Wood and bark structure in Buddleja: anatomical background of stem morphology

Abstract Bark (all tissues outside of the vascular cambium) has been extensively studied in recent years, especially its anatomy and physiology. Macromorphological bark characters can be important taxonomically for many plant groups, including the genus Buddleja (Scrophulariaceae). However, the relationship between macroscopic bark appearance and its microscopic structure remains obscure, hampering the use and interpretation of bark traits in plant taxonomy and phylogenetics as well as in other fields of botany. We studied micro- and macrostructure of bark in the species of Buddleja representing wide taxonomic and geographic diversity to identify general relationships between bark anatomy and morphology. We also examined Buddleja xylem and discussed the importance of anatomical traits for understanding the relationships between clades in this genus. The smooth bark surface in sect. Gomphostigma and the outgroup (Freylinia spp.) relates to the small number of periderms of superficial origin and limited sclerification. This allows for the retention of visible lenticels. In the rest of Buddleja, bark sloughs off and division of labour is present: collapsed phloem undergoes sclerification and acts as a protective layer, while thin-walled phellem forms the separation layers. A similar pattern is found in some groups (e.g. Lonicera), but in others (e.g. Vitis and the species of Eucalyptus with stringy bark), the pattern is inversed. Wood and bark anatomy supports a sister relationship between the southern African section Gomphostigma and the rest of Buddleja but is taxonomically uninformative among remaining clades. Limited development of periderms and sclerification allows for the retention of a smooth bark surface and conspicuous lenticels. Sloughing off of bark requires division of labour into a lignified protective layer and a thin-walled separation layer. These two functions are never served by a single tissue but are rather divided between phloem and periderm. How more subtle features (e.g. size and shape of fissures) are determined requires further study. Simultaneously, bark anatomy could be a useful source of data to complement molecular phylogenetic studies in a total evidence approach for systematics.


Introduction
Bark anatomy and physiology have recently received much attention (Angyalossy et al. 2016;Rosell 2016; Morris and Jansen 2017;Serra et al. 2022), but how anatomical traits translate to the staggering diversity of bark morphologies observed among angiosperms remains unexplored. This may be partly because unlike for anatomy, a terminology proposed for description of the bark morphology has not been commonly accepted (Trockenbrodt 1990;Junikka 1994). Among the few articles on the interdependence between the bark macroscopic appearance and microscopic structure (Chattaway 1953(Chattaway , 1955Kotina et al. 2012Kotina et al. , 2017Kopanina et al. 2022), Whitmore's studies of Dipterocarpaceae are probably the most important (1962a, 1962b; 1962c). He recognized seven bark types based on the features of the bark surface, sloughing modes, patterns of periderm formation, presence of expansion tissue, and structural traits. We used this classification as a framework and Buddleja (Scrophulariaceae) as a model to study the relationship between morphological and anatomical structural levels.
The genus Buddleja consists of 108 woody species (Chau et al. 2017). Most of the diversity is found in the New World section Buddleja (66 species) and Asian sect. Alternifoliae (24 species); eight species are Malagasy (together with B. polystachya from East Africa and the Arabian Peninsula, they form sect. Nicodemia), and the rest is from southern Africa (Fig. 1). Southern African species form a grade of successive sister groups to the rest of Buddleja: (1) monospecific sect. Salviifoliae (B. salviifolia), (2) sect. Gomphostigma (with two species: B. virgata and B. incompta), (3) sect. Chilianthus (with four species), (4) monospecific sect. Pulchellae (B. pulchella; some analyses resolved B. pulchella as the sister to the sect. Buddleja) and (5) Buddleja glomerata of uncertain position (most likely in sect. Chilianthus). The sister position to the rest of Buddleja is not fully resolved and occupied either by sect. Salviifoliae or sect. Gomphostigma (Chau et al. 2017).
A recent paper (Frankiewicz et al. 2020) reported that wood anatomy in Buddleja is of little taxonomic use as it is affected by species' maximal height and does not differ substantially among infrageneric sections. The same paper contained the first study of bark structure in Buddleja, including most of the southern African, some Asian and New World representatives, and two outgroups (Freylinia spp. from tribe Teedieae sister to Buddlejeae). The examined barks vary considerably (Fig. 1). In Freylinia and Buddleja virgata-the only representative of sect. Gomphostigma available in that study-the first-formed phellogen originates in the outer cortex and produces phellem and phelloderm; secondary phloem may be non-sclerified (B. virgata) or sclerification is only partial (Freylinia). In the remaining species of Buddleja, the first phellogen originates in the outer phloem and subsequent periderms cut off portions of phloem that undergoes solid sclerification; no phelloderm was observed. The similarity of bark structure between Buddleja virgata and Freylinia supports a sister position of sect. Gomphostigma with respect to the rest of Buddleja. The lack of phelloderm in all Buddleja species (except B. virgata) is particularly noteworthy as it implies a shift of cork cambium activity from bifacial to unifacial and would make a rare finding if confirmed (Evert 2006).
The present article aims to reignite interest in the anatomical-morphological relationship in bark which has received little attention since the 1960s. We undertake one of the very first attempts to describe bark macroscopic appearance in a systematic way, covering most of the taxonomic diversity and geographic range of Buddleja. Next, we examine wood and bark anatomy of 14 previously unstudied species and compare them with those previously reported in the literature. Finally, we discuss how anatomical traits interact to result in the staggering diversity of bark morphologies observed in woody plants and how anatomical traits contribute to current understanding of the systematics of Buddleja.

Origin of specimens
Sixteen species of Buddleja (Buddlejeae, Scrophulariaceae; 19 specimens) were sampled: 14 species (17 specimens) were obtained from plants cultivated in the National Buddleja Collection at Longstock Park Nursery (Stockbridge, Hampshire, United Kingdom), one specimen of B. incompta came from a natural population (Sutherland, Northern Cape, South Africa), and a sample of B. saligna was obtained from a cultivated tree (Johannesburg, Gauteng, South Africa).  Chau et al. (2017) that was supported by subsequent bark anatomical analyses by Frankiewicz et al. (2020), with sect. Gomphostigma marked in blue dashed line (representing uncertain position), and African taxa in bold. Only formally recognized clades are named, and clade triangle size approximates species richness, while monospecific clades are solitary lines. The most important anatomical differences between the two types of barks found in Buddleja and closely related Freylinia reported by Frankiewicz et al. (2020) are given at far right. The upper photo is from B. glomerata (sect. Chilianthus) and represents the more prevalent bark anatomy type. The lower photo is from B. virgata (sect. Gomphostigma) and depicts the bark anatomy type found only in this species and Freylinia in the previous analysis of Frankiewicz et al. (2020). The key differences in bark anatomy are listed. The bark anatomy of B. incompta, the second species in sect. Gomphostigma, was previously unknown. Frankiewicz et al. -Wood and bark structure in Buddleja: anatomical background of stem morphology Fourteen species were studied for the first time, two were re-examined (Frankiewicz et al. 2020).
The taxonomic coverage of new samples included: one species from sect. Gomphostigma (B. incompta), one species from sect. Chilianthus (B. saligna), three species from the New World sect. Buddleja, nine representatives of Asian sect. Alternifoliae, and two hybrids. Herbarium vouchers were deposited in JRAU, NBG or WA (Thiers 2013) and their photos were uploaded online (Bianchi and Gonçalves 2021), accession details are given in Appendix A (see 'Data' and 'Supporting Information').

Anatomical study
For wood anatomical studies, samples were obtained from the thickest lateral branch or a main stem. For bark examination, three pieces were cut: young twigs without visible periderm, lower parts of branches where periderm was starting to form, and thick branches with well-developed periderm. Material was immediately stored in 70% ethanol. Standard anatomical procedures were followed (Frankiewicz et al. 2020): we cut snippets of wood and bark 20-40 µm thick with sledge or freezing microtome, stained them with safranin and alcian blue, and mounted in Euparal. Histochemical tests for lignin and lipides presence were performed for the bark sections (Johansen 1940). To check the details of periderm structure, two specimens (B. incompta and B. saligna) were embedded in GMA, sectioned on an ultramicrotome, stained with toluidine blue, and mounted in Entellan (Feder and O'Brian 1968). Length of vessel elements and fibres was measured from macerated material. Wood descriptions and measurements follow the IAWA Committee (1989) and bark descriptions are according to Angyalossy et al. (2016). Measurements are available online (see 'Data').

Macroscopic bark descriptions
Aside from anatomical descriptions, we described macroscopic bark features (surface morphology). A universally accepted terminology of bark morphology is missing-we followed the most comprehensive nomenclature system presented by Junnika (1994).
In total, we studied 26 species (32 specimens) from tribe Teedieae (the sister to Buddlejeae, represented by Freylinia) and all sections within Buddleja except for sect. Nicodemia and sect. Pulchellae (available samples were too young): we included almost all specimens sampled for the present study (B. fallowiana KF037 and B. myriantha KF030 were excluded due to moderate bark development) and most specimens examined anatomically by Frankiewicz et al. (2020). Appendix A gives the full list of specimens. Bark morphologies were studied with the naked eye and with a dissecting microscope under low magnification; the barks were slashed with a pen knife to check for firmness. Figure 2 gives an overview of bark morphology of all examined species, and Fig. 3 shows close-ups-in both cases specimens are arranged taxonomically, the same colour-coding was applied, and accession numbers in the text and figures allow for cross-referencing. The major bark anatomical and morphological traits are summarized taxonomically in Fig. 4 (the data matrix used to generate this figure is available in Appendix B), and detailed descriptions follow below.

Macroscopic bark features of Buddleja
Bark texture is loose (crumbling when slashed) in most species. In B. albiflora (KF033), B. incompta (KF027), a thick stem of B. virgata (KF011) and Freylinia lanceolata (KF007) it is homogenous. In thin stems of B. forrestii (KF040) and B. tubiflora (KF045, very limited secondary growth), bark texture is also homogenous-likely because multiple periderms have not yet developed. In a thinner stem of B. virgata (KF010), the stem bark is rather homogenous and crumbles only slightly when slashing.
Bark patterns ( Fissuring is present in most species. In B. crotonoides (KF043) and B. tubiflora (KF045), fissures are macroscopically absent but observed under binoculars (possibly due to the specimens' limited secondary growth). In four species, they are absent or very rare: in B. lindleyana (KF015), the stem is smooth; in F. lanceolata (KF007), B. incompta (KF027) and a thick stem of B. virgata (KF011), the surface is only ruffled (in the two latter, it is sometimes broken transversely, not parallel to stem length      Figure 1 was retained. The studied traits and their states are: periderm origin: subepidermal (yellow) or in outer phloem (light red); arrangement and origin of sequent periderm: reticulate (yellow) or concentric to reticulate (light red); sclerification pattern of nonconducting phloem: none (yellow), partial with sclereids in small clusters (light red) or nearly solid with sclereids in bands (dark red); structures covering rhytidome surface: rhytidome absent (yellow), collapsed secondary phloem (light red), sclerified secondary phloem (dark red); bark texture: homogenous (yellow) or loose (light red); visual slashed patterns: missing (yellow) or ripple marks (light red); fissuring: absent (yellow), compound (light red), boat-shaped (dark red), oblique (light green), irregular (dark green), wavy (bluish); ridges: absent (yellow), flattened (light red), reticulate (dark red); scaling: absent (yellow), loose-hanged (light red), scaly flakes (dark red), irregular (light green). Missing values are in grey. Frankiewicz et al. -Wood and bark structure in Buddleja: anatomical background of stem morphology Young stem prior to initiation of periderm. Dome-like cells of epidermis are followed by a layer of isodiametric cells and a few layers of radially elongated cells with wide spaces between them. Pericycle is parenchymatous with discrete strands of fibres, and secondary phloem is homogenous. (B) Details of dome-and bottle-like epidermis cells covered with prominent cuticle, and possible remnants of a trichome in the centre. (C) Early stages of initiation of the first periderm (marked with red arrow on the left). The first phellogen forms just beneath the epidermis-which is still present and clearly visibleand produces phellem and phelloderm. Some sclerification occurs within the primary cortex, but not in the secondary phloem. (D) A subsequent periderm formed close to the secondary phloem. Despite the advanced stage, no phloem sclerification is present. (E) Formation of the first periderm in the outer cortex; the stem pith in this photo is on the right, the inset shows periderm detached from the stem with two abnormal periderms formed obliquely. (F) Formation of the first periderm in B. virgata studied in (Frankiewicz et al. 2020): the first phellogen forms in the outer cortex and no sclerification is present except in rare, solitary cells. (G) Rhytidome with three periderms in B. incompta. Scale bars: D = 500 µm, A-C & E-G = 100 µm.
The cortex is composed of ≤10 cell layers: the outermost layer, directly adjoining the epidermis, consists of isodiametric cells; it is followed by a few layers of radially elongated, palisade cells with large, intercellular spaces, and the innermost layers are made up of isodiametric, rounded cells (17-)27(-37) µm in tangential diameter (Fig. 5B). The pericyclic fibres are very thick-walled, mostly in small groups of 4-8 (rarely ≤17), sometimes solitary (Fig. 5C). No crystals were observed in cortical cells. Dilatation of the cortex is affected by tangential stretching of cortical cells and anticlinal divisions of parenchyma cells leading to tangential strands of 2 cells (Fig. 5B, C).
The first-formed periderm is initiated in the subepidermal layer of the outer cortex. The subsequent periderms are initiated in secondary phloem, and their arrangement is reticulate. Phellem is non-stratified and consists of ≤10 layers of isodiametric to tangentially elongated phelloid cells (Fig. 5D-G) with weakly lignified non-suberized walls. Phelloderm is unior biseriate (Fig. 6A, B). The rhytidome is present, its outer surface is covered by collapsed secondary phloem (Fig. 5G).
Conducting secondary phloem is very homogenous and consists of sieve tubes with companion cells (typically one per sieve tube member in cross section), axial parenchyma, and rays ( Fig. 5D-F). Sieve tube members are solitary or in small groups, (8-)13(-18) µm wide in tangential diameter and (86-)111(-165) µm long. Sieve plates are compound with 4-10 sieve areas on strongly inclined walls. Axial parenchyma forms the ground tissue and consists of fusiform cells and strands of ≤ 5 cells. Transition from conducting to nonconducting phloem is gradual, marked by tangential stretching of phloem elements. The nonconducting phloem consists of weakly obliterated and heavily collapsed elements; sclerified elements were not found.
Secondary phloem rays are uni-and biseriate. They are composed of square, procumbent and some upright cells forming multiple rows (≤10) flanked by upright cells, or all types of cells are intermixed throughout the ray body. No crystals, tannin deposits or secretory structure were observed.
Dilatation of secondary phloem is affected by tangential stretching of sieve tube members, axial parenchyma cells and ray cells, but no anticlinal divisions were found; possibly the broad intercellular spaces in the outer cortex are also the result of dilatation. No sclerification of any secondary phloem elements was observed.  (Fig. 7A). The epidermal cells are (14-)15-21(-23) µm in tangential diameter, their walls are thin, and cuticle is not prominent. Trichomes were not observed in B. albiflora (Fig. 7A) (Fig. 7A inset).

Bark anatomy of other Buddleja species
The cortex is composed of parenchymatous cells ( x weyeriana, pericyclic cells had thickened cell walls, but lignification was not detected. The first-formed periderm is initiated in the outermost layer of secondary phloem (Fig. 7C) with concentric to reticulate arrangement of subsequent periderms in deeper layers of secondary phloem (Fig. 7D, E). The mature periderm consists of easily identifiable phellem and phellogen (Fig. 7D-F). Uni-to biseriate phelloderm was recognised only in the youngest (innermost) periderm in GMA microsections of B. saligna; and it is obliterated in subsequent periderms (Fig. 6C, D). Therefore, phelloderm most likely is produced in all studied species and undergoes rapid obliteration (Fig. 7F). Phellem is composed of layers (4-9 in B. albiflora x wardii; ≤25 in B. x weyeriana) of isodiametric to radially elongated (Fig. 8B), thin-walled (phelloid) cells with weakly lignified non-suberized walls. The rhytidome is present, its outer surface is covered mostly by collapsed secondary phloem in B. forrestii, B. globosa (Fig. 7E), B. x weyeriana (Fig. 8A), or mostly by sclerified secondary phloem in other species.
Conducting secondary phloem is homogenous and consists of sieve tubes with companion cells (typically one per sieve tube member in cross section), axial parenchyma, and rays (Fig. 7G). Sieve tube members are solitary or in small clusters, rarely in tangential bands. Sieve tube members are Section quality did not allow for examination of the transition zone between conducting and nonconducting phloem in B. fallowiana and B. x wardii. In the remaining species, the transition is sharp, typically marked by periderm initiation. The nonconducting phloem consists of weakly obliterated to heavily collapsed elements, and of the sclerified elements. Zone of phloem sclerification is commonly adjacent to the underlying periderm. B. forrestii, B. globosa (Fig. 7E) and B. x weyeriana (Fig. 8A) show tangential bands and lens-like clusters of collapsed phloem elements, occasionally (rarely in B. x weyeriana) with sclereids in small groups and interrupted 1-2(-3)-seriate bands. Other species (except the juvenile sample of B. fallowiana) show more abundant (occasionally nearly solid) sclerification of nonconducting phloem with the sclereids in continuous bands ≤3 cells in width (Fig. 7B-D).
Secondary phloem rays are uni-to tetraseriate, most often bi-to triseriate (Fig. 9F, G). Ray bodies are composed of ≤10 rows of square (with some procumbent and upright) cells flanked by upright cells in one or two rows in B. albiflora (Fig. 9A)  Vessels are circular to oval in most species or mostly oval (in B. incompta and B. myriantha; Fig. 10A); slightly angular in outline in all species except B. myriantha where they are more rounded; narrow to very narrow [(16-)19-38(-50) µm wide] and numerous [(112-)125-375(-496) vessels mm 2 ] in all species except for B. incompta, where they are very numerous (mean 955 vessels mm -2 ). Vessels are partly solitary (in most species < 40% of solitary vessels; in B. incompta all vessels are in contact with other vessels). They are disposed in small clusters and radial multiples usually of 2-4. There is no distinct pattern of vessel disposition, except for a weak tendency towards diagonal pattern in latewood of B. albiflora (Fig. 10C), B. globosa and B. paniculata.  crotonoides, B. fallowiana, B. globosa, B. x weyeriana). Vessel-ray pits are similar in size and shape to intervessel pits, except for B. incompta where pitting is often scalariform or gash-like. Vessel-ray pits have clearly reduced borders (pitting in B. paniculata and B. x weyeriana could not be clearly observed). Very fine helical thickenings are present in most vessels and throughout their length in all species except B. x wardii (mostly in narrower vessels) and for  Gomphostigma) showing very numerous vessels almost always in contact with other vessels, many narrow rays, a single wider ray on the left, and weakly marked growth ring boundary in centre; (B) Growth ring boundary marked by 3-9 rows of radially flattened fibres and a shift from narrow latewood vessels to wide earlywood vessels in B. nivea (sect. Alternifoliae); (C) B. albiflora (sect. Alternifoliae) with growth ring marked in a similar fashion to B. nivea, but with a wider band of radially flattened fibres; (D) Few bi-to tetraseriate rays and very numerous short, uniseriate rays resembling axial parenchyma characteristic of B. incompta (sect. Gomphostigma); (E) B. albiflora (sect. Alternifoliae) with a similar proportion of uniseriate and multiseriate rays; (F) B. forrestii (sect. Alternifoliae) showing a few rays and multiple tracheids with fine helical thickenings. Scale bars: A = 500 µm, B-F = 100 µm. B. incompta (completely missing). Numerous vascular tracheids are present in B. albiflora, B. forrestii, and B. colvilei, while fibriform vessels (sensu (Carlquist 2001)) were observed in B. x wardii and B. x weyeriana; in all these cases the cells have helical thickenings similar to those in vessels.
Upright and square, rarely also procumbent, ray cells of B. globosa and B. tubiflora contained prismatic crystals, and in ray cells of B. fallowiana, B. forrestii, B. nivea, B. paniculata and B. x wardii droplets of tannins were found.

Relationships between micro-and macroscopic bark structure
In Buddleja, section Gomphostigma is like the sister group of the genus (Freylinia) in bark appearance and anatomy, and dissimilar to all remaining sections. Their species have a smooth, non-peeling, little or non-fissured bark surface, and homogenous texture. Instead, other Buddleja species share distinctly fissured and peeling bark with loose texture (Fig. 4). The barks of sect. Gomphostigma resemble the smooth or the shallow fissured type of Whitmore's classification (1962a), whereas the latter ones belong to the scaly type.
These contrasting bark appearances correspond to the different modes of periderm formation. Multiple narrow bands of thin-walled phellem cells formed by deeply initiated phellogens act as separation layers between the bark scales occurring in most Buddleja species (Figs. 4, 8; Appendix B). This mode of sloughing is associated with collapsing and sclerification of secondary phloem performing the protective role instead of the periderm. Similar anatomies are found in other taxa having stringy peeling bark: Lonicera (Caprifoliaceae), Melaleuca (Myrtaceae), Dodonaea (Sapindaceae) (Chiang and Wang 1984;Eryomin and Kopanina 2012;Crivellaro and Schweingruber 2013;Schweingruber et al. 2013aSchweingruber et al. , 2019. However, in Vitis (Vitaceae) (Eryomin and Kopanina 2012;Crivellaro and Schweingruber 2013;Schweingruber et al. 2013aSchweingruber et al. , 2019 and in Eucalyptus (Myrtaceae) with stringy bark (Chattaway 1955), the division of labour is inversed: expanded, thin-walled cells of secondary phloem make separation layers, while thicker-walled phellem cells perform the protective function (Chattaway 1955).
Thus, stringy/scaly barks of similar appearance can arise from two different microstructural ground plans: either with (1) periderms (phellem) acting as separation layers combined with protective secondary phloem (Lonicera, most Buddleja; Fig. 8), or with (2) the secondary phloem (phloem parenchyma) performing the separation function, and the periderm responsible for the protective function (Vitis). Theoretically two other modes are possible: (3) both functions can be performed exclusively by secondary phloem, or (4) only by periderm. We did not find, however, any data supporting the existence of these options.
In contrast, the smoothness of non-peeling barks in sect. Gomphostigma and in the genus Freylinia is associated with a limited number and superficial origin of the periderms serving a protective function (Fig. 5D-F). This pattern of periderm initiation is found in many plants with smooth to shallow fissured barks, e.g. Syringa and Fraxinus (Oleaceae). Also, the lenticels found in F. lanceolata and B. virgata are common in species with non-scaly barks (Eryomin and Kopanina 2012;Crivellaro and Schweingruber 2013). Among four studied species with smooth barks, F. tropica is distinctive in showing shallow fissures with a tendency to form chunky scales (Fig. 4). This species also has discontinuous tangential bands of sclereids in its nonconducting secondary phloem. Freylinia lanceolata however has fewer sclereids arranged into small clusters, whereas B. virgata and B. incompta show no sclerification in their nonconductive secondary phloem (Frankiewicz et al. 2020). Therefore, the shallow fissured bark of F. tropica is likely associated with the abundance and arrangement of sclereids in its secondary phloem.

Taxonomic significance of bark anatomy in Buddleja
Bark structure is different in the two constituents of sect. Gomphostigma than in other species. Buddleja incompta is very similar to B. virgata (Frankiewicz et al. 2020): the first periderm forms in the outer cortex, it produces uni-to biseriate phelloderm and thin-walled phellem, phloem does not undergo sclerification. The former species is distinctive only in the occurrence of rhytidome. In contrast, in all other Buddleja species, the first phellogen is deep-seated, it produces phellem and phelloderm (undergoing rapid obliteration), and cut-off phloem undergoes sclerification-a feature not reported outside Buddleja (Frankiewicz et al. 2020) (Fig.  4). Frankiewicz et al. (2020) incorrectly reported that the sclerification starts simultaneously or after initiation of periderm, but the order is reversed.
Stem and bark anatomy were used as taxonomic signals for Buddleja by Solereder (1908, pp. 538-547). Back then, the five genera presently classified in Buddleja (Buddleja, Chilianthus, Emorya, Gomphostigma, Nicodemia) were treated in the subfamily Buddleioideae within Loganiaceae. Solereder listed three diagnostic characters for Buddleioideae: (1) lack of intraxylary phloem, (2) presence of branched and glandular trichomes, and (3) pericyclic origin of the first periderm. The first feature differentiated Buddleioideae and Loganioideae -a group of species now known to be distantly related to Buddleja -and we can confirm that intraxylary phloem never occurs in Buddleja, although it is rarely found in other Scrophulariaceae (Scrophularia) (Doležal et al. 2018, pp. 379-380). Our results also corroborate the common presence of trichomes in Buddleja, and the few glabrous stems in our study may be incidental. It is also worth mentioning that Solereder distinguished Gomphostigma from other Buddleioideae using details of trichome cell wall structure: in the latter, the cell wall dividing trichomes in half is straight, in Gomphostigma it is undulating. We cannot agree with Solereder's statement that in all Buddleioideae the first phellogen originates internally from pericyclic fibres. This initiation site is typical for most Buddleja, but not the sect.

Gomphostigma.
Neither Solereder nor other early authors (Metcalfe and Chalk 1957) mentioned the abundant sclerification of phloem present in all Buddleja (save for sect. Gomphostigma). This feature was pointed out by Schweingruber et al. (2013b p. 263), who incorrectly identified sclerified cells as fibres, instead of sclerified cut-off phloem.
Scrophulariaceae are poorly studied anatomically: most species hitherto examined actually belong in Orobanchaceae or Plantaginaceae (Goldblatt and Manning 2002;Manning and Goldblatt 2012, pp. 731-763). Precise descriptions are absent, and detailed photos of bark anatomy are available only for Freylinia (Frankiewicz et al. 2020), Scrophularia (Makbul et al. 2006;Schweingruber and Landolt 2010;Schweingruber et al. 2013b pp. 264-266;Doležal et al. 2018, pp. 379-380), and Verbascum (Schweingruber and Landolt 2010;Schweingruber et al. 2013b, pp. 267-270). In these taxa, sieve tubes are often in small groups. The transition from conducting to nonconducting phloem may be marked by diffuse sclerification varying in intensity, from few (e.g. S. latifolia) to numerous, thick-walled sclerified cells (e.g. V. arcturus). Nevertheless, none of the examined species undergoes nearly solid phloem sclerification as reported in most Buddleja species, and the most similar condition is found in F. tropica (but not in F. lanceolata). Conversely, three of nine Scrophularia species and seven of nine Verbascum species in the Xylem Database (Büntgen et al. 2014) are completely devoid of phloem sclerification. Available sources do not allow for identification of the initiation site of the first phellogen in most taxa: it is the outer cortex in Freylinia, and probably likewise in at least some Scrophularia (S. parviflora) and Verbascum (V. arcturus). In all other studied members of Scrophulariaceae, phellem consists of thin-walled (phelloid) cells, and phloem and ray dilatation occurs commonly.
Based on this, admittedly limited, evidence, we hypothesise that moderate phloem sclerification and subepidermal periderm initiation may be plesiomorphic for Scrophulariaceae. Such anatomy makes a convenient starting point for evolution of both phloem completely devoid of sclerification in sect. Gomphostigma and phloem undergoing nearly solid sclerification in the remaining clades of Buddleja, supporting the position of sect. Gomphostigma as sister to the rest of the genus (Fig. 1). The alternative scenario resolved in certain molecular analyses (Chau et al. 2017(Chau et al. , 2018, with sect. Gomphostigma and sect. Salviifoliae swapped, requires evolution of abundant sclerification of phloem and deep-seated periderm initiation in the most recent common ancestor of Buddleja and then a complete loss of sclerification combined with a change in the phellogen initiation site in sect. Gomphostigma-a less parsimonious scenario. Our examination of semi-thin sections of B. saligna showed that uni-and biseriate phelloderm is present in this species (Fig. 6C, D). It is prominent in young periderms and becomes obliterated during bark transformations. Frankiewicz et al. (2020) did not recognize phelloderm in the periderm of Buddleja species (except sect. Gomphostigma) due to its obliteration.

Taxonomic significance of wood anatomy in Buddleja
The newly examined species fall within the range of anatomical diversity reported for Buddleja (Frankiewicz et al. 2020). Three earlier attempts to identify wood characters useful for delineation of infrageneric sections were unsuccessful: qualitative trait variation is limited in Buddleja (Carlquist 1997), and quantitative traits correlate with plant size rather than phylogenetic relationships (Terrazas et al. 2008;Frankiewicz et al. 2020).
Scarcity or absence of axial parenchyma is universal among Buddleja, helical thickenings were absent only in B. utahensis (Carlquist 1997), B. virgata (Frankiewicz et al. 2020) and B. incompta (this study). The two latter species-the sole members of sect. Gomphostigma-are also characterised by a markedly greater number of uniseriate rays mm -1 than in any other Buddleja (mean: 14.8 and 18.8, respectively; in most other species it typically is ≤5.6 mm -1 ). These rays are often short and resemble short strands or fusiform solitary cells of axial parenchyma. Both species of sect. Gomphostigma are also distinct in a very high number of vessels mm -2 (≤679 mm --2 in B. virgata, 955 mm -2 in B. incompta; in other species it rarely exceeds 400 mm -2 ), which in turn leads to the low percentage of solitary vessels. Consequently, the combination of high vessel density, vessels contacting other vessels, very numerous short and uniseriate rays resembling axial parenchyma, and lack of helical thickenings can distinguish sect. Gomphostigma from the other sections of Buddleja.

Consequences for taxonomy of Buddleja
The anatomical traits of southern African Buddleja sect. Gomphostigma may represent the plesiomorphic state for the genus, since they differ from those observed in the rest of the genus but share many similarities with traits in Freylinia species in tribe Teedieae (the sister to Buddleja). This supports sect. Gomphostigma as sister to the rest of Buddleja. Phylogenetic analyses of high-throughput sequencing of loci from targeted sequence capture resolved sect. Gomphostigma as sister to the rest of the genus with very strong support (Chau et al. 2018), although analyses with different smaller molecular datasets placed it in other positions with weak support (Chau et al. 2017(Chau et al. , 2018. The rather unique reproductive morphology of sect. Gomphostigma, including racemose inflorescences, cup-shaped corollas and exserted stamens (Chau et al. 2017), may represent autapomorphies for the section or ancestral trait states for the genus, thus being of little taxonomic use.

Conclusions
The relationship between the bark micro-and macrostructure is far from straightforward. Smooth bark surface is related to a small number of periderms, their superficial origin, and limited sclerification. This combination allows for the development and retention of conspicuous lenticels. Instead, fissured, stringy/scaly barks require division of labour: some tissues take a protective function, while others serve as separation layers. In the case of Buddleja, the former is done by sclerified phloem, while the thin-walled phellem serves the latter function. The literature shows that the reverse division of labour is also possible. Theoretically, both functions could be served by the same tissue, but no evidence of such cases could be found.
Bark-and to a lesser degree wood-anatomy support a sister relationship between South African Buddleja sect. Gomphostigma and all remaining Buddleja species. This shows that anatomy can be a useful source of data to complement molecular phylogenies, especially when molecular markers alone do not provide satisfactory resolution (total evidence approach).

Supporting Information
The following additional information is available in the online version of this article Appendix A -Accession information for specimens of Buddleja used in the study Appendix B -Tabulated summary of bark anatomical and morphological traits in Buddleja and Freylinia.

Data
Photos of herbarium vouchers, close-ups of bark surface, and measurements of quantitative anatomical traits are available on FigShare DOI: 10.6084/m9.figshare.14798034

Sources of funding
The study was supported by the Bekker NAWA Programme and Faculty of Biology (University of Warsaw) intramural grant to K.F. and A.O. was supported by the University of Johannesburg.

Author contributions
K.F. and A.O. designed, carried out the study, and wrote the manuscript. J.H.C. provided taxonomic expertise and edited the manuscript. A.T. and J.B. assisted with microscopy and preparation of figures.