. 4E). A higher degree of crystal and starch accu-
mulation is commonly observed in the non-conducting phloem
Non-conducting phloem The cessation of function in variant (Fig. 4F) and sclerification of parenchyma cells is also com-
phloem usually starts 8–12 files of cells away from the mon. In species in which the fibres are very abundant, no col-
cambium. The first sign of loss of function is seen as loss of lapse of sieve tube elements is seen (Fig. 4E). On the other
protoplast in the companion cells, followed by accumulation of hand, for species with fewer fibres, total or partial collapse is
definitive callose at the sieve plates (Fig. 4D) and/or the evident (Fig. 5A).
Ph

CZ
X
A B C
R R
D E F
FIG. 4. Variant phloem, general aspects. (A–C) Conducting phloem of Bignonia magnifica. (A) Transverse section. Sieve tube elements grouped in multiples, fibre
bands with rectangular-shaped fibres that are more thick-walled than the xylem fibres (arrows). Rays uniseriate to biseriate. (B) Longitudinal tangential section.
Sieve tube elements longer than 500 mm and with conspicuous P-protein near the sieve plates forming a slime plug, rays uniseriate to biseriate and shorter than
1 mm. (C) Transverse section. Whenever a ray borders a group of sieve elements, their companion cells tend to lie adjacent to the ray cells. (D–F) Non-conducting
phloem. (D) Transverse section of Xylophragma myrianthum. Massive definitive callose deposition at the sieve plates (arrows). (E) Transverse section of
Neojobertia sp. nov. Rays rarely dilate in the variant phloem, and when they dilate it occurs only in the outermost part of the phloem wedges (arrowheads). (F)
Longitudinal tangential section of Bignonia magnifica. Large amounts of starch accumulate in the parenchyma cells of the non-conducting phloem. Scale bars:
(A, F) ¼ 100 mm; (B) ¼ 200 mm; (C–D) ¼ 50 mm; (E) ¼ 500 mm.
Arrangement of sieve tube elements Three different types of

Variable characters of the variant phloem
arrangement can be found in Bignonieae: (1) predominantly
Sieve tube width Sieve tube area, a continuous character, was diffuse (Fig. 5A), such as that found in Amphilophium,
calculated and analysed for directionality, revealing absence of Callichlamys and Manaosella; (2) predominantly in radial mul-
directionality for this character within the tribe (Table 2). tiples (Fig. 5B), such as that found in Anemopaegma, Bignonia,
Furthermore, evolution of widths seems to have been independ- Dolichandra, Mansoa, Martinella, Pachyptera, Perianthomega,
ent of branch lengths, occurring in a punctuated fashion Pyrostegia and some Adenocalymma; and (3) predominantly in
(j ¼ 0), independently of the phylogeny (k ¼ 0), and with most tangential multiples, such as that found in the entire ‘Fridericia
characters evolving close to the terminals (species-specific and allies clade’ as well as in Pleonotoma, Stizophyllum and
adaptation; d ¼ 3). some Adenocalymma (Fig. 5C).

A B C
D 'Multiples of four clade' Fridericia and allies
Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata
Lundia corymbifera
Pyrostegia venusta
Bignonia magnifica
Cuspidaria pulchra
Tynanthus elegans
Fridericia speciosa
Martinella obovata
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Predominantly diffuse
Predominantly radial core Bignonieae
Predominantly tangential
FIG. 5. Sieve tube arrangement in variant phloem. (A) Transverse section of Amphilophium crucigerum, with sieve tube elements diffusely arranged (arrows). (B)
Transverse section of Mansoa difficilis, with sieve tube elements radially arranged (arrows). (C) Transverse section of Cuspidaria convoluta, with sieve tubes tangen-
tially arranged (arrows). (D) Ancestral character state reconstruction of sieve tube element arrangement. Scale bars ¼ 100 mm.
Ancestral character state reconstructions unambiguously ancestral state was unambiguously reconstructed as having a
reconstructed several character states at the most inclusive tangential arrangement of the sieve tubes (Fig. 5D).
nodes (e.g. the ‘Fridericia and allies clade’ and the ‘Multiples
of four clade’; Fig. 5D), but ambiguously reconstructed the Sieve plates As seen in radial section, sieve tube elements have
ancestral state of the tribe (Fig. 5D), assigning both radial and simple (Fig. 6A) to compound sieve plates (Fig. 6B–C).
tangential arrangements with 50 % probability. The node for Compound sieve plates have few sieve areas (from three to
the core Bignonieae, which excludes Perianthomega, assigned five; Fig. 6B) or many sieve areas (>20; Fig. 6C), or even up to
54 % probability that the ancestral state of this node had a tan- 40 sieve areas. Sieve tube elements with simple sieve plates are
gential arrangement of sieve tubes (Fig. 5D). Adenocalymma shorter than 500 mm, while those with compound sieve plates
was a very diverse genus in sieve tube arrangement, but its are longer than this (Fig. 6), an observation confirmed by the

A B C
D Perianthomega vellozoi
E Perianthomega vellozoi
Amphilophium magnoliifolium Amphilophium magnoliifolium
Amphilophium bracteatum Amphilophium bracteatum
'Multiples of four extended clade'

Amphilophium crucigerum Amphilophium crucigerum
Bignonia capreolata Bignonia capreolata
Bignonia prieurei
Bignonia prieurei
Bignonia corymbosa
Bignonia corymbosa
Bignonia binata
Bignonia binata
Bignonia magnifica
Bignonia magnifica
Anemopaegma laeve
Anemopaegma laeve
Pyrostegia venusta
Pyrostegia venusta
Mansoa difficilis
Mansoa difficilis
Mansoa standleyi
Mansoa standleyi
Pleonotoma albiflora Pleonotoma tetraquetra
Manaosella cordifolia Pleonotoma albiflora
'Fridericia and allies extended clade'

Pachyptera kerere Manaosella cordifolia
Tanaecium bilabiatum Pachyptera kerere
Tanaecium pyramidatum Tanaecium bilabiatum
Tynanthus elegans Tanaecium pyramidatum
Tynanthus cognatus Tynanthus elegans

Cuspidaria pulchra Tynanthus cognatus
Cuspidaria convoluta Cuspidaria pulchra
Lundia damazioi Cuspidaria convoluta
Lundia longa Lundia damazioi
Lundia corymbifera Lundia longa
Xylophragma myrianthum Lundia corymbifera
Fridericia chica Xylophragma myrianthum
Fridericia speciosa Fridericia chica
Fridericia conjugata Fridericia speciosa
Fridericia samydoides Fridericia conjugata
Fridericia platyphylla Fridericia samydoides
Martinella obovata Fridericia platyphylla
Stizophyllum riparium Martinella obovata
Adenocalymma bracteatum Stizophyllum riparium
Adenocalymma tanaecicarpum 0 to 4
Adenocalymma salmoneum 4 to 8 Adenocalymma tanaecicarpum
Adenocalymma nodosum 8 to 12 Adenocalymma salmoneum
Adenocalymma peregrinum 12 to 16 Adenocalymma nodosum
Adenocalymma neoflavidum 16 to 20 Adenocalymma peregrinum
Adenocalymma flaviflorum 20 to 24 Adenocalymma neoflavidum
Adenocalymma comosum 24 to 28 Adenocalymma flaviflorum
Adenocalymma divaricatum 28 to 32 Adenocalymma comosum
Mostly simple Neojobertia mirabilis 32 to 36 Adenocalymma divaricatum
Compound Neojobertia sp. nov. 36 to 40 Neojobertia mirabilis
40 to 44 Neojobertia sp. nov.
FIG. 6. Sieve plate type in variant phloem. (A) Longitudinal radial section of Amphilophium crucigerum. Sieve tube elements bearing simple sieve plates. Detail of a
simple sieve plate with large sieve pores, as seen in transverse section. (B) Longitudinal radial section of Bignonia campanulata. Sieve tube elements bear compound
sieve plates with few sieve areas. (C) Longitudinal radial section of Fridericia samydoides. Sieve tube elements bear compound sieve plates with many sieve areas.
(D) Ancestral character state reconstruction of the sieve plate types. (E) Ancestral character state reconstruction of the average number of sieve areas per sieve plate.
Scale bar ¼ 50 mm.

A B C
D
Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata
Lundia corymbifera
Pyrostegia venusta
Bignonia magnifica
Cuspidaria pulchra
Tynanthus elegans
Fridericia speciosa
Martinella obovata
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
One per sieve element

Two–three per sieve element at the same side
Two per sieve element at different sides
FIG. 7. Number of companion cells per sieve tube element in the variant phloem. (A) Transverse section of Perianthomega vellozoi. Sieve tube elements are accom-
panied by one companion cell (arrows). (B) Transverse section of Tynanthus cognatus. Sieve tube elements are accompanied by two or three companion cells lying
at the same side of the sieve tube (arrows). (C) Transverse section of Adenocalymma flaviflorum. Sieve tube elements are accompanied by two companion cells, each
at one corner of the sieve tube element (arrows). (D) Ancestral character state reconstruction of the number of companion cells per sieve tube element. Scale
bars ¼ 50 mm.
correlation analysis (see Correlation analyses section). (Fig. 6E). Tree-scaling parameters (Table 2) indicated that evo-
Ancestral character state reconstructions unambiguously indi- lution in this clade was punctuational (j ¼ 00), with a high
cated compound sieve plates as the ancestral state in the tribe phylogenetic signal (k ¼ 094) and with most changes occurring
(Fig. 6D), with two independent origins for simple sieve plates, early in the phylogeny (adaptive radiation; d ¼ 054).
one in Perianthomega and another in Amphilophium (Fig. 6D). In the clade that contains the ‘Fridericia and allies extended
When the average number of sieve areas (Table 1) was clade’ (Fig. 6E), no directional evolution was detected in the
mapped as a continuous character, the ancestral condition for number of sieve areas (Table 2). However, here we observed
the tribe was reconstructed as having approximately three sieve the genera with higher numbers of sieve areas per sieve plate,
areas per sieve plate. We found no signal of directional evolu- with the ancestor of Manaosella and Pleonotoma showing 20
tion when the tribe was considered as a whole. However, two sieve areas per sieve plate and Pleonotoma tetraquetra with
major clades exhibited contrasting patterns. In the ‘Multiples of sieve plates having >30 sieve areas (Table 1). Tree-scaling
four extended clade’, sieve areas in the sieve plates decreased parameters (Table 2) indicated that the evolution of this charac-
in number towards most terminal nodes (Fig. 6E; Table 2), with ter was more conspicuous in the longer branches (j ¼ 228),
Amphilophium bearing mostly simple sieve plates and an ances- with high phylogenetic signal (k ¼ 10) and occurring early in
tor inferred as bearing sieve plates with two sieve areas the phylogeny (adaptive radiation; d ¼ 030).
Pc
Pc
Pc Pc
Pc
Pc Pc
Pc

Pc
A B
C Fridericia and allies
Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata
Lundia corymbifera
Pyrostegia venusta
Bignonia magnifica
Cuspidaria pulchra
Tynanthus elegans
Fridericia speciosa
Martinella obovata
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Diffuse
Sieve-tubecentric
FIG. 8. Phloem parenchyma type in the variant phloem. (A) Transverse section of Perianthomega vellozoi. Diffuse phloem parenchyma. Radial rows were also found
in the phloem (here shown with asterisks). (B) Transverse section of Neojobertia sp. nov. Sieve-tubecentric phloem parenchyma. (C) Ancestral character state recon-
struction of phloem parenchyma type. Pc, axial parenchyma cell. Scale bar ¼ 50 mm.
Companion cell Companion cells always accompany the sieve sieve tube elements with two or three companion cells lying at
tube elements in one or more of their corners. However, the the same side of the sieve tube element in the ‘Fridericia and
number and position of the companion cells are variable. In allies clade’ (except from Lundia), Pleonotoma and
most Bignonieae a single companion cell is found per sieve Stizophyllum (Fig. 7D), but a single origin of two companion
tube element (Fig. 7A, D), while two or three companion cells cells lying at the two opposing sides of the sieve element in
lying at the same corner of the sieve tube element are found in Adenocalymma, although the ancestor of Adenocalymma was
Cuspidaria, Fridericia, some Lundia, Pleonotoma, reconstructed as having a single companion cell lying on the
Stizophyllum, Tanaecium, Tynanthus and Xylophragma corner of the sieve tube (Fig. 7D).
(Fig. 7B) or, as in some Adenocalymma, two companion cells
per sieve tube element, each lying on an opposite corner of the
element (Fig. 7C). Perianthomega is polymorphic, sometimes Axial phloem parenchyma Axial parenchyma generally occupies
having just one and sometimes two companion cells, lying at <50 % of the transverse area (Table 1). Short radial rows of
opposite sides of the sieve tube. three to seven cells are ubiquitous in the variant phloem, inter-
Ancestral character state reconstructions revealed that the secting other cell types. In addition to these radial rows, two
ancestral state in the tribe was a single companion cell per sieve types of arrangements were delimited: (1) diffuse (Fig. 8A) and
element (Fig. 7D), with at least three independent origins of (2) sieve-tubecentric (Fig. 8B). Sieve-tubecentric axial

A B C
Fridericia and allies

D
Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata
Lundia corymbifera
Pyrostegia venusta
Bignonia magnifica
Cuspidaria pulchra
Tynanthus elegans
Fridericia speciosa
Martinella obovata
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Absent
Present when touching the fibres
Present in random portions
FIG. 9. Ray lignification on the variant phloem. (A) Transverse section of Amphilophium crucigerum. Rays are never lignified (arrows). (B) Transverse section of
Adenocalymma peregrinum. Rays lignify only when touching the fibre bands (arrowheads). (C) Transverse section of Tanaecium pyramidatum. Rays are lignified
(arrowhead) or non-lignified (arrow), randomly distributed. (D) Ancestral character state reconstruction of ray lignification. Scale bars (A) ¼ 200 mm; (B,
C) ¼ 50 mm.
parenchyma forms a sheath of cells around each sieve tube (k ¼ 028), accelerating towards the terminals (species-specific
element (Fig. 8B). adaptation; d ¼ 3).
Ancestral character state reconstruction revealed that the
ancestral state for the tribe was the diffuse axial parenchyma
type (Fig. 8C), with multiple independent origins of the sieve- Lignification of the phloem rays Rays are predominantly uniseri-
tubecentric: once in Pleonotoma, once in the ‘Fridericia and ate to biseriate in the variant phloem (Figs 4B and 9), except for
allies clade’, once in Neojobertia and probably more than once the limiting rays. The diversity of rays is related to their state of
in Adenocalymma (Fig. 8C). lignification. Three different states of lignification are encoun-
When the axial parenchyma of the variant phloem was ana- tered: (1) non-lignified, where the rays are never lignified, not
lysed for directional evolution, we encountered no directional- even when crossing the fibre bands (Fig. 9A); (2) rays always
ity (Table 2). Furthermore, tree-scaling parameters indicated lignified when crossing the fibre bands (Fig. 9B); and (3) rays
that the axial parenchyma evolved independently of branch randomly lignified, sometimes lignified when touching the fibre
lengths (gradually, j ¼ 11) and independently of phylogeny bands and sometimes lignified far from them (Fig. 9C).

A B C
D Amphilophium elongatum E Amphilophium elongatum
'Multiples of four extended clade'

10 % Bignonia capreolata
Bignonia capreolata
Bignonia prieurei
Bignonia prieurei
Bignonia corymbosa
Bignonia corymbosa
Bignonia binata
Bignonia binata
Bignonia campanulata 21 %
Bignonia magnifica
Bignonia magnifica
Anemopaegma laeve Anemopaegma laeve
Anemopaegma chamberlaynii Anemopaegma chamberlaynii
Anemopaegma robustum Anemopaegma robustum

Pyrostegia venusta
Pyrostegia venusta 15 %
Mansoa difficilis Mansoa difficilis
Mansoa onohualcoides 23 % Mansoa onohualcoides
Mansoa standleyi Mansoa standleyi
Dolichandra unguiculata Dolichandra unguiculata

Dolichandra unguis cati Dolichandra unguis cati
Dolichandra quadrivalvis Dolichandra quadrivalvis
Pleonotoma stichadenia Pleonotoma stichadenia
Pleonotoma tetraquetra Pleonotoma tetraquetra
Pleonotoma albiflora Pleonotoma albiflora
Manaosella cordifolia Manaosella cordifolia
Pachyptera kerere Pachyptera kerere
Tanaecium bilabiatum Tanaecium bilabiatum
Tanaecium pyramidatum Tanaecium pyramidatum
Tynanthus elegans Tynanthus elegans
Tynanthus cognatus 24 % Tynanthus cognatus

Cuspidaria pulchra Cuspidaria pulchra
Cuspidaria convoluta Cuspidaria convoluta
Lundia damazioi Lundia damazioi
Lundia longa Lundia longa
Lundia corymbifera Lundia corymbifera
Xylophragma myrianthum Xylophragma myrianthum
Fridericia chica 32 % Fridericia chica
Fridericia speciosa Fridericia speciosa
Fridericia conjugata Fridericia conjugata
Fridericia samydoides Fridericia samydoides
Fridericia platyphylla 34 % Fridericia platyphylla
Martinella obovata Martinella obovata
Stizophyllum riparium Stizophyllum riparium
Adenocalymma bracteatum Adenocalymma bracteatum
Adenocalymma tanaecicarpum Adenocalymma tanaecicarpum
1·7 to 7·4
Adenocalymma salmoneum Adenocalymma salmoneum
7·4 to 13·1
Adenocalymma nodosum Adenocalymma nodosum
13·1 to 18·8
Adenocalymma peregrinum Adenocalymma peregrinum
18·8 to 24·5
Adenocalymma neoflavidum Adenocalymma neoflavidum
24·5 to 30·2
30·2 to 35·9 Adenocalymma flaviflorum
35·9 to 41·6 Adenocalymma comosum
41·6 to 47·3 Adenocalymma divaricatum
Scarcely fibrous Neojobertia mirabilis
47·3 to 53 Neojobertia mirabilis
Semi fibrous 53 to 58·7 Neojobertia sp. nov.
Fibrous 58·7 to 64·4
FIG. 10. Phloem types according to their fibre arrangement in variant phloem. (A) Transverse section of Callichlamys latifolia, a scarcely fibrous species, with more
than ten cells of phloem parenchyma and sieve tube elements between fibre bands (arrows). Note that the phloem fibres (arrows) are thicker than the xylem fibres
(arrowheads). (B) Transverse section of Adenocalymma comosum, a semifibrous species, with fewer than ten cells of phloem parenchyma and sieve tube elements
between fibre bands (arrows). Note the undulated arrangement of the fibre bands. (C) Transverse section of Xylophragma myrianthum, a fibrous species, with the
fibres forming a background tissue where other cell types are embedded. (D) Ancestral character state reconstruction of phloem type evolution. (E) Ancestral charac-
ter state reconstruction of the percentage of fibres per tranverse area. Scale bars (A, B) ¼ 200 mm; (C) ¼ 120 mm.

A B C

Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus

Bignonia capreolata
Lundia corymbifera
Tynanthus elegans
Bignonia magnifica
Martinella obovata
Pyrostegia venusta
Cuspidaria pulchra
Pachyptera kerere
Fridericia speciosa
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Absent
Present in phloem parenchyma and sieve elements
Present for axial and radial elements
FIG. 11. Storied elements in the variant phloem. (A) Longitudinal tangential section of Callichlamys latifolia, without a storied structure. (B) Longitudinal tangential
section of Amphilophium elongatum, with axial elements storied. (C) Longitudinal tangential section of Dolichandra unguis-cati, with radial and axial elements
storied. (D) Ancestral state reconstruction of the storied structure evolution. Scale bars ¼ 200 mm.
Ancestral character state reconstruction revealed that the Fibre Phloem fibres are generally more thick-walled than xylem
ancestral state for the tribe was having rays that lignify only fibres, as seen in transverse section (Fig. 10A, C). Depending on
when touching the fibre bands (Fig. 9D). Six independent ori- the abundance of fibres, it is possible to qualitatively divide the
gins of rays that never lignify have occurred: once in variant phloem into three types: (1) scarcely fibrous; (2) semifi-
Perianthomega, once in Amphilophium, once in Bignonia brous; and (3) fibrous. Scarcely fibrous phloem is marked by the
magnifica, once in Dolichandra, once in Pleonotoma (except presence of fibre bands two to five cells wide that alternate with
P. albiflora) and once in Lundia corymbifera. The ancestral more than ten cells of phloem parenchyma and sieve tube ele-
state for both Bignonia and Lundia was having rays that lignify ments (Fig. 10A). Semifibrous phloem has undulated fibre bands
when touching the fibre bands (Fig. 9D). The ‘Fridericia and that alternate with fewer than ten cells of sieve elements and
allies clade’ is marked by the exclusive presence of rays that phloem parenchyma between them (Fig. 10B). Finally, fibrous
lignify in random portions, except for a reversal in Lundia phloem has fibres that are closely arranged, forming a back-
corymbifera (Fig. 9D). ground tissue in which all other cells are embedded (Fig. 10C).

A B C

Anemopaegma laeve

Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata
Tynanthus elegans
Bignonia magnifica
Martinella obovata
Lundia corymbifera
Pyrostegia venusta
Fridericia speciosa
Cuspidaria pulchra
Pachyptera kerere
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Acicular and navicular

Prismatic
Styloid
Absent
FIG. 12. Crystal types in the phloem and ray parenchyma. (A) Transverse section of Perianthomega vellozoi: acicular crystals (arrows). (B) Transverse section of
Pleonotoma tetraquetra: prismatic crystals (arrows). (C) Transverse section of Neojobertia sp. nov.: elongate crystal (arrow) and acicular crystals (arrowhead). (D)
Ancestral character state reconstruction of crystal type in the phloem and ray parenchyma. Scale bars (A, B) ¼ 30 mm; (C) ¼ 20 mm.
Ancestral state reconstruction of fibre band types revealed four extended clade’, while other lineages exhibited directional
that the ancestral state for the tribe was the scarcely fibrous evolution towards an increase in fibre abundance, such as the
type, with well-spaced fibre bands (stratified phloem), and mul- ‘Fridericia and allies extended clade’ (Table 2). Fibres in the
tiple parallel origins of the two other types (Fig. 10D). The tribe as a whole seems to be evolving toward higher stasis in
entire ‘Fridericia and allies clade’, Stizophyllum, Neojobertia the long branches (Table 2), in a more punctuated fashion
and Pleonotoma have fibrous phloems. (j ¼ 027), with closely related taxa resembling each other
When the percentage of fibres per total phloem area (k ¼ 081), within a mode compatible with Brownian motion
(Table 1) was mapped as a continuous character (Fig. 10E), fol- (default gradualism; d ¼ 133).
lowed by directional analysis (Table 2), it was seen that the When the two major lineages of the tribe were analysed for
tribe as a whole exhibited no sign of directional evolution. parameters of evolution, contrasting models were demonstrated
However, when certain lineages were considered separately again. In the ‘Fridericia and allies extended clade’, the parame-
within the tribe, two contrasting results were found. Some line- ter showed that this lineage differed from the tribe in having
ages exhibited directional evolution towards a decrease in the minimal effect of phylogeny (k ¼ 033) and accumulating
amount of fibres (Table 2; Fig. 10E), such as the ‘Multiples of changes in the long branches (species-specific adaptation;

A
A B
B C
C

Anemopaegma laeve
Bignonia corymbosa
Tynanthus cognatus
Bignonia capreolata

Pyrostegia venusta
Lundia corymbifera
Bignonia magnifica
Tynanthus elegans
Cuspidaria pulchra
Fridericia speciosa
Pachyptera kerere
Martinella obovata
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Absent
Present in non-lignified cells
Present in lignified cells
Present in both lignified and non-lignified cells
FIG. 13. Location of crystals according to the lignification state of the parenchyma cells. (A) Transverse section of Bignonia magnifica. Acicular crystals are present
in non-lignified axial and ray parenchyma cells. (B) Transverse section of Pleonotoma tetraquetra. Prismatic crystals are present in the non-lignified ray cells. (C)
Transverse section of Adenocalymma divaricatum. Prismatic crystals are present in lignified ray cells. (D) Ancestral character state reconstruction of the presence
and place of occurrence of the crystals. Scale bars (A–C) ¼ 50 mm.
d ¼ 28). The ‘Multiples of four extended clade’ differed only Ancestral character state reconstruction showed that the
in the fact that most changes occurred early in the phylogeny, ancestral state in the tribe was unambiguously a non-storied
followed by stasis, a scenario compatible with adaptive radia- structure (Fig. 11D), with three independent origins of a storied
tion (d ¼ 068). structure: once in Amphilophium, once in Perianthomega and
once in Dolichandra (Fig. 11D).
Storied structure A storied structure is absent in most genera
(Fig. 11A), being present exclusively in Amphilophium,
Dolichandra and Perianthomega (Fig. 11B–D). In both
Crystal inclusions in the secondary phloem
Amphilophium and Perianthomega, only the axial elements are
storied (parenchyma, sieve tube elements and fibres; Fig. 11B), The presence of crystals is widespread in Bignonieae.
while in Dolichandra both axial and radial elements are storied However, their type and location are variable. Three distinct
(Fig. 11C). types of crystals are found in the axial and ray parenchyma: (1)

A B
C
Multiples of four + Dolichandra

Anemopaegma laeve
Tynanthus cognatus
Bignonia capreolata
Bignonia corymbosa

Tynanthus elegans
Pyrostegia venusta
Cuspidaria pulchra
Lundia corymbifera
Bignonia magnifica
Pachyptera kerere
Martinella obovata
Fridericia speciosa
Mansoa standleyi
Bignonia prieurei
Lundia damazioi
Mansoa difficilis
Bignonia binata
Fridericia chica
Lundia longa
Absent
Acicular
Prismatic
Styloid
FIG. 14. Types of crystal in fibre-associated crystalliferous parenchyma. (A) Transverse section of Perianthomega vellozoi crystalliferous parenchyma sheath bearing
acicular crystals (arrows). (B) Transverse section of Mansoa standley: crystalliferous parenchyma sheath bearing prismatic crystals (arrows). (C) Ancestral character
state reconstruction of type of crystals in fibre-associated crystalliferous parenchyma. Scale bar ¼ 50 mm.
acicular (Fig. 12A), (2) prismatic (Fig. 12B) and (3) styloid/ Pleonotoma (Fig. 12D). Two Adenocalymma species (A. laeve
elongate (Fig. 12C). and A. robustum) are polymorphic, with prismatic and acicular
Ancestral character state reconstruction revealed that the crystals co-occurring (Fig. 12D). Styloid and elongate crystals
ancestral state for the tribe was the presence of acicular crystals are found exclusively in four of the analysed species:
(Fig. 12D), with two to four independent origins of phloem and Adenocalymma comosum, Neojobertia sp. nov., Bignonia cam-
ray parenchyma bearing prismatic crystals, once in the clade panulata and Bignonia capreolata. However, except for
formed by Adenocalymma and Neojobertia and another in B. capreolata, the other species are polymorphic, always having
Overall phloem anatomy Morphology of sieve elements Storied structure

Scarcely to semi-fibrous
Stratified phloem

Non-stratified phloem
Fibrous
FIG. 15. Drawing summarizing the two contrasting phloem anatomies present in Bignonieae (Bignoniaceae).
acicular crystals mixed with the styloid/elongate crystals, and clade’, except Amphilophium, in Manaosella cordifolia, in
co-occurring with prismatic crystals in the case of Neojobertia Fridericia and in Adenocalymma (Fig. 13D).
sp. nov. (Fig. 12C). In species with scarcely fibrous and semifibrous phloem,
Crystals are found exclusively in the non-lignified axial and parenchyma sheaths bearing crystals are common around the
ray parenchyma cells (Fig. 13A, B), exclusively in lignified fibre bands (Fig. 14A, B). Ancestral state reconstruction
cells (Fig. 13C), and in both lignified and non-lignified cells. revealed unequivocally that the ancestral state for the tribe is
Only rarely are crystals absent altogether. the absence of crystalliferous sheaths (Fig. 14C). Three
Ancestral state reconstructions indicated that the ancestor of lineages, however, have such crystalliferous sheaths,
all Bignonieae likely had crystals in the non-lignified paren- Perianthomega, Pachyptera and the members of the ‘Multiple
chyma (Fig. 13D) and further suggest a single origin in of four’ extended clade, except for punctual reversals
Adenocalymma for species with crystals exclusively in the lig- (Fig. 14D), such as that of Amphilophium magnoliifolium,
nified cells (Fig. 13D) and at least five independent origins for Bignonia capreolata, B. corymbosa and Anemopaegma
the presence of crystals in both lignified and non-lignified axial chamberlaynii. A single origin for the appearance of prismatic
and ray parenchyma, as observed in the ‘Multiples of four crystals was inferred for the ‘Multiples of four clade’ and
TABLE 1. Anatomical characters of the variant phloem of Bignonieae (Bignoniaceae)
Secondary phloem characters and character states Cell inclusions in the phloem
Taxon Lateral Limiting ray Storied Sieve Sieve Sieve Number of Percentage of Phloem Ray Phloem type Percentage Crystals in Type of Crystalliferous Type of
steps on lignification2 structure3 tube plate areas companion parenchyma per parenchyma lignification8 according to of fibres phloem and crystals in parenchyma crystal in the
the phloem arrangement4 type5 per sieve cells6 transverse area type7 fibre ray phloem and sheath crystalliferous
wedges1 plate frequency9 parenchyma10 ray parenchyma
(average) parenchyma11 sheath11
Adenocalymna bracteatum A X – R C 7 1 ? D þF SCF 108 L A – –

Adenocalymma comosum A X – T C 14 1 297 D þR SCF 117 L A/S – –
Adenocalymma divaricatum A X – T C 8 1 106 STC þR F 466 L P – –
Adenocalymma flaviflorum A X – R C 8 2D 322 D þF F 233 L P – –
Adenocalymma neoflavidum A X – T C 6 2D 209 STC þR SF 238 L A/P – –
Adenocalymma nodosum A na – R C 7 2D 421 D þF SCF 159 L P – –
Adenocalymma peregrinum A X – R C 6 2D 323 D þF SF 194 – – – –
Adenocalymna salmoneum A XP – T C 7 1 212 STC þR SF 325 L/N P – –
Adenocalymma tanaecicarpum A X – C 6 1 ? D þF SF 301 L P – –
Amphilophium bracteatum A X A D S ? 1 ? D – SCF 21 N A þ ?
Amphilophium crucigerum A X A D S 0 1 663 D – SCF 36 N A þ P
Amphilophium elongatum A X A D S 1 1 172 D – SCF 19 N A þ S
Amphilophium magnoliifolium A X – D S 2 1 ? D – SCF 17 N A þ ?
Amphilophium paniculatum A X A D S 0 1 252 D – SCF 20 N A þ P
Anemopaegma chamberlaynii A X A R S 5 1 299 D þF SF 318 – – – –
Anemopaegma laeve A X – R C 4 1 254 D þF SCF 208 B A/P þ A
Anemopaegma robustum A X – R C 5 1 283 D þF SCF 282 B A/P þ A
Bignonia binata A X – R C 10 1 265 D þF SCF 199 – – þ P
Bignonia campanulata A X – R C 8 1 225 D þF SCF 200 B A/S þ S
Bignonia capreolata A X – R C 5 1 ? D þF SCF 120 B S – –
Bignonia corymbosa A X – R C ? 1 ? D ? SCF 150 ? ? þ ?
Bignonia magnifica A X – R C 7 1 43 D – SCF 100 B A þ A
Bignonia prieurei A X – R C ? 1 ? D ? SCF 60 B A þ ?
Cuspidaria convoluta S XP – T C 26 2S 180 STC þR SF 321 N A – –
Cuspidaria pulchra S XP – T C 17 2S 167 STC þR SF 552 N A – –
Dolichandra quadrivalvis A – AR R C 4 1 31 D – SCF 100 N A þ A
Dolichandra unguiculata A – AR R C 4 1 293 D – SCF 64 N A þ A
Dolichandra unguis-cati A – AR R C 6 1 401 D – SCF 114 N A þ A
Fridericia chica A XP – T C 18 2S 259 STC þR SF 362 B A – –
Friderica conjugata A XP – T C 15 2S 353 STC þR SF 173 N A – –
Fridericia platyphylla A XP – T C 14 2S 168 STC þR F 387 N A – –
Fridericia samydoides A XP – T C 26 2S 234 STC þR F 363 B A – –
Fridericia speciosa A XP – T C 18 2S 190 STC þR SF 328 B A – –
Lundia corymbifera A X – T C 16 1 314 STC – SF 360 ? A – –
Lundia damazioi A X – T C 18 1 189 STC þR F 326 N A – –
Lundia longa A X – T C 15 1 116 STC þR F 587 – – – –
Manaosella cordifolia S X – D C 12 1 306 D þF SCF 147 B A – –
Pace et al. — Secondary phloem diversity and evolution in Bignonieae
Mansoa difficilis A X – R C 5 1 374 D þF SCF 81 B A þ P

Mansoa onohualcoides A X – R C 5 1 ? D þF SCF 70 B A þ P
Mansoa standleyi A X – R C 9 1 361 D þF SCF 182 B A þ P/S
Martinella obovata ? XP – R C ? 1 305 D þF SF 300 ? ? – –
Neojobertia mirabilis A X – T C 8 1 215 STC þF F 401 L A/P – –
Neojobertia sp. nov. A X – T C 10 1 188 STC þF F 394 N A/S/P – –
Pachyptera kerere A X – R C 7 1 374 D þF SCF 130 N A þ A
Perianthomega vellozoi A X – R S 0 1/2D 374 D – SCF 84 N A þ A
Pleonotoma albiflora A XP – T C 40 2S ? STC þ F 442 N P – –
Pleonotoma stichadenia A x – T C 34 2S 222 STC – SF 458 ? ? – ?
(continued)
351

multiple origins for both the acicular crystals and the
A; asymmetrical; S, symmetrical. 2X, to the xylem face; XP, to both faces. 3A, present in axial parenchyma; AR, present in axial and ray parenchyma. 4R, radial; T, tangential; D, diffuse. 5S, mostly sim-
þF, present when touching the fibre bands. 9SCF, scarcely fibrous; SF, semi-fibrous; F, fibrous. Fibres: N, narrow; W, wide. 10L, present in lignified cells; N, present in non-lignified cells; B, present in
ple; C, compound. 61, one per sieve tube; 2D, two per sieve tube at different sides; 2S, two or more per sieve element at the same side. 7STC, sieve-tubecentric; D, diffuse. 8þR, present in random portions;
styloid/elongate crystals within the crystalliferous parenchyma
(Fig. 14C).
crystalliferous
crystal in the
parenchyma
sheath11
Type of
P
–
–
–
–
–
–
–
Correlation analyses
Crystalliferous
Cell inclusions in the phloem
parenchyma
In the literature, some characters were hypothesized to

sheath
evolve in correlation (see Introduction). Here we tested the cor-

þ
–
–
–
–
–
–
–
relations between (1) sieve tube length and sieve plate type, and
(2) sieve tube length and storied structure. Also, we tested sev-
parenchyma11
phloem and
crystals in
eral characters in relation to fibre abundance, such as (1) sieve

Type of
ray
?
?
A
A
A
A
A
P
plate type, (2) sieve tube arrangement, (3) parenchyma type, (4)

ray lignification and (5) two or three companion cells lying at
parenchyma10
the same side of the sieve tube. The results from correlation
phloem and
Phloem type Percentage Crystals in
analyses are summarized in Table 3.

ray
B
B
N
–
–
N
N
N
Sieve tube length showed positive correlation with both sieve

plate type and storied structure (P < 001; LRTs 1344 and
companion parenchyma per parenchyma lignification8 according to of fibres
2717, respectively). Therefore, short sieve tubes usually bear

348
153
369
447
356
266
351
410
simple sieve plates, while long sieve tubes bear compound

sieve plates. Whenever a storied structure is present the sieve
frequency9
tubes are short.

SCF
fibre
SF
F
F
SF
F
F
F
Regarding fibre abundance, we encountered a positive corre-

lation with sieve plate type, sieve tube arrangement, paren-
chyma type and number of companion cells (P < 001, LRTs
þR
þR
þR
þR
þR
þR
Ray
þF
–
2500, 2501, 1754 and 872, respectively), indicating that the

sieve tubes have a compound sieve plate, tangential arrange-
ment of the sieve tubes, sieve-tubecentric phloem parenchyma
Phloem
STC
STC
STC
STC
STC
STC
STC
7
type
D
Secondary phloem characters and character states
and two or three companion cells lying at the same side of the
sieve tube whenever the phloem has a higher abundance of
transverse area
fibres. Correlation between lignification of rays and fibre abun-

Number of Percentage of
dance was rejected (P ¼ 079; LRT 066).

325
376
119
210
284
226
206
164
DISCUSSION
6
cells
2S
2S
2S
2S
2S
2S
2S
1
both lignified and non-lignified cells. 11A, acicular; P, prismatic; S, styloid or elongate.
Growth dynamic of the phloem wedges

For continuous variables, ‘Number’ represents the mean of an overall sample of 30.
(average)
arrangement type per sieve
Sieve
Stems of Bignonieae have a very particular growth dynamic. At

areas
plate
37
17
19
19
21
25
8
6
the onset of development, the stem has the exact same pattern
of growth as that of most woody plants, with the vascular
Sieve
5
plate
C
C
C
C
C
C
C
C
cambium producing more xylem to the inside and less phloem

4
to the outside and with equal activity across its entire girth
Sieve
tube
R
T
T
T
T
T
T
T
(Dobbins, 1969; Pace et al., 2009). Soon in development, how-

ever, four equidistant portions of the cambium switch from a
þ, present; –, absent; ?, not sampled; na, not applicable.
regular to a variant activity, with these variant cambia ceasing

steps on lignification2 structure3
Limiting ray Storied
–
–
–
–
–
–
–
–
to divide anticlinally and starting to produce less secondary

xylem and more secondary phloem, eventually producing deep
phloem wedges that furrow the xylem (Dobbins, 1971; Pace
XP
XP
XP
XP
XP
XP
X
X
et al., 2009, 2015). Because the variant cambia do not divide

anticlinally and remain at the same limited size, while all the
the phloem
tissues around them progress normally the secondary growth,

wedges1
Lateral
S
S
na
na
A
A
A
A
these four variant portions must accommodate their growth in

order to keep up with the stem thickening without risking the
occurrence of ruptures. In Bignonieae, this occurs by means of
TABLE 1. Continued
two different mechanisms: production of tall multiseriate limit-

ing rays and lateral phloem steps.

Tynanthus cognatus
Pyrostegia venusta
Tynanthus elegans
Ray enlargement to accommodate secondary growth has

long been known to occur in the secondary phloem, being
extremely conspicuous in e.g. the Malvaceae s.l. (Metcalfe and
Chalk, 1950). However, these limiting rays, term coined by
Taxon
Schenck (1893), are not all equal and vary in their lignification
TABLE 2. Scaling parameters of evolution for the continuous characters analysed following Pagel (1999), and their
implications for the pattern of evolution of these characters
Character j Branch length k Contribution of d Total path scale Model A Model B LR

scale phylogeny
Sieve-tube areas 00*

Punctuational 00* Star phylogeny 30* Temporally later change –22565 –22622 –114
evolution (species-independent) (species-specific adaptation)
Variant parenchyma (%) 11 Default 028* Minimal effect of 30* Temporally later change –17679 –17766 –175
gradualism phylogeny (species-specific adaptation)
Fibres in variant phloem 027* Stasis in longer 081 Default phylogeny 133 Default gradualism –20764 –20803 –078
of the entire tribe (%) branches
Fibres in variant phloem of 044* Stasis in longer 033* Minimal effect of 28* Temporally later change –7728 –8730 –2004
‘Fridericia and allies branches phylogeny (species-specific adaptation)

extended clade’ (%)
Fibres in variant phloem of 016* Stasis in longer 099 Default phylogeny 068* Adaptive radiation –6935 –7043 –217
‘Multiples of four branches
extended clade (%)
Number of sieve areas in 228* Longer branches 10 Default phylogeny 030* Adaptive radiation –6074 –6077 –006
variant phloem of accumulate
‘Fridericia and allies more changes
extended clade’
Number of sieve areas in 00* Punctuational 094 Default phylogeny 054* Adaptive radiation –4675 –5415 –1480
variant phloem evolution
of ‘Multiples of four
extended clade
*Significant deviations from 10 for the scaling parameters values kappa (j), lambda (k), and delta (d), assessed with the likelihood ratio (LR) test.
LR values greater than 2 indicate directional evolution.
TABLE 3. Pagel’s 1994 correlation analyses of discrete phloem characters, from 1000 simulations. Continuous characters
were made categorical and their respective character states are presented in parentheses
Log likelihood Log likelihood Difference P value

under under between
four-parameter eight-parameter log likelihoods
model model
Length of sieve tube (shorter or longer than 500 mm) 3886 2541 1344* <001*
and sieve plate type (simple or compound)
Length of sieve tube (shorter or longer than 500 mm) 3618 901 2717* <001*
storied structure (absent or present)
Percentage of fibres (non-fibrous or semi-fibrous to fibrous) 5281 2780 2500* <001*
compound sieve plates (absent or present)
tangential arrangement of sieve tubes (absent or present)
sieve-tubecentric parenchyma (absent or present)
Percentage of fibres (non-fibrous or semi-fibrous to fibrous) 4359 4292 066 0798
ray lignification (absent or present)
2 or 3 companion cells on the same side (absent or present)
*Significant correlations.
state. The limiting rays may be entirely non-lignified, only lig- African Hippocrateoideae (Celastraceae s.l.), which also have
nified next to the xylem face, or lignified at both sides but with disjunct included cambia (Obaton, 1960). Limiting rays are
a core middle part of non-lignified ray cells. Given that the var- also present in other plant families in which an included
iant cambia are included within the phloem wedges and sur- cambium produces phloem wedges, such as Icacinaceae
rounded by a matrix of stiff secondary xylem, a non-lignified (Bailey and Howard, 1941; Lens et al., 2008) and some lianas
ray portion, even if just a central core, is critical to allow cell of Mimosa (Leguminosae; Angyalossy et al., 2015). Other
division in order to guarantee the possibility of cell dislocation authors, like Schenck (1893), on the other hand, believed that
from within the wedges. Thus, while cambial derivatives are normal ruptures would be formed periodically at these limiting
periodically formed and pushed outwards from within the rays, while new secondary phloem was produced from inside
phloem wedges, the limiting rays divide along with these newly the wedge, given its mechanical fragility. Careful developmen-
formed phloem cells. This mechanism was first proposed for tal analyses with fresh material have shown, however, that these
ruptures are only artefacts and, hence, non-existent (Ozório- sieve plates with many sieve areas are the most common, with
Filho, 2002). some species bearing >30 sieve areas per sieve plate (e.g.
As another possible mechanism to accommodate secondary Pleonotoma tetraquetra). Compound sieve plates evolving
growth, some Bignonieae develop successive lateral steps on from ancestors with fewer sieve areas is a pattern opposite to
the sides of the original phloem wedges. These steps are formed that proposed for the angiosperms as a whole (Hemenway,
by portions of cambium on the sides of phloem wedges that 1913; Esau, 1969). These two sister clades are thought to have
switch from regular to variant activity; these steps can be sym- split from an ancestral lineage inhabiting lowland Amazon,
metrical, asymmetrical or, more rarely, lacking altogether, with diversifying in the Amazon and (re)colonizing the Atlantic rain-
much wider limiting rays present in these cases, such as in forest (Lohmann et al., 2013). A smaller clade nested within
Tanaecium. While most Bignonieae have asymmetrical phloem the ‘Fridericia and allies clade’, formed by Fridericia and
steps at the wedges, three genera (two clades), Cuspidaria þ Xylophragma, occupies the South American dry areas
Tynanthus, which together form a clade, and Manaosella, (Lohmann et al., 2013). One could envisage a physiological

exhibit symmetrical phloem wedges. The steps are so conspicu- implication for the compound sieve plates with many sieve
ous and regularly spaced in Tynanthus species that studies on areas if Fridericia and Xylophragma exclusively had this sieve
seasonal formation of xylem show the boundaries of growth plate type. However, since other species of this clade occupy
rings in the variant xylem of Tynanthus cognatus as appearing more moist areas and also have the same anatomical feature, a
in a stair-step fashion (Lima et al., 2010). All other features dis- correlation between habitat occupation and diverse sieve tube
cussed below address the variant secondary phloem in detail. element morphologies is debatable.
A few ecological and functional studies have been carried
out for phloem, and they did find some correlations between
Phylogenetic and evolutionary comparison of the secondary sieve element morphology and environmental or organographic
phloem of Bignonieae variables. In monocots, simple and supposedly more efficient
Overall pattern Analysing and comparing the variant secondary sieve plates are found in the source organs (i.e. rhizomes full of
phloem of Bignonieae in detail shows us that the phloem in this starch, and leaves), while compound sieve plates are found in
tribe is extremely diverse; however, two main phloem anato- sink or consuming organs (i.e. stems and roots; Cheadle and
mies can be described (see summary in Fig. 15). The first is Witford, 1941; Cheadle, 1948). In the eudicot Styrax, sieve ele-
illustrated by a stratified structure, with evenly spaced fibre ments bear transverse and simple sieve plates in the roots and
bands, sieve tubes in radial or diffuse arrangement, simple or inclined and compound sieve plates in the stem of species
compound sieve plates with few sieve areas, and abundant growing in the Cerrado, a strong seasonally dry environment
intermingling parenchyma. This phloem anatomy is similar to (Machado et al., 2005, 2007), while always exclusively com-
that described for arboreal Bignoniaceae (Roth, 1981) (Fig. 15) pound in species growing in more mesic habitats (Machado
and the regular interwedge phloem of Bignonieae (Pace et al., et al., 2007), suggesting a potential functional role of different
2011). The other phloem anatomy type in the tribe is described sieve plate morphologies. More studies are needed to test possi-
for the first time for the family, being marked by the presence ble functional hypotheses.
of a background matrix of fibres where all other cell types are Meanwhile, the evolutionary significance of sieve area num-
embedded, with sieve tubes in tangential arrangement, com- ber in the sieve plates remains elusive. We do know that sieve
pound sieve plates with many sieve areas and the axial paren- tube elements with simple sieve plates are usually encountered
chyma surrounding the sieve tubes (here called ‘sieve- in shorter sieve tube elements, while compound sieve plates are
tubecentric parenchyma’; Fig. 15). normally found in the longer and wider sieve tube elements
(MacDaniels, 1918; Parthasarathy, 1968; Esau, 1969; Den
Diversity and evolution of individual phloem characters Outer, 1983). This same pattern holds true in Bignonieae,
Sieve tube elements in Bignonieae were studied here for their within which the sieve tube elements bearing simple sieve
width, sieve plate type, sieve element length, and arrangement. plates are much shorter than those bearing compound sieve
Regarding their width and length, our analyses indicate that plates, an observation confirmed by correlation analyses. In
sieve tubes are not undergoing directional evolution, towards Bignonieae, the presence of shorter sieve tube elements bearing
either an increase or a decrease in their overall size within simple sieve plates is additionally associated with the presence
Bignonieae. This result diverges from the general trend pro- of storied structure, as seen in Perianthomega, Amphilophium
posed by earlier authors considering sieve tube element length and Dolichandra. Storied cambia have already been shown to
and width on a broader scale, to vascular or seed plants as a have shorter fusiform initials and, therefore, to produce shorter
whole, which were suggested to undergo a decrease in length axial derivatives, both in the xylem and in the phloem (Eames
and increase in width along time (Hemenway, 1913; Zahur, and MacDaniels, 1947; Esau, 1969; Evert, 2006).
1959; Esau, 1969; Den Outer, 1993). Phloem fibres also exhibit contrasting evolutionary patterns
Sieve tube elements in Bignonieae have either simple or within the Bignonieae. In the ‘Multiples of four extended clade’
compound sieve plates. Two major sister clades within the we observe a reduction in the abundance of fibres along the
tribe, the ‘Multiples of four extended clade’ and the ‘Fridericia phylogeny, while in its sister group, the ‘Fridericia and allies
and allies extended clade’, are marked by a contrasting differ- extended clade’, the opposite scenario is observed, with an aug-
ence. In the ‘Multiples of four extended clade’, a reduction in mentation of fibre abundance, which also holds true for
the number of sieve areas per sieve plate is observed along the Stizophyllum and some species of Adenocalymma. The multiple
phylogeny, something confirmed by directional analyses. In origins for an increase in fibre abundance may be related to the
contrast, in the ‘Fridericia and allies extended clade’ compound mechanical safety of the conductive cells produced by the
variant cambia within the phloem wedges, similar to what has where a matrix of parenchyma has been substituted for a matrix
been proposed for the fibrous secondary phloem of Carya cor- of fibres. Fibres in Bignonieae as a whole seem to be evolving
diformis (Juglandaceae; Eames and MacDaniels, 1947), possi- in a punctuated fashion. When the ‘Fridericia and allies
bly avoiding damage from natural girdling. Collapse of the extended clade’ is considered alone, phylogenetic statistics
sieve tube elements has never been recorded in the phloem of indicate that most changes in this lineage occur at the terminal
fibrous species, not even in the non-conducting phloem, as branches, which agrees with a species-specific type of adapta-
opposed to the ‘Multiples of four extended clade’, where most tion. Meanwhile, its sister group, the ‘Multiples of four
species experience total collapse of their sieve tube elements in extended clade’, exhibits the opposite pattern, with most
the non-conducting phloem. Evolution towards a decrease in changes occurring early in the evolution of the lineage and with
fibre frequency in the ‘Multiples of four extended clade’, on the stasis in the terminal branches, a hypothesis consistent with a
other hand, may be enhancing the flexibility of these stems to scenario of ancient diversification.
climb, as some species of this group were suggested as the Other characters encountered in this study were shown to be

most flexible in the tribe (e.g. Dolichandra; Gentry, 1980). taxon-specific and therefore diagnostic of some groups, such as
Thus, the combination of lower fibre percentage and multiple the presence of crystalliferous parenchyma sheaths in the
phloem wedges (Dos Santos, 1995; Pace et al., 2009) seems to ‘Multiples of four extended clade’ and Pachyptera. The pres-
increase stem flexibility, a hypothesis that is currently being ence of crystalliferous parenchyma surrounding the fibre bands
tested biomechanically (Gerolamo et al., 2014). The increase in is a quite common feature in phloem and has been related to
fibre abundance undergone by at least three independent line- the presence of true fibres (Roth, 1981; Evert, 2006). However,
ages of Bignonieae illustrates the opposite pattern in relation to true fibres, differentiating directly from cambial derivatives
what had been proposed for the angiosperms as a whole close to the cambial zone, are found here even in the absence of
(Hemenway, 1913; Zahur, 1959; Den Outer, 1993), challenging crystalliferous parenchyma. The multiple origins of different
the one-way directionality proposed by these earlier theories. types of crystals and the fact that more than one type of crystal
On the other hand, in lineages undergoing an overall decrease may occur in a single species illustrate how labile might be the
in the amount of fibres, we never see their substitution for scler- conversion of one type of crystallization to another, with the
eids in the variant phloem of Bignonieae studied here, contrary acicular crystals being the most common in this tribe. Crystals
to what was predicted by Den Outer (1993), although more have been found in nearly all species, and these have been sug-
studies with different families in which fibres occur are needed gested to play a key role in plant metabolism, being involved in
to further explore Den Outer’s proposal. cell division, growth, physiological maintenance and mechani-
Several traits that were variable in Bignonieae have shown cal support (Arnott, 1976; Franceschi, 1989; Marcati and
an evolutionary correlation with the increase in fibre abun- Angyalossy, 2005; Evert, 2006).
dance, such as the tangential arrangement of sieve tube ele-
ments, abundant number of sieve areas, the presence of sieve-
tubecentric phloem parenchyma and the presence of multiple
companion cells. These correlations probably result from archi-
CONCLUSIONS
tectural constraint (sensu Gould and Lewontin, 1979).
Bignoniaceae as a whole have a stratified phloem, i.e. a phloem This study shows that the phloem is extremely diverse in the
with regularly spaced tangential fibre bands (Roth, 1981; this speciose tribe Bignonieae (Bignoniaceae), with two main types
work). When fibres increase in abundance, these fibre bands of phloem anatomy: a fibrous and a scarcely fibrous phloem
become more closely arranged, leading the elements towards (stratified). These different fibre abundances have, in addition,
tangential disposition, while the axial parenchyma reduces in evolved in correlation with sieve elements and axial paren-
abundance and becomes sieve-tubecentric. The presence of this chyma. Ancestral character state reconstructions and phyloge-
sieve-tubecentric axial parenchyma in the fibrous species may netic statistical analyses have shown that directional evolution
contribute fundamentally to phloem transport by creating the of fibre abundance is present in Bignonieae, and with different
osmotic pressure known to be necessary to maintain turgor lineages having evolved in contrasting directions, creating the
pressure for phloem loading and unloading (Aloni et al., 1986; dissimilar types of phloem anatomy seen today in the tribe.
Sjölund, 1997; Van Bel, 2003). Also, in some instances func- What has likely triggered the appearance in evolution of such
tioning integration between the axial parenchyma and the com- contrasting phloem types in Bignonieae remains to be eluci-
panion cells has been shown, with the parenchyma contributing dated. Further ecophysiological studies interpreted in the light
to the maintenance of sieve elements (Esau, 1969), which may of the anatomical findings encountered here would be critical in
well explain the close association of these cells around the sieve order to address such questions. Moreover, our results challenge
elements in the fibrous species of Bignonieae. A positive corre- the hypotheses of general trends in phloem character evolution,
lation was also found between abundant fibres and the presence such as decreasing size of the sieve tube elements, increasing
of more than one companion cell per sieve element. The pres- sieve tube element diameter, increasing amount of parenchyma
ence of two companion cells, one lying at each side of the sieve and decreasing number of fibres, during angiosperm evolution,
tube element, has already been reported in other plant groups which have historically pervaded the anatomical literature. The
(Cheadle and Esau, 1958; Chavan et al., 1983, 2000). However, findings of such contrasting evolutionary patterns even on a
the presence of two or three companion cells lying on the same narrow taxonomic scale, like the tribe Bignonieae, makes it still
side of the sieve tube element has, to the best of our knowledge, more exciting to pursue a holistic understanding of phloem
never been mentioned in the literature and may contribute to diversification to expand our knowledge on phloem diversifica-
the maintenance of these long and wide sieve elements in taxa tion in angiosperms as a whole.
ACKNOWLEDGEMENTS Cheadle VI, Gifford EM, Esau K. 1953. A staining combination for phloem
and contiguous tissues. Stain Technology 28: 49–53.
We thank Alexandre Zuntini, André Amorim, André Lima, Costa CG, Coradin VTR, Czarneski CM, Pereira BAS. 1997. Bark anatomy
Anselmo Nogueira, Daniel Villavoel, Diana Sampaio, of arborescent Leguminosae of Cerrado and gallery forest of central Brazil.
IAWA Journal 18: 385–399.
Geraldo Damasceno, Gregório Ceccantini, Juliana El Ottra, Den Outer RW. 1983. Comparative study of the secondary phloem of some
Luzmilla Arroyo, Márdel Lopes, Mariane S. de Sousa-Baena, woody dicotyledons. Acta Botanica Neerlandica 32: 29–38.
Milton Groppo Jr, Renata Udulutsch and Rosani Arruda for Den Outer RW. 1993. Evolutionary trends in secondary phloem anatomy of
field assistance or for sending us specimens for the present trees, shrubs and climbers from Africa (mainly Ivory Coast). Acta Botanica
study; Harry Lorenzi for allowing us to collect in the Neerlandica 42: 269–287.
Dobbins DR. 1969. Studies on the anomalous cambial activity in Doxantha
Plantarum Institute and Botanical Garden; Antonio C. F. unguis-cati (Bignoniaceae). I. Development of the vascular pattern.
Barbosa for assistance with anatomical procedures, especially Canadian Journal of Botany 47: 2101–2106.
at the beginning of this work; and André Lima, José R. Pirani, Dobbins DR. 1971. Studies on the anomalous cambial activity in Doxantha
unguis-cati (Bignoniaceae). II. A case of differential production of second-

Neusa Tamaio and two anonymous reviewers for invaluable
ary tissues. American Journal of Botany 58: 697–705.
comments on earlier versions of this manuscript. Funding for Dos Santos GMA. 1995. Wood anatomy, chloroplast DNA, and flavonoids of the
this study was provided by a fellowship to M.R.P. from tribe Bignonieae (Bignoniaceae). PhD Thesis, University of Reading, UK.
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APPENDIX Paulo, Brazil. Amphilophium magnoliifolium (Kunth) L.G.

Lohmann, Lohmann 851, Rio Negro, Amazonas, Brazil; Dos
Taxa, collectors and localities. Vouchers for all specimens Santos 272, Porto de Moz, Pará, Brazil, received from the
were deposited in SPF herbarium, unless otherwise indicated MADw wood collection, vouchers in MAD, MO and MG.
after the plant information. Amphilophium paniculatum (L.) Kunth, Pace 46, Livings
Adenocalymma bracteatum (Cham.) DC., Castanho 153, collection Plantarum Institute, Nova Odessa, São Paulo,
Lohmann 861, Rio Negro, Amazonas, Brazil. Adenocalymma Brazil. Anemopaegma chamberlaynii (Sims) Bureau & K.
comosum (Cham.) DC., Pace 53, Living collection Plantarum Schum., Zuntini 15, Vale do Rio Doce Forest Reserve,
Institute, Nova Odessa, São Paulo, Brazil. Adenocalymma Espı́rito Santo, Brazil. Anemopaegma laeve DC., Pace 338,
divaricatum Miers, Udulutsch 2808, Lençóis, Bahia, Brazil. Fazenda Bom-Recreio, Boa Nova, Bahia, Brazil.
Adenocalymma flaviflorum (Miq.) L.G. Lohmann, Sousa- Anemopaegma robustum Bureau & K. Schum, Pace 74,
Baena 2, Vale do Rio Doce Forest Reserve, Espı́rito Santo, Reserva Ducke, Manaus, Amazonas, Brazil. Bignonia binata
Brazil. Adenocalymma neoflavidum L.G. Lohmann, Zuntini Thunb, Galvanese 22, Rio Negro, Amazonas, Brazil.
23, Vale do Rio Doce Forest Reserve, Espı́rito Santo, Brazil. Bignonia campanulata Cham., Pace 39, Livings collection
Adenocalymma nodosum (Silva Manso) L.G. Lohmann, Pace Plantarum Institute, Nova Odessa, São Paulo, Brazil.
20, Uberlândia, Minas Gerais, Brazil. Adenocalymma pere- Bignonia corymbosa (Vent.) L.G. Lohmann, Zuntini 2,
grinum (Miers) L.G. Lohmann, Pace 26, Living collection Zuntini 17, Vale do Rio Doce Forest Reserve, Espı́rito Santo,
Plantarum Institute, Nova Odessa, São Paulo, Brazil. Brazil. Bignonia magnifica W. Bull, Pace 51, Livings collec-
Adenocalymma salmoneum J.C. Gomes, Lohmann 658, Vale tion Plantarum Institute, Nova Odessa, São Paulo, Brazil.
do Rio Doce Forest Reserve, Espı́rito Santo, Brazil. Bignonia prieurei DC., Zuntini 13, Vale do Rio Doce Forest
Adenocalymma tanaeciicarpum (A.H. Gentry) L.G. Reserve, Espı́rito Santo, Brazil. Callichlamys latifolia (Rich.)
Lohmann, Dos Santos 263, Porto de Moz, Pará, Brazil, K. Schum, Zuntini 175, Vale do Rio Doce Forest Reserve,
received from the MADw wood collection, vouchers in MAD, Espı́rito Santo, Brazil.; Pace 42 Livings collection Plantarum
MO and MG. Amphilophium bracteatum (Cham.) L.G. Institute, Nova Odessa, São Paulo, Brazil; Pace 63, Reserva
Lohmann, Ozório-Filho 8, São Paulo, São Paulo, Brazil. Ducke, Manaus, Amazonas, Brazil. Cuspidaria convoluta
Amphilophium crucigerum (L.) L.G. Lohmann, Pace 1, Pace (Vell.) A.H. Gentry, Pace 48 Livings collection Plantarum
2, Pace 3, Pace 34, São Paulo, São Paulo, Brazil. Institute, Nova Odessa, São Paulo, Brazil. Cuspidaria pulchra
Amphilophium elongatum (Vahl) L.G. Lohmann, Pace 45, (Cham.) L.G. Lohmann, Pace 24, Pace 25, Uberlândia, Minas
Livings collection Plantarum Institute, Nova Odessa, São Gerais, Brazil. Dolichandra quadrivalvis (Jacq.) L.G.
Lohmann, Pace 105, Corumbá, Mato Grosso do Sul, Brazil. Brazil, analysed at MADw wood collection, voucher in MAD,
Dolichandra unguiculata (Vell.) L.G. Lohmann, Zuntini 176, MO, MG; Dos Santos 317, Gurupa, Moju riverside, tributary
Vale do Rio Doce Forest Reserve, Espı́rito Santo, Brazil. of the Amazon River, Pará, Brazil, analysed at MADw,
Dolichandra unguis-cati (L.) L.G. Lohmann, Ceccantini voucher in MAD, MO, MG. Neojobertia mirabilis (Sandwith)
2687, Matozinhos, Minas Gerais, Brazil; Groppo 322, São L.G. Lohmann, Dos Santos 48, Buriticupu Forest Reserve,
Paulo, São Paulo, Brazil. Fridericia chica (Bonpl.) L.G. Maranhão, Brazil, received from the MADw wood collection,
Lohmann, Pace 50, Livings collection Plantarum Institute, vouchers in the MAD, MO, and MG. Neojobertia sp. nov.,
Nova Odessa, São Paulo, Brazil. Fridericia conjugata (Vell.) Zuntini 18, Vale do Rio Doce Forest Reserve, Espı́rito Santo,
L.G. Lohmann, Pace 44, Livings collection Plantarum Brazil. Pachyptera kerere (Aubl.) Sandwith, Castanho 143,
Institute, Nova Odessa, São Paulo, Brazil. Fridericia platy- Lohmann 834, Rio Negro, Amazonas, Brazil. Perianthomega
phylla (Cham.) L.G. Lohmann, Pace 22, Pace 23, Uberlândia, vellozoi Bureau, Pace 10, Pace 15, Viçosa, Minas Gerais,
Minas Gerais, Brazil. Fridericia samydoides (Cham.) L.G. Brazil; Pace 28, Pace 29, Santa Cruz de la Sierra, Santa Cruz,

Lohmann, Pace 49, Livings collection Plantarum Institute, Bolivia. Pleonotoma albiflora (Salzm. ex DC.) A.H. Gentry,
Nova Odessa, São Paulo, Brazil. Fridericia speciosa Mart., Pace 78, Reserva Ducke, Manaus, Amazonas, Brazil.
Pace 40, Livings collection Plantarum Institute, Nova Odessa, Pleonotoma tetraquetra (Cham.) Bureau, Ozório-Filho 11,
São Paulo, Brazil. Lundia damazioi C. DC., Pace 55, Pace São Paulo, São Paulo, Brazil. Pleonotoma stichadenia K.
56, São Paulo, São Paulo, Brazil. Lundia longa (Vell.) DC., Schum., Zuntini 7, Vale do Rio Doce Forest Reserve, Espı́rito
Zuntini 1, Vale do Rio Doce Forest Reserve, Espı́rito Santo, Santo, Brazil; Dos Santos 187, Parauapebas, Pará, Brazil, ana-
Brazil; Pace 227, Reserva Biológica de Poço das Antas, Silva lysed at the MADw wood collection, vouchers in MAD, MO
Jardim, Rio de Janeiro, Brazil. Manaosella cordifolia (DC.) and MG. Pyrostegia venusta (Ker Gawl.) Miers, Pace 17,
A.H. Gentry, Pace 41, Brazil, Livings collection Plantarum Campinas, São Paulo, Brazil; Pace 36, São Paulo, São Paulo,
Institute, Nova Odessa, São Paulo, Brazil. Mansoa difficilis Brazil. Stizophyllum riparium (Kunth) Sandwith, Pace 16,
(Cham.) Bureau & K. Schum., Pace 35, São Paulo, São Paulo, Pace 33, São Paulo, São Paulo, Brazil; Zuntini 9, Vale do Rio
Brazil; Zuntini 4, Vale do Rio Doce Forest Reserve, Espı́rito Doce Forest Reserve, Espı́rito Santo, Brazil. Tanaecium bila-
Santo, Brazil. Mansoa onohualcoides A.H. Gentry, Zuntini biatum (Sprague) L.G. Lohmann, Lohmann 850, Rio Negro
276, Vale do Rio Doce Forest Reserve, Espı́rito Santo, Brazil. Amazonas, Brazil. Tanaecium pyramidatum (Rich.) L.G.
Mansoa standleyi (Steyerm.) A.H. Gentry, Pace 43, Livings Lohmann, Pace 14, Pace 35, São Paulo, São Paulo, Brazil.
collection Plantarum Institute, Nova Odessa, São Paulo, Tynanthus cognatus (Cham.) Miers: Pace 9a, Pace 9b, São
Brazil. Martinella obovata (Kunth) Bureau & K. Schum., Paulo, São Paulo, Brazil. Tynanthus elegans Miers, Zuntini
Zuntini 7, Vale do Rio Doce Forest Reserve, Espı́rito Santo, 147, Vale do Rio Doce Forest Reserve, Espı́rito Santo, Brazil.
Brazil; Dos Santos 237, Porto de Moz, Xingu riverside, Pará, Xylophragma myrianthum (Cham. ex Steud.) Sprague.

Secondary Phloem Diversity

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Annals of Botany 116: 333–358, 2015

doi:10.1093/aob/mcv106, available online at www.aob.oxfordjournals.org

Secondary phloem diversity and evolution in Bignonieae (Bignoniaceae)

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Most studies on phloem diversity have been descriptive

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D Fridericia and allies

Neojobertia sp. nov.

To the xylem face

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Arrangement of sieve tube elements Three different types of

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D 'Multiples of four clade' Fridericia and allies

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'Multiples of four extended clade'

Manaosella cordifolia Pleonotoma albiflora

'Fridericia and allies extended clade'

Tanaecium bilabiatum Pachyptera kerere

Tanaecium pyramidatum Tanaecium bilabiatum

Tynanthus elegans Tanaecium pyramidatum

Tynanthus cognatus Tynanthus elegans

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One per sieve element

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C Fridericia and allies

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Fridericia and allies

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'Multiples of four extended clade'

Anemopaegma chamberlaynii Anemopaegma chamberlaynii

Anemopaegma robustum Anemopaegma robustum

Mansoa onohualcoides 23 % Mansoa onohualcoides

Mansoa standleyi Mansoa standleyi

Dolichandra unguiculata Dolichandra unguiculata

Fridericia and allies

Fibrous 58·7 to 64·4

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Dolichandra unguis cati

Neojobertia sp. nov.

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Dolichandra unguis cati

Neojobertia sp. nov.

Acicular and navicular

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Dolichandra unguis cati

Neojobertia sp. nov.

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Dolichandra unguis cati

Neojobertia sp. nov.

Overall phloem anatomy Morphology of sieve elements Storied structure

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Adenocalymna bracteatum A X – R C 7 1 ? D þF SCF 108 L A – –

Mansoa difficilis A X – R C 5 1 374 D þF SCF 81 B A þ P

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multiple origins for both the acicular crystals and the