international journal of plant sciences

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E 182 NUMBER 7

SEPTEMBER 2021

PAGES 577 –662

A NEW INTERPRETATION OF THE SUCCESSIVE CAMBIA OF SOMENYCTAGINACEAE AS INTERXYLARY PHLOEM

Israel L. Cunha Neto,1,* Marcelo R. Pace,† and Veronica Angyalossy*

*Departamento de Botânica, Laboratório de Anatomia Vegetal, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, São PauloSP 05508-090, Brazil; and †Departamento de Botánica, Laboratorio de Botánica Estructural, Instituto de Biología, Universidad Nacional

Autónoma de México, Circuito Zona Deportiva s/n de Ciudad Universitaria, 04510, Delegación Coyoacán, Mexico City, Mexico

Editor: Alexandru M.F. Tomescu

Premise of research. The alternative patterns of secondary growth (vascular cambial variants) in stems ofNyctaginaceae are outstanding and have been widely investigated since the late nineteenth century. However,there are controversial interpretations in the literature regarding the existence of either one or two types of cam-bial variants in this family (successive cambia vs. interxylary phloem). We aim to explore the anatomical diver-sity of stems in Nyctaginaceae to document the real nature of the cambial variant present in most species of thefamily.

Methodology. We analyzed 60 species, focusing on 18 species from 12 genera, for developmental studies. Ana-tomical and ontogenetic features were characterized from images produced by standard plant techniques for macro-and microscopic analyses.

Pivotal results. Our analyses reveal that most species of Nyctaginaceae present stems with polycyclic eusteles,which later develop a single cambium that produces secondary xylem and secondary phloem at unequal ratesaround the stem circumference. This unusual activity results in the absence of a regular cylinder of secondary vas-cular tissues and in the formation of secondary phloem strands (surrounded by variable amounts of sheathing axialparenchyma) embedded within the secondary xylem. In cross section, adult stems can exhibit different tissue ar-rangements (i.e., phloem islands/strands, patches, or concentric bands) that result from differences in rates of pro-duction of phloem and associated sheathing axial parenchyma forming the strands. The cambial variant in thesestems is described as interxylary phloem, as similarly observed in other eudicot lineages.

Conclusions. Our examination of the stem development of Nyctaginaceae confirms the presence of interxylaryphloem, which has been overlooked in the family as most previous studies have reiterated descriptions of successivecambia as the common cambial variant within the family. These findings emphasize the importance of develop-mental studies encompassing a representative number of genera to further our understanding of stem macro-morphologies and to highlight the complexity and diversity of stem architectures in Nyctaginaceae.

Keywords: cambial variant, Caryophyllales, ontogeny, polycyclic eustele, secondary growth, stem anatomy.

Online enhancement: supplemental table.

Introduction

The prevalence of families with cambial variants in the orderCaryophyllales, compared with other angiosperm orders, hasbeen reported since the first treatises on the subject (de Bary1884; Schenck 1893; Pfeiffer 1926). Cambial variants are re-ported in at least 19 of the 39 families currently recognized inthe Caryophyllales (Gibson 1994; Carlquist 2010; Hernández-

Ledesma et al. 2015). Until the late twentieth century, themajor-ity of papers on the formation of alternative vascular anatomiesin this order followed the traditional view (Schenck 1893;Pfeiffer 1926) that regarded themain cambial variant in the fam-ily as successive cambia (i.e., additional increments of vasculartissue through the formationof new cambia outside thefirst cam-bium). More recently, Carlquist’s outstanding contributionsto the understanding of vascular anatomies in Caryophyllales(Carlquist 1991, 1999, 2000, 2001, 2003, 2004, 2007, 2010)have concurred with earlier views that successive cambia is thecambial variant occurring in the order. This view has been sub-sequently shared by other authors (Rajput and Rao 1998; Raj-put et al. 2009, 2012; Hernández-Ledesma et al. 2011, 2015;Rajput and Marcati 2013; Rajput 2015; Myśkow et al. 2019;Zumaya-Mendoza et al. 2019).

1 Author for correspondence; email: [email protected],[email protected].

Manuscript received October 2020; revised manuscript received January 2021;electronically published July 7, 2021.

Int. J. Plant Sci. 182(7):620–637. 2021.q 2021 by The University of Chicago. All rights reserved.1058-5893/2021/18207-0004$15.00 DOI: 10.1086/715505

620

Nyctaginaceae is one of the most emblematic families in thecore Caryophyllales, and the anatomy of the stem and cambialvariants has been widely investigated. For the family, de Bary(1884) and Schenk (1893) were some of the first to characterizethe presence of successive cambia in stems of some species (e.g.,in the generaBougainvillea,Mirabilis). This topic was further in-vestigated by Esau and Cheadle (1969) and Carlquist (2004).The former authors undertook detailed ontogenetic analyses ofstems and roots inBougainvillea spectabilis, which demonstrateda pattern of successive cambia originating from the pericycle andproducing multiple additional bifacial cambia. Carlquist’s studyinvestigated different genera and species (including B. spectabilis),interpreting all of them as having successive cambia that origi-nated from an independent meristem arising in the cortex, whichCarlquist termed a “master cambium.”Despite these interpreta-tions, the pattern of secondary growth in some species of Nycta-ginaceae has also been recurrently interpreted as interxylaryphloem in a number of studies (Chalk and Chattaway 1937;Metcalfe and Chalk 1950; Studholme and Philipson 1966; Lopeset al. 2008; Sonsin et al. 2014).Nevertheless,most of these studieseither focused on a single species or compared multiple speciesbut did not explore in detail the origin, development, and anat-omy of the vascular system of Nyctaginaceae, where situationsintermediate between successive cambia and interxylary phloemhave been hiding in some species, especially nonornamentalones. As a result, given the widespread distribution of cambialvariants in Nyctaginaceae and their complex macromorphol-ogies, different names and classifications have been proposedto describe their cambial variants. These different classificationsemphasize different structural aspects, including the origin andtype of meristem that forms the tissues and the arrangement ofcell types in stem cross section (stem topologies; see table S1,available online).

Successive cambia and interxylary phloem are recognizedas two entirely different types of cambial variants, whose cor-rect identification can be hindered by their common (althoughontogenetically different) characteristic (i.e., the presence ofsecondary phloem amid the secondary xylem in stem cross sec-tion; Carlquist 2001, 2013; Angyalossy et al. 2012, 2015).Overall, in the case of successive cambia, new bands of xylem,phloem, and sometimes conjunctive parenchyma are producedby the formation of subsequent cambia (and their products) out-side of the original cambium. Species with successive cambiagenerally initiate their secondary growth, forming a regular vas-cular cylinder of secondary xylem and phloem, and only laterare additional rings or concentric increments of vascular tissueproduced, the first of which originates either from the cortex,pericycle, and phloem parenchyma (Angyalossy et al. 2015;Pace et al. 2018) or from procambium remnants (Myśkowet al. 2019). On the other hand, the appearance of phloemwithinthe xylem in species with interxylary phloem results from asingle cambium (Carlquist 2001, 2013; Angyalossy et al. 2016),and three subtypes of interxylary phloem can be distinguishedaccording to how the phloem cells are produced by the cambium(see the IAWA Bark Committee; Angyalossy et al. 2016). Re-gardless of the mode of phloem production in stems with thiscambial variant, the interxylary phloem generally forms smallgroups of conducting cells, for which reason it has been referredto as “included phloem” or “phloem islands” (IAWA Commit-tee 1989; van Veenendaal and den Outer 1993; Lens et al. 2008;

Rajput et al. 2009; Carlquist 2013; Gondaliya and Rajput 2017).As a result, when stems with cambial variants display long orcontinuous rings of vascular increments in cross section, authorsare prone to identify them as successive cambia, while if second-ary phloem is observed within the xylem forming small strands,they are typically recognized as interxylary phloem. However,this distinction becomes difficult to apply when all gradationsbetween these two extremes are present, which is the case forNyctaginaceae.Here, we performed a comparative analysis between species

of Nyctaginaceae with a clear-cut aspect of interxylary phloemand those with intermediate forms using a broad sampling ofthe family to understand the real nature of the cambial variantpresent in most species of the Nyctaginaceae.

Material and Methods

Plant Collection and Sampling

We analyzed 60 species of the Nyctaginaceae (see the appen-dix), focusing on 18 species from genera distributed in five ofseven tribes to understand whether successive cambia or inter-xylary phloem better explain the cambial variants in these spe-cies (table 1). Most of the specimens were collected by us in nat-ural populations, while some were studied in slides deposited inwood collections (appendix). Some representatives were usedfor the ontogenetic study, while well-developed stems of all spe-cies/specimens were analyzed to observe the entire range of an-atomical variability.Thematerial collected included different portions of the stems,

from the stem apex to the plant base in the case of scandentplants and herbs and fragments from branches and the maintrunk in the case of trees and shrubs. During fieldwork, the spec-imens were immediately fixed in FAA 70 (formaldehyde–aceticacid–ethanol or 70% isopropanol) and then stored in 70% eth-anol (Johansen 1940).

Anatomical Procedures

Samples of the stems in different stages of development wereembedded in polyethylene glycol 1500 (Ruup 1964) and sec-tioned in transverse, longitudinal radial, and longitudinal tan-gential planeswith a Leica slidingmicrotome (Nussloch, Eisfeld,Germany) using a Styrofoam resin coat (Barbosa et al. 2010).Sections were typically 16–22 mm thick and sectioned with per-manent steel knives sharpened with sandpaper (Barbosa et al.2018). Anatomical sections were double stained with safrablau(Bukatsch 1972; modified by Kraus and Arduin 1997) andmounted in Canada balsam to make permanent slides.To describe the cambium, we embedded small portions of

the stem containing the cambium in HistoResin (Leica) to ob-serve its activity during primary and secondary growth. Sections,typically 5–10 mm thick, were cut with the rotary microtome,then stained with toluidine blue (O’Brien et al. 1964). The slideswere observed and photographed using a lightmicroscope (LeicaDMLB).

CUNHA NETO ET AL.—INTERXYLARY PHLOEM IN NYCTAGINACEAE 621

Results

Transition from Primary to Secondary Growth: Fromthe Polycyclic Eustele to a Single Cambium

In primary growth, most species of Nyctaginaceae have apolycyclic eustele, which consists of medullary bundles anda continuous cylindrical procambium (CCP; fig. 1A–1C). Sub-sequently, the CCP divides into fascicular and interfascicularsectors, giving rise to a ring of vascular bundles (primary vas-cular tissue) that delimits the pith (fig. 1B, 1C). Circumscribingthe vascular tissue, a parenchymatous pericycle that later be-comes lignified and an endodermis (starch sheath or innermostcortical layer) characterized by larger cells sometimes con-taining starch are usually present (figs. 2A, 2B, 2D–2F, 3, 4A–4C, 5B, 5D, 5E). Later, the CCP undergoes structural changeswith several periclinal and anticlinal divisions around its en-tire extent, initiating the transition from primary to secondary

growth (fig. 1D). Thus, the cambium develops in continuitywith the CCP, since the continuous ring of fascicular andinterfascicular procambium becomes the continuous fascicularand interfascicular cambium (figs. 1C, 1D, 2). The transitionfrom primary to secondary growth is also marked by the ligni-fication of several cells in the periphery of the pith (figs. 2C,2E, 3A, 3B).

In early stages of secondary growth, additional secondary xy-lem and phloem conducting cells are produced by the fascicularcambium within the vascular bundles, while the interfascicularcambium gives rises mostly to secondary xylem fibers internallyand phloem parenchyma cells externally (figs. 1D, 2A, 2B, 2E,2F). Therefore, at this stage, a single cambium that produces sec-ondary xylem to the inside and secondary phloem to the outsideis formed (fig. 2A). Soon, this cambium initiates an irregular ac-tivity (described in detail below) whereby secondary xylem andsecondary phloem are produced at unequal rates around the cir-cumference of the stem (figs. 2C, 2E, 2F, 3C, 3E). Because of this

Table 1

List of Studied Species from Nyctaginaceae Used for Developmental Analyses

Tribe, species Habit Source/collector Location

Nyctagineae:Acleisanthes chenopodioides (A.Gray)

R.A.Levin.Herb Douglas 2289, 2293 (FLAS) Las Cruces, New Mexico

Commicarpus scandens (L.) Standl. Liana Acevedo-Rodríguez 16250 (US),Douglas 2291 (FLAS)

Tonalá, Oaxaca, Mexico; New Mexico

Cyphomeris gypsophiloides(M. Martens & Galeotti) Standl.

Herb Douglas 2287 (FLAS) Organ Mountains–Desert Peaks NationalMonument, Las Cruces, New Mexico

Mirabilis cf. albida (Walter) Heimerl. Herb Douglas 2286 (FLAS) New MexicoMirabilis jalapa L. Subshrub Acevedo-Rodríguez 16480 (US) Veracruz, Mexico

Pisonieae:Grajalesia fasciculata (Standl.) Miranda Scandent tree Pace 765 (MEXU, SPF, US) Chiapas, MexicoGuapira laxa (Netto) Furlan. Tree Cunha Neto 08, 09 (HURB) Universidade Estadual de Feira de Santana,

Feira de Santana, BahiaGuapira pernambucensis (Casar.)

Lundell.Scandent shrub Cunha Neto 04, 05 (HURB) Alagoinhas, Bahia

Pisonia aculeata L. Liana Acevedo-Rodríguez 16549 (US),Aw 1073

Bonito, Mato Grosso do Sul, Brazil;Soledad, Cuba

Pisoniella glabrata (Heimerl) Standl. Liana/scandentshrub

Nee 64137, 64151 Parque Nacional Amboró, Vallegrande,Santa Cruz, Bolivia

Pisoniella arborescens (Lag. & Rodr.)Standl.

Liana Pace 738, 739 Hidalgo, Mexico

Neea hermaphrodita S.Moore Shrub Nee 64112 (USZ) Living Collection Jardín Botánico Municipalde Santa Cruz de la Sierra, Santa Cruzde la Sierra, Bolivia

Bougainvilleae:Bougainvillea berberidifolia Heimerl. Shrub Nee 64140 (USZ) Parque Nacional Amboró, Comarapa,

Santa Cruz, BoliviaBougainvillea campanulata Heimerl. Shrub Acevedo-Rodriguez 16772 (US),

Nee 64142 (USZ)Mato Grosso do Sul, Brazil; Parque NacionalAmboró, Comarapa, Santa Cruz, Bolivia

Bougainvillea spectabilis Willd. Scandent shrub Rossetto 453 (RB), Aw 24470 Estrada Carlos Chagas-Teófilo Otoni,Minas Gerais, Brazil

Colignonieae:Colignonia glomerata Griseb. Liana Nee 64157-64159 Parque Nacional Amboró, Samaipata,

Santa Cruz, BoliviaColignonia rufopilosa Kuntze Liana Nee 64061 (USZ) Cochabamba, Bolivia

Boldoeae:Salpianthus purpurascens (Cav. ex Lag.)

Hook. & ArnShrub Pace 774 El Cobanal, Chiapas, Mexico

622 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 Overview of young stems showing polycyclic eustele. A, Colignonia glomerata. B, C, Salpianthus purpurascens. The yellow triangle in Bindicates a vascular bundle developed from the continuous cylindrical procambium (CCP; shown in detail in C, ellipse). D, Cyphomerisgypsophiloides, early secondary growth (see details in fig. 2). Thick arrows indicate CCP, thin arrows indicate medullary bundles, and yellowtriangles indicate bundles derived from the CCP. co p cortex; ccpf p continuous cylindrical procambium, fascicular; ccpi p continuous cylindricalprocambium, interfascicular; mb p medullary bundles. Stained with astrablue (A). Stained with toluidine blue (B–D). Scale bars p 100 mm.

Fig. 2 Details of the onset of secondary growth in young stems. A, B, Acleisanthes chenopodioides. Detail of early secondary activity (i) withinthe bundles by the fascicular cambium and (ii) in the interfascicular cambium (center) producing mostly xylem fibers internally and parenchymaticcells in the secondary phloem, beneath the pericycle. In A, note the secondary xylem (white double arrow) and secondary phloem (black doublearrow). In B, note the lignified (pe) and parenchymatous (asterisks) pericycle. C, D, Cyphomeris gypsophiloides. Note the production of additionalconducting cells within the bundles (ellipses) and, in the interfascicular region, the production of mostly xylem fibers internally and parenchymaticcells in the secondary phloem. In D, the irregular activity of the cambium has produced xylem fibers delimiting the first phloem strand (black doublearrow), which is constituted of the phloem derived from the bundle of the continuous cylindrical procambium (ellipse). Also note the irregularity ofthe cambium (wavy appearance, arrows) producing xylem fibers and phloem derivatives in different rates and the developing coalescent cambium

irregular activity of the cambiumand because the first secondaryconducting cells are formed within the vascular bundles (by thefascicular cambium), the primary and secondary phloem of thebundles, derived from the CCP and the fascicular cambium, re-spectively, become the first interxylary phloem immersed withinthe secondary xylem, near the pith, as seen in stem cross sections(figs. 2C, 2E, 2F, 3A, 3B). Therefore, a regular cylinder of second-ary xylem and phloem is not formed in these species (figs. 2B, 3A,3B).

A Single Cambial Zone Maintains a Dynamic IrregularActivity Leading to Complex Stem Anatomies

In the studied Nyctaginaceae species, the cambium maintainsan irregular activity due to differential rates and arrangementsof its derivatives. To the inner side, the cambiumproducesmostlyxylem fibers, except for the regions where conducting elements(vessels) are formed, which are coradial with the phloem strands(figs. 2E, 2F, 3C, 3D, 4B–4E). In the secondary phloem, the axialparenchyma is predominant and usually forms a set of radiallyoriented cells (fig. 4). These layers of parenchyma vary from afew rows (2–10; figs. 2E, 2F, 3A, 3C, 3D, 4A–4C) to several rows(110; figs. 4E, 4F, 5A). The conducting cells of the phloem ap-pear as strands (fig. 4C, 4D), which are surrounded by the inner-most layers of the phloem axial parenchyma, named here“sheathing axial parenchyma” (figs. 4A, 4B, 4D, 4E, 5A, 5C).Externally to the phloem, parenchymatous and lignified pericy-clic cells may also be observed (fig. 2A, 2B).

As secondary growth progresses, from time to time the cam-bium reduces the production of xylem and increases the pro-duction of phloem, forming discrete concavities around thecambial girth (figs. 2D, 3A, 3D, 3E, 4A, 4C–4F). Later, theresulting phloem strands are included by the formation ofan arc of coalescent cambium external to these concavities(figs. 3E, 4C, 4E). The coalescent cambium is formed in con-tinuity with the rest of the cambial layer (i.e., the cambiumsectors that have grown in the regular mode, in terms of typesand rates of secondary tissues produced). The coalescent cam-bium differentiates from the inner layers of axial phloem pa-renchyma located external to the sheathing axial parenchymathat enclose the phloem strands (figs. 2E, 2F, 3E; 4C, 4E, 5E).The coalescent cambium produces xylem and phloem in theusual polarity and overarches the developing strand of phloem,enclosing it within the secondary xylem (figs. 4D, 5D, 5E).Through the activity of the coalescent cambium, mostly xylemfibers are produced internally, while only phloem parenchymaare formed externally (fig. 4D). By this whole process, all re-gions with conducting cells of the secondary phloem with theiroriginal cambial segment are embedded within the secondary

xylem (figs. 4D, 4F, 5B–5F, 6). Thus, in these species, the con-ducting phloem is restricted to the strands immersed within thesecondary xylem, and the secondary phloem external to the sin-gle cambium is composed only of nonconductive phloem cells(i.e., phloem parenchyma; figs. 4A–4C, 4E, 5). The cambiumenclosedwithin each strand is functional for some time, and bothconducting and nonconducting phloem can be distinguished(fig. 4A, 4F).Despite the irregular activity of the cambium forming phloem

strands throughout the stem and over the entire life span of theplant (see the cambium in mature stems in fig. 4), the secondaryxylem and phloemproduced by this cambial activity also exhibitfeatures associated with typical cambial growth. This is demon-strated by features such as the formation of rays (figs. 4A, 4E, 5A–5D, 7A, 7B),which are continuousbetween thexylemand phloemin the external portion of the phloem (fig. 5A, 5B); the alterna-tion of thick-walled and thin-walled fibers (fig. 4F); and the for-mation of growth rings formed by either thick-walled fibers oraxial parenchyma in lines (fig. 7A, 7B, 7D). In some species, someaxial phloem parenchyma cells become sclerified (figs. 4F, 5A,5B, 5D), while other species exhibit conspicuous ray dilatationin the secondary phloem and sclerification of axial and radialcells of the nonconducting secondary phloem (fig. 5A).

Different Arrangements of Interxylary PhloemOccur in Nyctaginaceae

The type of secondary growth resulting from the developmen-tal sequence described above indicates that the stems can beinterpreted as having interxylary phloem. In Nyctaginaceae,the ontogenetic pathway generating this pattern gives rise todistinct macromorphologies (figs. 6, 7A–7C). The different ar-rangements are distinguished in cross sections: type 1, strandsof phloem and sheathing axial parenchyma confined to smallislands (figs. 6A, 6B, 7A); type 2, strands of phloem with thesheathing axial parenchyma forming a patchy arrangementdue to tangential confluences of the sheathing axial parenchymaconnecting two or more islands (figs. 6C, 6D, 7B); and type 3,long concentric tangential bands of sheathing axial parenchymawith small phloem strands within them (figs. 6E–6H, 7C). Thethree different arrangements result from variation in the extentof the coalescent cambium: the longer the coalescent cambiumis, the longer the islands or bands of sheathing axial parenchymaproduced are. It is observed that in species with a type 3 arrange-ment, the concentric aspect is given by the continuity of conflu-ent sheathing axial parenchyma, while the sieve tube elementsand associated cells are still concentrated in small segments ofthese bands, in positions opposite the xylem vessels (figs. 6E,6F, 7C, 7E). In some cases, the rays are usually wider and may

(arrowheads) overarching the phloem that will form another phloem strand. Red double arrow indicates primary xylem, and green double arrowindicates secondary xylem. E, F, Mirabilis jalapa. Detail of the single cambium (thick arrows) producing cell types at differential rates. Note thatonly phloem parenchyma remains at the periphery, while sieve tube elements and companion cells are exclusive to the strands. In E, note the phloemparenchyma undergoing cell division (gray arrows) to form the coalescent cambium that will overarch the early produced phloem. In F, note thepresence of cambial cells between the xylem inside (vessel element, white arrow) and phloem outside (sieve tube element, green arrow), evidencingthe cambium bifacial activity. These phloem conducting cells will be later overarched by a new coalescent cambium. Yellow arrows indicate cambialinitials, and black double arrows indicate first phloem strand. co p cortex; en p endodermis; mb p medullary bundles; pa p phloem parenchyma;pe p lignified pericycle; ph p phloem; pi p pith; sph p secondary phloem; sxy p secondary xylem; xy p xylem. Stained with toluidine blue (A, C–F). Stained with safrablau (B). Scale bars p 100 mm (A–E), 50 mm (F).


Fig. 3 Cross view of early secondary growth in young stems. A, Pisoniella glabrata. B, Bougainvillea spectabilis. In A and B, note the absenceof a regular cylinder. Note also lignified cells from periphery of the pith (asterisks) and that the first interxylary phloem (black double arrow) wasformed from the primary phloem of the vascular bundles (originated from the continuous cylindrical procambium) along with some secondaryphloem produced by the fascicular cambium within the bundles; new phloem strands (yellow double arrows) are produced by the coalescent cam-bium (arrowheads) and the irregular activity of the main cambium (thick arrows). C–E,Pisoniella arborescens. Note the irregularity (wavy appear-ance) of the cambium (thick arrows), which produces cell types at differential rates, producing less secondary xylem andmore secondary phloem (yellowdouble arrow). Also note that the developing coalescent cambium (arrowhead) will overarch the phloem strand, which maintains part of the main cam-bium. enp endodermis; mbpmedullary bundles; pap phloem parenchyma; pep lignified pericycle; scp sclerified parenchyma; sxyp secondaryxylem. Stained with safrablau (A), stained with safranine (B), stained with toluidine blue (C–E). Scale bars p 100 mm.

form a networking arrangement with the tangential sheathingaxial parenchyma (figs. 6D, 6F, 6H, 7C–7E).

Some taxa are characterized by starting with one anatomicalarrangement, such as members of Bougainvillea that begin withtype 1 anatomy (discrete phloem islands), followed by type 2anatomy (patches of phloem and sheathing axial parenchyma),and eventually mature to a typical type 3 anatomy (fig. 7C). Interms of adult forms, some genera have predominantly one sin-gle arrangement (e.g., Bougainvillea and Colignonia, both withtype 3), while other genera have species with different adult ar-rangements. For instance,Guapira,Neea, and Pisonia predom-inantly show phloem islands (type 1), but some species, suchas Guapira pernambucensis and Pisonia obtusata, also havephloem in patches (type 2; see table 2). The genus Pisoniella, withonly two recognized species, is interesting because each specieshas a different arrangement: P. glabrata has type 1 anatomy(phloem islands), while P. arborescens exhibits type 3 anatomy(concentric bands; table 2). More generally, the species withinterxylary phloem are noteworthy for possessing conductingelements of both xylem and phloem grouped along narrowsegments of the cambium, which produce a cluster-like arrange-ment in the overall patterning of the secondary tissues (fig. 4A,4B). However, the fibrous tissue around these clusters of con-ducting cells is also xylem, produced by the cambium, as demon-strated by the formation of growth rings delimited by radiallynarrowfibers (fig. 7A) and alsomarginal lines of axial parenchyma(fig. 7B, 7D).

Discussion

Interxylary Phloem in Angiosperms

Interxylary phloem (i.e., strands of phloem embedded in thesecondary xylem) is a type of cambial variant observed in morethan 10 angiosperm families distributedmostly within the rosidsand asterids, such asAcanthaceae, Apocynaceae, Combretaceae,Icacinaceae, Loganiaceae, and Thymeleaceae (Chalk and Chat-taway 1937; van Veenendaal and den Outer 1993; Carlquist2001, 2013; Lens et al. 2008; Patil and Rajput 2008; Angyalossyet al. 2012, 2015; Gondaliya and Rajput 2017; Luo et al. 2020).In most of these families, interxylary phloem seems to be presentin only a few genera or species (e.g., Strychnos, Loganiaceae;someCombretum, Combretaceae;Erisma, Vochysiaceae), whilesome taxa have the regular mode of vascular cambial growth(Carlquist 2013). In somefamilies, interxylaryphloemis not the onlycambial variant to occur, but other types might also be present,including successive cambia (e.g., Acanthaceae, Icacinaceae; Lenset al. 2008; Angyalossy et al. 2012; Carlquist 2013).

The formation of interxylary phloemdoes not follow the sameontogeny in all families. To date, at least three main types havebeen characterized. One is the Strychnos type, produced bysegments of vascular cambium that temporarily form less sec-ondary xylem and more secondary phloem, generating depres-sions or concavities that later are overarched by a flanking cam-bium that encloses the phloem and starts producing vasculartissue in the usual way, with xylem to the inside and phloem tothe outside. In particular, this pattern creates interxylary phloeminwhich the phloem strands aremaintainedwith a portion of thecambium (van Veenendaal and den Outer 1993; Rajput et al.2010). The second is the Thunbergia type, in which small seg-

ments of the cambium produce secondary phloem (instead ofxylem) to the inside for a short period of time and then resumeformation of xylem, embedding phloem islandswithin thewood.In this type of interxylary phloem, no cambium remains insidethe phloem islands. Similar development has been observed inCombretum (van Veenendaal and den Outer 1993) and Aqui-laria (Thymelaeaceae; Luo et al. 2018, 2020). The third typeof interxylary phloem is the Ixanthus type, formed by redif-ferentiation of axial parenchyma into strands of phloem. Thispattern also occurs in other taxa of Gentianaceae (e.g.,Orphium)as well as in the families Onagraceae (e.g., Pseudolopezia) andConvolvulaceae (e.g., Turbina; Carlquist 2013).The terminology applied to interxylary phloem, sometimes

called “included phloem,” has undergone critical reexaminationas a result of improved understanding of anatomical and devel-opmental diversity of cambial variants. Currently, the term“interxylary phloem” is preferred instead of “included phloem”

because in the past some studies used the latter term to describestems with similar macromorphologies that originated from dif-ferent ontogenies (Carlquist 2013). For example, the presence ofincluded phloem was used to define groups within Icacinaceae,but it has been demonstrated by Lens et al. (2008) that both suc-cessive cambia and interxylary phloem can occur in the familyand that some species of Sarcostigma were inaccurately identi-fied as having successive cambia instead of interxylary phloem.Here, we showed that a similar case seems to have occurredfor Nyctaginaceae.

Interxylary Phloem of Nyctaginaceae

In Nyctaginaceae, just as in other families, interxylary phloemresults from a single cambium, which has an unequal activity,switching on and off in different sectors to generate interxylaryphloem. These phloem strands are formed in such a way that aportion of the original cambium is maintained, similar to whatis observed in Strychnos (Loganiaceae), as reported in severalstudies (Scott and Brebner 1889; van Veenendaal and den Outer1993; Rajput et al. 2010; Moya et al. 2018). However, thedynamics of the cambium in Nyctaginaceae species generatephloem strands consisting of conducting cells associated withthe sheathing axial parenchyma (remaining phloem parenchymacells) in several amounts and arrangements, which makes thistype of interxylary phloem novel and unique.The formation of interxylary phloem in Nyctaginaceae seems

to be intrinsically related to the type of eustele, since virtuallymost of the species with polycyclic eustele (but not all, e.g.,Allionia; Cunha Neto et al. 2020b) have this type of cambialvariant. As a rule, the ontogeny of stems with this cambial var-iant in Nyctaginaceae involves three steps that deviate from thetypical developmental pattern found in plants with the regularmode of vascular cambial growth: (i) the cambium is formedin a continuum with a CCP; (ii) the activity of the cambium isregular for a short period of time and soon becomes irregular,leading to the absence of a complete regular cylinder in the stem;and (iii) the unusual dynamics of the cambium lead to the pro-duction of phloem strands embedded within the secondary xy-lem throughout the stem body.In Nyctaginaceae, the formation of this cambial variant,

which generates phloem strands, is based on modifications atthe cellular level, especially the pattern and/or rate of cell division


Fig. 4 Details of cambium in mature stems. A, Neea hermaphrodita. Note the lignified pericycle delimiting the vascular system and the irreg-ular cambium (thick arrow) producing new phloem strands (yellow double arrows). Also note the developed phloem strands (black double arrow),which are formed by conducting and nonconducting cells in the center and surrounded by sheathing axial parenchyma, also originated from thecambium. B, Grajalesia fasciculata. Detail of cambium (thick arrow) and phloem strands (black double arrow). C, D, Guapira pernambucensis.C, Detail of cambium (thick arrow) and the coalescent cambium (arrowhead) overarching the developing phloem strands (yellow double arrow).Note that in the center, only xylem fibers are produced internally, and parenchymatic cells are formed to the outside. Vessels and sieve tubeelements are formed to the left and to the right (see detail in D). Yellow asterisk indicates crystal. D, On the left, vessels and sieve tube elementsare produced by the cambium (thick arrow). On the right, note a phloem strand (black double arrow) with included cambium (yellow arrows), sievetube elements with their companion cells in the center, and sheathing axial parenchyma surrounding them. As the coalescent cambium becomes

and differentiation of cambium initials and its derivatives, indifferent sectors of the cambium. There is evidence that themeristematic activity of the vascular cambium is regulated bygenetic mechanisms that can independently control distinct de-velopmental modules that consist of distinct sets of processes,such as periclinal divisions, the control of radial polarity, orthe independent differentiation of xylem and phloem (Du andGroover 2010; Etchells et al. 2013; Bossinger and Spokevicius2018; Tomescu and Groover 2019). The interplay of such inde-pendent modules regulating these major biological processesmight explain the asynchronous development of vascular tissuesresulting in disparate morphologies, as is the case of interxylaryphloem in Nyctaginaceae. Future transcriptomic studies of thevascular meristems in Nyctaginaceae can further our under-standing of the molecular developmental mechanisms underly-ing procambial identity in the CCP, the functioning of the cam-bium as a bifacial meristem, and its activity in distinct sectorsthat produce xylem and phloem at unequal rates.

The ontogeny of interxylary phloem as described here is aunique developmental trajectory likely present not only inNycta-ginaceae but also in related families. In some Amaranthaceae,for example, we note stems (e.g., Celosia argentea; Myśkowet al. 2019) that seem to have the same polycyclic eustele withmedullary bundles and a CCP (“continuous concentric procam-bium” of Cunha Neto et al. 2020a), while in secondary growth,some species also initiate vascular cambial growth without aregular cylinder of cambium but form phloem strands with var-ious arrangements (patches, concentric) in mature stems (e.g., Sim-mondsia chinensis [Schweingruber et al. 2011];Hebanthe eriantha[Rajput andMarcati 2013]; Iresine spp. [Zumaya-Mendoza et al.2019]; Camissoa altissima [I. L. Cunha Neto, personal observa-tion]). In the case of Hebanthe eriantha (Rajput and Marcati2013), the stem has phloem islands in the inner region and radi-ally longer phloem strands as they reach the periphery. This ob-servation indicates that even stage-dependent polymorphism inthemode of vascular cambial growth similar to that documentedhere in some Nyctaginaceae, where two types of arrangementsoccur in the same stem, is present in other Caryophyllales. How-ever, more studies are needed to confirm these hypotheses. Al-thoughmost taxa with interxylary phloem in other families havephloem islands (e.g.,Combretum, Strychnos), concentric arrange-ment seems to not be limited to Nyctaginaceae or Caryophyllales(see Carlquist 2013). In their study of Icacinaceae, Lens et al.(2008) mentioned species of Pleurisanthes flava with interxylaryphloem that is more or less arranged in rings (not illustrated bythose authors), while Sarcostigma, another genus with this cam-bial variant, is described as having scattered phloem islands. InMalpighiaceae, the genus Dicella has interxylary phloem (Chodatand Vischer 1917), and in Dicella macrocarpa, this interxylaryphloem forms patches that are also surrounded by differentamounts of axial parenchyma (Pace 2015).

The abundance and distribution of axial parenchyma asso-ciated with the phloem strands in Nyctaginaceae are remark-able. In previous studies that classified these species as havingsuccessive cambia, this tissue surrounding the conducting ele-ments of each vascular increment was termed “conjunctive tis-sue,” whether it was parenchymatous or fibrous (Rajput andRao 1998; Carlquist 2007, 2010; Hernández-Ledesma et al.2011). Instead of “conjunctive parenchyma,” we use the term“sheathing axial parenchyma” for these large parenchyma cellssurrounding the phloem, because in the literature the term“conjunctive tissue” has been used originally for products ofthe secondary thickening meristem in the monocots (Cheadle1937; Diggle and DeMason 1983) as well as in the context ofsuccessive cambia (Carlquist 2007, 2010). More importantly,it is reported that in most species with successive cambia, theconjunctive tissue originates from the pericycle (master cambiumof Carlquist [2007] or procambium remnants of Myśkow et al.[2019]), while the sheathing axial parenchyma ofNyctaginaceaeoriginate directly from the cambium. Within the Nyctaginaceae,in species with type 2 and type 3 arrangements, neighboringstrands can be connected by lateral or radial confluences of sheath-ing axial parenchyma, contributing to the different arrange-ments seen in cross section. These radial connections have beennamed “radial plates of conjunctive tissue,” “raylike radial sheetsof conjunctive parenchyma” (Metcalfe and Chalk 1950), and“radial panels of conjunctive parenchyma” (Esau and Cheadle1969). Given the multiple physiological functions of paren-chymatic tissue, the relative amounts and distribution of thesheathing axial and radial parenchyma in specieswith interxylaryphloem are key features that still need experimental investigation.The physiological impact of the conducting phloem confinedwithin the secondary xylem in an apparent regular arrangementis another aspect that deserves to be fully investigated in these taxa.

Classification of the Cambial Variant: Why InterxylaryPhloem and Not Successive Cambia

Some cambial variants are readily identifiable on the basis oftopological aspects, as in the case of phloem wedges (commonin Bignoniaceae) or axial xylem and phloem elements in plates(common in Aristolochiaceae, Piperaceae). However, the dis-tinction between successive cambia and interxylary phloem hasbeen problematic in several cases (Lens et al. 2008; Carlquist2013), including for Nyctaginaceae (table S1). Most of the pre-vious studies with Nyctaginaceae did not document in detailthe development of the stems, using instead only themature stemanatomy. In addition, themajority of these studies examined onlya few species frommore common genera, such asGuapira,Neea,and Pisonia, whose timbers can sometimes be found in the mar-ket; these are taxa with remarkably similar anatomy, showingin most cases small phloem strands (phloem islands; i.e., our

active, new xylem fibers (asterisks) are produced and enclose the phloem strand. Yellow asterisk indicates crystal. E, Pisonia aculeata. Detail ofcambium (thick arrow) and coalescent cambium (arrowheads) overarching the future phloem strand (yellow double arrow). Yellow asterisks indi-cate crystals. F, Bougainvillea spectabilis. Detail of irregular cambium producing phloem strands (yellow double arrow). Note that both thick-walled xylem fibers (green double arrow) and thin-walled xylem cells (blue double arrow) are both produced by the same cambial initials—theyarise in a succession in radial files. co p cortex; cph p conducting phloem; pa p phloem parenchyma; pe p lignified pericycle; ncph p noncon-ducting phloem; pa p phloem parenchyma; ra p vascular ray; sap p sheathing axial parenchyma; sc p sclerified parenchyma; ste p sieve tubeelement; sxy p secondary xylem; ve p vessel element. Stained with safrablau (A), stained with toluidine blue (B–E), stained with safranine (F).Scale bars p 250 mm (A), 200 mm (B, E), 100 mm (C, F), 50 mm (D).


Fig. 5 Details of cambiumand secondary tissues inmature stems.A,BCross sections.C–F, Longitudinal radial sections.A, Pisonia ligustrifolia. Notethe continuity of the rays (red arrows) between the xylem and phloem and the ray dilatation (left) and sclerified parenchyma, common features of the de-veloped nonconducting phloem (white double arrow). B, Pisonia aculeata. Note the cambium (thick arrow) and the coalescent cambium (arrowheads)enclosing the phloem strand (yellow double arrow). C, D, Guapira laxa. Rays (red arrows) crossing the xylem and the phloem and reaching the lignifiedpericycle. Note that some phloem parenchyma become sclerified (red asterisks), intermixing with the lignified pericycle, and the xylem fibers (asterisks)originated from the coalescent cambium (arrowhead), which encloses the phloem strand (yellow double arrow). Thick arrow indicates single cambium.E,Colignonia glomerata. Secondary xylem, phloem strand (yellowdouble arrow) and fibers (asterisks) derived from the coalescent cambium (arrowheads).Green arrow indicates sieve tube element. F, Bougainvillea campanulata. Note the secondary xylem, the developing phloem strand (yellow double arrow),and the coalescent cambium (arrowheads). cop cortex; enp endodermis; pap phloemparenchyma; pep pericycle; rap vascular ray; sapp sheathingaxial parenchyma; scp sclerified parenchyma; sxyp secondary xylem. Stainedwith safrablau (A). Stainedwith toluidine blue (B–F). Scale barsp 200 mm(A, C, E), 100 mm (B, D, F).

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Fig. 6 Diversity of stem vascular architectures of Nyctaginaceae species with interxylary phloem. A, C, E, G, Macroscopic view. B, D, F, H,Microscopic view. A, Neea hermaphrodita. B, Pisonia aculeata. C, D, Pisoniella glabrata. E, Colignonia glomerata. F, Colignonia rufopilosa.G, Bougainvillea berberidifolia. H, Bougainvillea campanulata. Scale bars p 5 mm (A, C, E, G), 2 mm (B, D, F, H).

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Fig. 7 Cross section of mature stems showing different arrangements of interxylary phloem.A,Guapira laxa (type 1, phloem islands). Black arrowindicates growth rings delimited by thick fibers. B, Pisonia obtusata (type 2, patches formed by longer phloem strands). Yellow arrows indicate growthrings delimited by parenchyma in lines.C,Colignonia rufopilosa (type 3, continuous concentric). Note that at the beginning of secondary growth, smallphloem strands are formed (type 1, yellow double arrow), and later, various tangential bands are produced (type 3, black double arrow). Arrowheadsindicate rays, and yellow pointers indicate tangential bands of phloem and sheathing axial parenchyma.D, Pisoniella glabrata (type 1, phloem islands;type 2, patches). Note the connection between neighboring phloem strands (blue cells) through the larger abundance of sheathing axial parenchyma(tangential whitish tissue). Arrowheads indicate rays, and yellow arrows indicate growth rings delimited by parenchyma in lines. E, Pisoniella arbor-escens (type 3, tangential bands). Darker blue cells represent the phloem conducting cells, while the lighter cells are sheathing axial parenchyma. Ar-rowhead indicates rays, and yellow pointers indicate tangential bands of phloem and sheathing axial parenchyma. mbpmedullary bundles; pip pith;sap p sheathing axial parenchyma. Stained with safrablau (A, C–F). Stained with safranine (B). Scale bars p 500 mm.

type 1). Chalk and Chattaway (1937, p. 20) described the inter-xylary phloem inNyctaginaceae as “strands of included phloemisolated, wider tangentially than radially,” without any furtherdevelopmental information. Although these authors recognizedthat tangential bands could also be formed in some taxa (e.g.,Pisonia), other genera with concentric arrangement (e.g., Bou-gainvillea,Colignonia) were classified as having successive cam-bia, which is inconsistent with our findings. For Studholme andPhilipson (1966, p. 355), who studied the anatomy of a singlespecies of Pisonia, the stem diverges from a regular mode of vas-cular cambial growth by “the differentiation of longitudinalstrands of phloem within the zone of meristematic cells.” Thisidea of a single cambium or a meristematic zone that moves out-ward and produces phloem “within” it instead of outside it hadbeen defended by some authors and neglected by others (table S1).Our results also show that the original cambium maintains its ac-tivity throughout, producing phloem outward and xylem inwardand being, therefore, a bifacial cambium. Sectors of this cambiumremain with the phloem strands, and their activity is maintainedfor some time (andnonconducting, collapsedphloem is often seenin some species), providing additional evidence for this bifacialcambial activity.

The presence of a single vascular cambium is the main aspectthat distinguishes interxylary phloem from the phloem producedby successive cambia (Carlquist 2001, 2013; Angyalossy et al.2015). Therefore, interpretations proposing successive cambiafor most species of Nyctaginaceae are not supported, since mul-tiple cambia are not observed. Stemswith true successive cambiagenerally initiate secondary growth with a single cambium pro-ducing a complete cylinder of secondary xylem and secondaryphloem, and only after some period of such regular secondarygrowth, the first additional cambium is formed outside the initialcambium and independent of it. These ontogenetic steps charac-terize the formation of successive cambia in distantly relatedlineages, including gymnosperms (e.g., Gnetales,Gnetum; Carl-quist and Robinson 1995; Carlquist 1996, 2012) and severaleudicot families (e.g., Convolvulaceae [Terrazas et al. 2011];Fabaceae [Dias-Leme et al. 2020]; Menispermaceae [Tamaioet al. 2009]; Sapindaceae [Cunha Neto et al. 2018]; Vitaceae[Pace et al. 2018]), as well as in someNyctaginaceae not coveredhere, which we will present in a future paper (Cunha Neto et al.,unpublishedmanuscript). In contrast, in species with interxylary

phloem like those we have presented here, secondary growthstarts with a variant cambium and continues with productionof secondary phloem and secondary xylem at differential ratesthroughout its girth. This developmentalmode prevents the stemfrom forming a typical cylinder of secondary vascular tissues,and phloem strands immersed within the secondary xylem areformed starting in the early stages of secondary growth. In addi-tion, the coalescent cambium overarching the phloem strands isusually continuouswith the original cambium. To the best of ourknowledge, the development of interxylary phloem with thesecharacteristics has not been reported previously.Why the vascu-lar cambium has this differential activity throughout its girth isstill unknown, but given the regularity in the distribution ofthe phloem strands within the xylem, a robust, canalized regula-tory mechanism is expected to exist. Such a mechanism explain-ing the distribution of phloem strands could involve interactionssimilar to those of the Turing-like mechanism in the stochasticreaction-diffusion model proposed by Hearn (2019).

Systematic and Phylogenetic Implications ofInterxylary Phloem in Nyctaginaceae

The observation of interxylary phloem in Nyctaginaceae rep-resents a new piece of information with phylogenetic impor-tance, since it indicates the evolution of this feature within thelarge and diverse order Caryophyllales. The presence of inter-xylary phloem in Nyctaginaceae is also significant at the familylevel. We encountered this cambial variant in at least 16 generaof Nyctaginaceae, distributed in five of seven tribes. Undoubt-edly, this is the most widespread type of cambial variant in thefamily. At the genus level, we found an interesting example ofhowdifferent vascular arrangements can beuseful as a taxonomicfeature to distinguish related species. By investigating the twospecies of the genus Pisoniella, we found that they have differentarrangements of interxylary phloem: P. glabrata shows phloemislands (type 1 and/or 2), whereas P. arborescens has stems withconcentric arrangement of interxylary phloem (type 3). Possiblyrelated to this difference in vascular cambial growth, we showedin an earlier study that these two species also differ strikingly inthe architecture of their primary vascular system, since P. gla-brata has a polycyclic eustele characterized by concentric rings

Table 2

Characteristics and Distribution of the Different Arrangements of Interxylary Phloem in Nyctaginaceae

Arrangement Taxa Observations

Type 1—phloem islands: small strandsof secondary phloem embedded withinconjunctive parenchyma

Acleisanthes, Anulocaulis, Boerhavia,Commicarpus, Cryptocarpus, Grajalesia,Guapira, Neea, Pisonia, Pisoniellaglabrata, Mirabilis

The genera Abronia and Nyctaginia probably be-long to this category, but they are herbs with fewsecondary growths and with very slender stems

Type 2—patches: longer tangential strands,usually forming confluences betweentwo or more phloem islands

Commicarpus, Guapira, Neea, Pisonia,Pisoniella glabrata, Phaeoptilum

In Guapira, Neea, and Pisonia, some species mayshow patches in later secondary growth and/orin scandent shrubs, as observed in G.pernambucensis.

Type 3—continuous concentric: long tan-gential bands of sheathing axial paren-chyma with secondary phloem embeddedin it

Bougainvillea, Colignonia, Cyphomeris,Pisoniella arborescens, Salpianthus

Belemia may fall into this category, since it is amember of the Bougainvilleae tribe; however,the samples from this genus were obtained fromthe herbarium and were too young


of medullary bundles (more than 20 bundles), while P. arbor-escens has only one ring with eight bundles (Cunha Neto et al.2020a).

Conclusions

Our examination of the stem development of Nyctaginaceaeconfirms the presence of interxylary phloem in the family. Thistype of cambial variant has been overlooked in Nyctaginaceae,as most previous studies have reiterated descriptions of succes-sive cambia as the common pattern of secondary growth withinthe family. Here, we not only confirm the presence of interxylaryphloem but also demonstrate that these species combine an un-common set of features (e.g., polycyclic eustele, absence of regularcambial cylinder, conducting phloem confinedwithin the second-ary xylem) that clearly differentiates them from the regular modeof vascular cambial growth ofmost eudicots and from stemswithsuccessive cambia. In addition, our results show that within thissame type of cambial variant, there are different stem architec-tures resulting from similar ontogenetic pathways. These findingsemphasize the importance of developmental studies in furtheringour understanding of stemmacromorphologies and highlight thecomplexity and diversity of stem architectures in Nyctaginaceae.Broader future studies including species of other lineages are

planned to shed light on the distribution and the evolution of de-velopment of all cambial variants found inNyctaginaceae (CunhaNeto et al., unpublished manuscript).

Acknowledgments

This work was carried out as part of the PhD dissertation ofI. L. CunhaNeto,whowas funded by the Fundação deAmparo àPesquisa do Estado de São Paulo (2017/17107-3) and Coor-denação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (finance code 001). M. R. Pace was funded by DirecciónGeneral de Asuntos del Personal Académico (Papiit IA200319,IA200521). We are grateful to Cyl Farney C. de Sá, MichaelH.Nee, andNormanA.Douglas for assistance in field collectionand identificationofplants.AlejandroMantúfar,ÁlvaroCampos,and Camille Truong are also acknowledged for field assistance.We thank the staff of the Plant Anatomy Laboratory of the Uni-versity of São Paulo for assistance with laboratory work.We areindebted to Associate Editor Alexandru M.F. Tomescu and twoanonymous reviewers of the manuscript for valuable commentsand corrections. We have no conflicts of interest for this article.Supplemental material has been deposited in the Dryad DigitalRepository (https://doi.org/10.5061/dryad.w9ghx3fn1).

Appendix

List of species examined and site of collection/wood collection where specimens were obtained for all studied Nyctaginaceae (or-ganized by tribes).Herbaria: A, Harvard University Herbaria; FLAS, Florida Museum of Natural History; HURB, Universidade Federal do

Recôncavo da Bahia; MEXU, Universidad Nacional Autónoma de México; MW, Moscow State University; RB, Jardim Botânicodo Rio de Janeiro; SPF, Universidade de São Paulo; US, Smithsonian Institution; USZ, Museo de Historia Natural Noel KempffMercado, Universidad Autónoma Gabriel René Moreno.Species name, collector/collector number (herbarium), collection site.

Tribe Nyctagineae

Abronia fragrans Nutt. ex Hook., Douglas 2290 (FLAS), Las Cruces, New Mexico, USA. Abronia neealleyi Standl., Douglas2281, (FLAS) Eddy County, Yeso Hills, New Mexico, USA. Acleisanthes chenopodioides (A.Gray) R.A.Levin., Douglas 2289,2293 (FLAS), Las Cruces, New Mexico, USA. Acleisanthes lanceolata (Wooton) R.A.Levin, Douglas 2277 (FLAS), MaloneMountains, Sierra Blanca, Texas, USA. Acleisanthes longiflora A.Gray., Douglas 2279 (FLAS), Malone Mountains, Sierra Blanca,Texas, USA. Allionia choisyi Standl., US 498327, New Mexico, USA; Allionia incarnata L., Nee 64124–64126, (USZ), ParqueNacional Amboró, Pampa Grande, Santa Cruz, Bolivia; Douglas 2293 (FLAS), Las Cruces, New Mexico, USA. Anulocaulisleiosolenus var. gypsogenus (Waterf.) Spellenb. & Wootten, Douglas 2280 (FLAS) Eddy County, Yeso Hills, New Mexico,USA; Douglas 2283 (FLAS), Crest of 7 Rivers Hills, New Mexico, USA. Boerhavia diffusa L., Pace 753 (MEXU, US), Veracruz,Mexico. Boerhavia linearifolia A.Gray., Douglas 2284 (FLAS), New Mexico, USA. Boerhavia torreyana (S. Watson) Standl.,Douglas 2294 (FLAS), Las Cruces, New Mexico, USA. Boerhavia wrightii A.Gray., Douglas 2288 (FLAS), Las Cruces, NewMexico, USA. Commicarpus scandens (L.) Standl., Acevedo-Rodríguez 16250 (US), Tonalá, Oaxaca, Mexico; Douglas 2291(FLAS), New Mexico, USA. Cyphomeris gypsophiloides (M. Martens & Galeotti) Standl., Douglas 2287 (FLAS), OrganMountains-Desert Peaks National Monument, Las Cruces, New Mexico, USA. Mirabilis cf. albida (Walter) Heimerl., Douglas2286 (FLAS), New Mexico, USA. Mirabilis jalapa L., Acevedo-Rodríguez 16480 (US), Veracruz, Mexico. Nyctaginia capitataChoisy., Douglas 2282 (FLAS), New Mexico, USA. Okenia hypogea Schltdl. & Cham., Pace 749 (MEXU, SPF, US), Veracruz,Mexico


Tribe Pisonieae

Grajalesia fasciculata (Standl.) Miranda., Pace 765 (MEXU, SPF, US), Chiapas, Mexico. Guapira bracei Britton, USw 23138,Monroe, Florida, USA. Guapira cuspidata (Heimerl) Lundell, USw 35378, Mérida, Venezuela. Guapira linearibracteata (Heimerl)Lundell., USw 29941, Belize. Guapira pernambucensis (Casar.) Lundell., Cunha Neto 04–05 (HURB), Alagoinhas, Bahia, Brazil.Guapira graciliflora (Mart. ex J.A.Schmidt) Lundell., Cunha Neto 06 (HURB), Alagoinhas, Bahia, Brazil. Guapira laxa (Netto)Furlan., Cunha Neto 08 (HURB), Universidade Estadual de Feira de Santana, Feira de Santana, Bahia, Brazil. Guapira ligustrifolia(Heimerl) Lundell, USw 1886, San Gabriel Island, Dominican Republic; USw 1982, Hispaniola Island, Dominican Republic.Guapira longifolia (Heimerl) Little, Hw 29374. Neea cauliflora Heimerl in Engl. & Prantl, USw 9134, Acre, Brazil. Neeadelicatula Standl., Pace 689 (US), Reserva Biológica La Selva, Sarapiquí, Heredia, Costa Rica. Neea hermaphrodita S.Moore,Nee 64112 (USZ), Living Collection Jardín Botánico Municipal de Santa Cruz de la Sierra, Santa Cruz de la Sierra, Bolivia. Neealaetevirens Standl., Pace 713, 716 (US), Reserva Biológica La Selva, Sarapiquí, Heredia, Costa Rica; USw 16154, Los Santos, Pan-ama. Neea macrophylla Britton ex Rusby., USw 40851, San Martín, Peru. Neea ovalifolia Spruce ex J.A. Schmidt, USw 42783,Régina, French Guiana. Neea psychotrioides Donn. Sm., Pace 763 (MEXU), Estación de Biología Tropical Los Tuxtlas, Veracruz,Mexico; USw 29939, Belize. Pisonia aculeata L., Acevedo-Rodríguez 16549 (US), Bonito, Mato Grosso do Sul, Brazil; Aw 1037.Pisonia brevipetiolata (Heimerl) Urb., USw 1793, Fond Parisien, Etang Saumatre, Haiti. Pisonia fragrans Dum.-Cours., USw35502, Republica Dominicana. Pisonia ligustrifolia Heimerl, USw 1886, San Gabriel Island, Dominican Republic. Pisoniaobtusata Jacq., Aw8322. Pisonia rotundata Griseb., USw 21834, Monroe, Florida, USA; Aw 8323. Pisoniella arborescens(Lag. & Rodr.) Standl., Pace 738–739 (MEXU, SPF, US), Alfajayucan, Hidalgo, Mexico. Pisoniella glabrata (Heimerl) Standl.,Nee 64137, 64151 (USZ), Parque Nacional Amboró, Vallegrande, Santa Cruz, Bolivia.

Tribe Bougainvilleeae

Belemia fucsioides Pires., Farney 4887, 4888 (RB). Bougainvillea sp1., Nee 64176 (USZ), Rio Grande, Santa Cruz de la Sierra,Bolivia. Bougainvillea sp2., Nee 64182 (USZ), Rio Grande, Santa Cruz de la Sierra, Bolivia. Bougainvillea berberidifolia Heimerl.,Nee 64140 (USZ), Parque Nacional Amboró, Comarapa, Santa Cruz, Bolivia. Bougainvillea campanulata Heimerl., Acevedo-Rodríguez 16772 (US), Mato Grosso do Sul, Brazil; Nee 64142 (USZ), Parque Nacional Amboró, Comarapa, Santa Cruz, Bolivia.Bougainvillea modesta Heimerl., Nee 64115 (USZ), Living Collection Jardín Botánico Municipal de Santa Cruz de la Sierra, SantaCruz de la Sierra, Bolivia. Bougainvillea stipitata Griseb., Nee 64121 (USZ), Parque Nacional Amboró, Samaipata, Santa Cruz,Bolivia. Bougainvillea spectabilis Willd., Rossetto 453 (RB), Estrada Carlos Chagas-Teófilo Otoni, Minas Gerais, Brazil.Phaeoptilum spinosum Radlk., MADw 37340, Mocamedes, Angola.

Tribe Colignonieae

Colignonia glomerata Griseb., Nee 64157–64159 (USZ), Parque Nacional Amboró, Samaipata, Santa Cruz, Bolivia. Colignoniarufopilosa Kuntze, Nee 64061 (USZ), Cochabamba, Bolivia.

Tribe Boldoeae

Cryptocarpus pyriformis Kunth, US 2833648, Galapagos. Salpianthus macrodontus Standl., US 2219249, Sinaloa, Mexico.Salpianthus purpurascens (Cav. Ex ag.) Hook. & Arn., Pace 774 (MEXU, SPF, US), El Cobanal, Chiapas, Mexico.

Tribe Leucastereae

Andradea floribunda Allemão, US 2627753, Espírito Santo, Brazil; Rossetto 445 (RB), Linhares, Espírito Santo, Brazil. Leucastercaniflorus (Mart.) Choisy, US 2839822, Jacarepaguá, Rio de Janeiro, Brazil; Rossetto 447 (RB), Linhares, Espírito Santo, Brazil;Rossetto 455 (RB), Teófilo Otoni, Minas Gerais, Brazil. Ramisia brasiliensis Oliv., US 2947296, Nova Venécia, Espírito Santo,Brazil; Rossetto 448 (RB), Nanuque, Minas Gerais, Brazil. Reichenbachia hirsuta Spreng., Nee 64109 (USZ), Living CollectionJardín Botánico Municipal de Santa Cruz de la Sierra, Santa Cruz de la Sierra, Bolivia; Nee 64169 (USZ), Rio Grande, Santa Cruzde la Sierra, Bolivia.

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