Wood and Bark of Buddleja: Uniseriate Phellem, and Systematic and Ecological Patterns Kamil E

IAWA Journal 42 (1), 2021: 3–30

Wood and bark of Buddleja: uniseriate phellem, and systematic and ecological patterns Kamil E. Frankiewicz1,⁎, John H. Chau2, and Alexei A. Oskolski3,4 1Institute of Evolutionary Biology, Faculty of Biology, University of Warsaw, Biological and Chemical Research Centre, Żwirki i Wigury 101, 02-089 Warsaw, Poland 2Centre for Ecological Genomics and Wildlife Conservation, Department of Zoology, University of Johannesburg, PO Box 524, Auckland Park 2006, Johannesburg, South Africa 3Department of Botany and Plant Biotechnology, University of Johannesburg, PO Box 524, Auckland Park 2006, Johannesburg, South Africa 4Botanical Museum, Komarov Botanical Institute, Prof. Popov 2, 197376 St. Petersburg, Russia *Corresponding author; email: [email protected]

Accepted for publication: 20 June 2020

ABSTRACT Wood anatomy of Buddleja is well-explored but not in many southern African members, which form a grade of species and small clades that form successive sister groups to the rest of the genus, and its bark structure has not been studied at all. We provide new descriptions of wood anatomy for twelve species, including nearly all Buddleja from southern Africa and two species of Freylinia in the sister group of Buddleja. We also describe bark structure from fifteen species. To assess if wood anatomy provides phylogenetic and/or ecological signal, we compiled data on wood traits and climatic variables from the distributions of 53 species. Wood traits counteracting cavitation correlated with higher temperature and precipitation seasonality; simultaneously they were better expressed in species with smaller maximal plant height. It is likely that hotter and drier areas harbour smaller plants which have traits conveying higher conductance safety. Bark structure varies considerably. In bark of Buddleja section Gomphostigma, periderm is initiated in the outer cortex and develops thin-walled phellem, and sclerification of their phloem does not occur. This resembles bark in Freylinia, supporting the position of section Gomphostigma as sister to the rest of Buddleja.In the remaining Buddleja species, bark is characterised by formation of periderm with phelloid cells in the secondary phloem. The phellem is often uniseriate, a condition not reported elsewhere. Its formation occurs close in time to solid sclerification of the cut-off phloem, suggesting a possible novel ontogenetic mechanism. Keywords:cavitation; Freylinia; phelloid cells; phloem sclerification; phylogenetic signal; Scrophulariaceae; uniseriate phellem; wood ecology.

In memory of Prof. Fritz Hans Schweingruber

INTRODUCTION

Although genus Buddleja (tribe Buddlejeae, Scrophulariaceae) has been recognised since 1753 (Linnaeus) its phylogenetic relationships were resolved only recently. The traditional narrow circumscription of the genus was shown to be paraphyletic, and four allied genera (Chilianthus, Emorya, Gomphostigma and Nicodemia) were subsequently merged into Buddleja (Chau etal. 2017).This new circumscription resulted in a genus of ca. 108 species from North and South America (66 species), Asia (24 species), and Africa including Madagascar (18 species). They are characterised by a diversity of life forms (shrubs, trees, lianas; Fig. 1) and wide ecological preferences (including montane tropical, subtropical and warm-temperate habitats; Table 1). Most of the southern African species (B. auriculata, B. dysophylla, B. glomerata, B. incompta, B. loricata, B. saligna, B. salviifolia and B. virgata) were determined to form a grade of small clades that are successive sister groups to the rest of Buddleja.The precise relationships among these lineages are still uncertain, including which taxon is sister to the rest of the genus. Two conflicting topologies were resolved by Chau et al. (2017): (a) section Gomphostigma, sister to the remaining Buddleja; and (b) section Salviifoliae, sister to the remaining Buddleja (Fig. 2). In either case, it is clear that these southern African species are crucial for understanding patterns of evolution within the genus.

© The authors, 2021 DOI 10.1163/22941932-bja10020 This is an open access article distributed under the terms of the CC BY-NC-ND 4.0 license. Downloaded from Brill.com10/06/2021 12:43:59AM via free access 4 IAWA Journal 42 (1), 2021

Figure 1. Habit diversity of exemplar Buddleja species. (A) B. virgata (shrub); (B) B. loricata (shrub); (C) B. salviifolia (intermediate between shrub and tree); (D) B. coriacea (tree).

The first detailed descriptions of Buddleja wood anatomy were provided by Mennega (in: Leeuwenberg 1980, p. 112–161) and were further elaborated by Quintanar et al. (1996), Carlquist (1997), and Aguilar-Rodríguez & Terrazas (2001). Additional species are also included in the InsideWood database (2004; Wheeler 2011). Altogether, to date the wood anatomy of 41 Bud- dleja species have been studied. Carlquist (1997) concluded that only the occurrence of crystals in ray cells might be of any taxonomic value in Buddleja, while other wood anatomical traits reflect species-specific ecological preferences. Aguilar- Rodríguez et al. (2006) studied patterns in wood traits with respect to plant size, latitude, altitude, soil type, and climate (e.g., annual temperature amplitude, precipitation). They examined multiple specimens of a single species (B. cordata) and showed that within the species range, plant size and several wood traits (vessel frequency, fibre length, ray size) are affected by local climatic conditions, namely rainfall and temperature. Terrazas et al. (2008) explored correlations between selected wood traits, habit, and latitude. Unlike Aguilar-Rodríguez et al. (2006), they investigated multiple species and showed that most of the studied wood traits correspond with plant size rather than distribution. Thus, it seems that Buddleja wood can be affected by species-specific habit and/or ecology, but it is unknown if there are phylogenetic patterns. Regardless of whether wood anatomy has any taxonomic value in Buddleja, previous studies have focused mainly on Asian and American species in sections Alternifoliae and Buddleja, respectively. Of the eight species in the southern African grade comprising clades sister to the rest of the genus, only two (B. saligna and B. salviifolia) have been studied, leading to a gross underrepresentation of this highly diverse group (found in grassland, desert, riparian, and, most commonly, montane forest habitats; Fig. 1A–C; Table 1). Simultaneously, to the best of our knowledge, bark anatomy of Buddleja remains mostly unexplored — it was passingly mentioned by Moeller (1882, p. 182), and a single photo depicting bark structure of B. davidii is shown by Schweingruber et al. (2013, p. 263). Our study had three main goals focusing on wood and bark anatomy in Buddleja and representatives of the sister group to Buddleja in the genus Freylinia for comparison. First, we described wood anatomy of several BuddlejaDownloadedand fromFreylinia Brill.com10/06/2021species, 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 5 3 3 2 5 2 2 3 2 2 , or on the banks in sand and mud; ostly at edges or in open places; 300–1100 m Cloud forests; 1700–2000 m a.s.l. ; 1200–2500 m a.s.l. (near Cape from 150 m a.s.l.) 7 4 3 tional Botanical Institute, Cape Town, p. 511–655. 8 5–31. 3 3 . Wageningen 79: 1–158. 1 r courses, or in damp sheltered gullies; 1600–2700 m a.s.l. treambank or riverbank bush ins in mountainous country nning water among boulders or on banks in sand or mud es; 200–2700 m a.s.l. ded valleys, forest margins and along rivers ve 2000 m a.s.l. in dongas and isolated bushes on hillsides 3 , oak forests, montane mesophilic forests, tropical deciduous forests, 2 3 arts Pinus , ; 2900–4000 m a.s.l. 3 6 near riverbanks in the mountains; 2000–4000 m a.s.l. Abies e list is provided below. 9 1 ence Press, Beijing, p. 329–337. 3 otanical Garden, New York, NY. Montane forests or thickets, often in gullies; 600–2000 m a.s.l. Forest edges or scrub; 0–2600 m a.s.l. Among the rocks on the hills and mountains of the sandy karroid areas Bush on road sides, mostly in the mountains; 300–1800 m a.s.l. Woodland or light forest in the mountains; 1200–2000 m a.s.l., in forest, m In coastal bush at river mouth; 0–2000 m a.s.l. Forest edges, rocky slopes, along water courses, and in montane grassland Along and in watercourses and rivers, only in running water among boulders a.s.l. 600–2000 m a.s.l. xerophilic matorral, mostly in secondary vegetation; 1400–3200 m a.s.l. soamericana 5: 379–385. te 115: 1–31. (Buddlejeae, Scrophulariaceae) in Brazil. Phytotaxa 379: 187–226. he northern provinces of South Africa, keys and diagnostic characters. Na a. Struik, Cape Town, p. 763–825. Buddleja Forst. XVI Gomphostigma Turcz. Meded. Landbouwhogesch. Wageningen 77: 1 L. II Revision of the African and Asiatic species. Meded. Landbouwhogesch Buddleja Geniostoma Africa (South Africa) In rocky ravines, on mountain sides or at forest marg North America (Mexico, Guatemala) Very diversified habitat including Africa (South Africa) Slopes of high mountains, among boulders along wate South America (Bolivia, Argentina) Rocky areas and semi-arid thorn scrub South America (Bolivia) 3000–4350 m Africa (Madagascar) Bush in the mountains; 600–2000 m a.s.l. Africa (South Africa) Along or near watercourses and rivers, growing in ru Africa (South Africa) Forest margins, rocky slopes, along watercourses, Africa (South Africa, east Africa) In forest, mostly on margins or in open p Africa (South Africa)Asia (China, Bhutan, India, Myanmar) Forest, forest edges, scrub, mostly On forest margins, in patches of bush in kloofs or in s Asia (China, Japan) Mountains beside trails, scrub by streams, forest edg Africa (South Africa) Among rocks on hills and mountains South America (Brazil) Rocky grasslands and shrublands at elevations abo Africa (South Africa) Dry hillsides, mixed bushveld, mountainsides, woo species included in this study as presented in original sources. Referenc Buddleja o L & Leeuwenberg AJM. 1996. Loganiaceae. In: Flora of China, Volume 15. Sci Norman EM. 2000. Buddlejaceae. Flora Neotropica, volumeRetief 81. The E. New & York Herman B PP. 1997. Loganiaceae, Scrophulariaceae. In Plants of t Ocampo Acosta G. 2011. Buddlejaceae. Flora delNormal bajío EM, y Christenhusz de MJM, regiones Davidse adyacen G. 2011.Palgrave Scrophulariaceae. KC. Flora 1977. Me Loganiaceae. In: Moll EJ (ed.),Ping-ta Trees of Southern Afric Coelho GP & Miotto STS. 2018. ALeeuwenberg taxonomic AJM. revision 1977. of Loganiaceae the of genus Africa XV . Leeuwenberg AJM. 1979. Loganiaceae of Africa XVIII 1 2 3 4 5 6 7 8 9 SpeciesBuddleja aromatica Buddleja auriculata Buddleja cordata Buddleja coriacea Buddleja dysophylla Buddleja forrestii Buddleja glomerata DistributionBuddleja lindleyana Buddleja longiflora Buddleja loricata Buddleja madagascariensis Buddleja pulchella Buddleja saligna Buddleja salviifolia Buddleja virgata Habitat Table 1. Habitat descriptions of Downloaded from Brill.com10/06/2021 12:43:59AM via free access 6 IAWA Journal 42 (1), 2021

Figure 2. Two phylogenetic trees for Buddleja inferred in Chau et al. (2017). Clades of uncertain position are marked with dashed lines: yellow: sect. Gomphostigma, blue: sect. Salviifoliae, pink: sect. Pulchellae, black: B. glomerata (in tree A, this species is nested within sect. Chilianthus). including previously neglected species from the southern African grade. Second, we evaluated if wood anatomy provides phylogenetic and/or ecological information in Buddleja. Third, we studied bark anatomy in multiple species representing all sections recognised within Buddleja and Freylinia, and assessed what systematic information it conveys.

MATERIALS AND METHODS Wood and bark structure Sampling —Twenty-two samples from seventeen species were obtained from nature, botanic gardens, and herbaria (Table A1 in the Appendix). Samples represent seven Buddleja species from the southern African grade (B. auriculata, B. dysophylla, B. glomerata, B. loricata, B. saligna (section Chilianthus); B. virgata (section Gomphostigma); and B. salviifolia (section Salvi- ifoliae)), eight additional Buddleja species from other groups (B. forrestii, B. lindleyana (section Alternifoliae); B. aromatica, B. cordata, B. coriacea, B. longiflora (section Buddleja); B. madagascariensis (section Nicodemia); and B. pulchella (section Pul- chellae)), and Freylinia lanceolata and F. tropica from tribe Teedieae, the sister group of Buddleja, to serve as outgroups for comparison. Voucher specimens for newly collected samples were deposited in J and WTU (acronyms after Thiers 2013). Anatomical study — For twelve species, wood anatomy was newly studied and their wood descriptions are provided here. Additionally, we measured quantitative wood traits of five species with previous descriptions in the literature. For nine species, complete bark anatomy descriptions were prepared, and for six further species, only the general structure of mature bark was investigated due to poor sample preservation. For two species, bark could not be studied at all in our samples (Table A1 in the Appendix). Fresh samples with mature bark from main stems or thick branches, portions of young stems without visible periderm, as well as wood samples were collected and stored in 70% ethanol. Samples from herbaria were rehydrated before sectioning. Sections of wood were prepared with a sledge microtome, and sections of bark were obtained with a freezing microtome. All sections were stained with 1:1 aqueous alcian blue/safranin mixture. Wood maceration was carried out in 1:1 acetic acid/hy- drogen peroxide solution, and macerated material was stained with toluidine blue. Descriptive terminology follows IAWA Committee (1989) for wood, and Angyalossy et al. (2016) for bark anatomy. Histochemical tests with vanillin for tannins (Gardner 1975), Sudan III staining for suberin (Angyalossy et al. 2016), and phloroglucinol for lignin (Speer 1987) were performed on thin, razor-cut wood and bark sections. The use of two latter tests allowed us to determine if non-conducting phloem underwent lignification and/or deposition of suberin.

Evaluation of systematic and ecological effects on wood traits Obtaining wood trait data — We compiled quantitative wood trait data from this study and the literature (Carlquist 1997; Aguilar-Rodríguez & Terrazas 2001; Aguilar-Rodríguez et al. 2006; Terrazas et al. 2008). This resulted in a matrix with data on 91 specimens representing 53 species (51 Buddleja spp. and 2 Freylinia spp.). Minimal and maximalDownloaded values from were Brill.com10/06/2021 not available 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 7 for all taxa and all traits, so we performed subsequent analyses with the means of the 11 wood traits with the highest cover- age (10 wood traits and fibre length to vessel element length ratio; see https://doi.org/10.6084/m9.figshare.12066717.v3). The specimen-level data matrix was converted into a species-level data matrix by computing mean trait values across specimens for each species. In addition, we gathered data on maximal plant height for each species from the literature (see https://doi.org/10.6084/ m9.figshare.12066717.v3 and references therein). Obtaining bioclimatic data — Geographic coordinates of species occurrences were retrieved from the Global Biodiversity Information Facility web portal (GBIF 2020; DOIs of particular species datasets are provided; see https://doi.org/10.6084/ m9.figshare.12066717.v3). We used the ‘gbif’ function from the R package dismo (Hijmans et al. 2017; R Core Team 2019) to download species occurrence data for all 53 species in our wood trait dataset. Duplicate occurrences and records from outside the natural range of species were removed to produce a clean occurrence dataset for each species. The ‘getData’ function from the R package raster (Hijmans 2020) was used to obtain bioclimatic (bio1–bio19) and altitude data from WorldClim (Fick & Hijmans 2017). For each species, each variable was standardised by dividing their mean value by the standard deviation of a given variable. Statistical analyses — The compiled data were used to conduct three types of analyses: (1) scatterplots of all wood traits in pairs to assess if any trait pair is useful in discrimination of sections in Buddleja recognised by Chau et al. (2017) or genera, (2) linear regressions between wood traits and principal components from principal component analysis of bioclimatic data with (pPCA) and without (PCA) phylogenetic correction to assess if any wood trait is correlated with climatic variables, and (3) linear regressions between wood traits and maximal plant height to assess if any wood trait is correlated with plant size. (1) We prepared scatterplots of all quantitative wood traits in pairs, which we assessed to determine if points for species in different sections of Buddleja or different genera segregate to allow for discrimination among taxa. (2)To reduce the dimensionality of bioclimatic variables, we performed principal component analysis (PCA) on the matrix of standardised mean values of bioclimatic variables for all 53 species, and phylogenetic principal component analysis (pPCA) on a reduced matrix with the 46 species that were included in the phylogeny of Buddleja in Chau et al. (2017). For PCA, we used the ‘PCA’ function from the FactoMineR package in R (Lê et al. 2008; R Core Team 2019). For pPCA, we used the phylogenetic tree from Bayesian multispecies coalescent analyses from Chau et al. (2017) and employed the function ‘phyl.pca’ from the R package phytools (Revell 2019). For both PCA and pPCA analyses, we extracted first and second principal components. Phylogenetic signal in the 11 quantitative wood traits and maximal plant height were assessed by calculating Pagel’s lambda and Blomberg’s K using the same phylogenetic tree used as above and the function ‘phylosig’ in the R package phytools (Revell 2019). After finding that phylogenetic signal in all traits was either low or non-existent, we performed regression analyses with the first two principal components of bioclimatic variables against the 11 quantitative wood traits and maximal plant height. To assess which correlations were statistically significant, we employed a two-tier test: first, we calculated the 95% confidence limits (CL) of the slope and its P-value; and second, we obtained the coefficient of determination (R2) and its 95% confidence limits. These calculations were executed with the functions ‘confint’, ‘geom_point’ and ‘geom_smooth’ from the ggplot2 package and ‘CI.Rsqlm’ from the psychometric package in R (Wickham 2009; Fletcher 2010). Correlations with slope CL excluding 0 (i.e., with P-value <0.05) and with exclusively positive (i.e., excludes 0) R2 CL were considered significant, while correlations with a slope P-value <0.05 but a R2 CL including 0 were considered weakly significant. (3)We conducted linear regressions between wood traits and maximal plant height employing the same methods as above to assess the significance of correlation.

RESULTS

Wood anatomy of Buddleja Wood descriptions of Buddleja (Fig. 3) are based on samples of B. aromatica, B. auriculata, B. coriacea, B. dysophylla, B. forrestii, B. glomerata, B. lindleyana, B. longiflora, B. loricata and B. virgata. Measurements of quantitative characters were also done for B. cordata, B. madagascariensis, B. puchella, B. saligna and B. salviifolia, which are provided at https://doi.org/10.6084/m9.figshare.12066717.v3. Growth ring boundaries are indistinct (B. longiflora) or distinct, marked by tangential rows of radially flattened fibres (1–3 rows in B. auriculata and B. glomerata,upto:4inB. dysophylla and B. forrestii,5inB. lindleyana,6inB. virgata,and8inB. aromatica and B. coriacea; Fig. 3A–D) and/or by a difference in vessel diameter between early- and latewood (B. aromatica, B. glomerata, B. lindleyana, B. loricata, B. virgata; Fig. 3B). In B. auriculata (KF012), a ‘false growth ring’ was observed. In B. auriculata (KF003), ray cells crossing the growth ring boundary and in the vicinity thereofDownloaded have brownish from Brill.com10/06/2021 occlusions, 12:43:59AM and the via free access 8 IAWA Journal 42 (1), 2021

Figure 3. Wood anatomy of exemplar Buddleja species. (A) B. virgata; diffuse-porous wood (TS); (B) B. loricata; ring-porous wood (TS); (C) B. auriculata; a growth ring boundary marked by ca. 3 layers of radially flattened fibres (TS); (D) B. aromatica; a growth ring boundary marked by ca. 6–8 layers of radially flattened fibres (lower half of the photo; TS). (E) B. virgata; numerous short, uniseriate rays and few multiseriate rays (TLS); (F) B. glomerata; helical thickenings visible in vessel elements (TLS); (G) B. forrestii; droplets of tannins in ray cells (RLS). (H) B. longiflora; prismatic crystals in ray cells and helical thickenings in vessel elements (RLS).

same material is deposited in fibre lumens in this area (which lends a darker appearance in this zone under lower magnifi- cations). Wood is ring-porous to semi-ring-porous (B. loricata; Fig. 3B), mostly semi-ring-porous (B. aromatica, B. lindleyana), or diffuse-porous (Fig. 3A). Vessels are circular to oval (B. auriculata, B. coriacea, B. dysophylla, B. forrestii, B. glomerata, B. lindleyana, B. longiflora, B. virgata; Fig. 3A,C), mostly oval (B. loricata; Fig. 3B), or mostly circular (B. aromatica); mostly angular (B. coriacea, B. lindleyana, B. virgata; Fig. 3A) or rounded to slightly angular in outline; narrow to very narrow (mean tangential diameters range 13–56 μm; https://doi.org/10.6084/m9.figshare.12066717.v3); and numerous to very numerous (379–679 vessels/mm2 in B. virgata, and 84–319 vessels/mm2 in other species). Vessels are partly solitary (19–60%, except for B. dysophylla (KF009) and B. virgata (KF010), where virtually all vessels are in contact with other vessels; Fig. 3A). They are disposed in small clusters, radial multiples, and interannual tangential bands of 2.2(−15), but most often they are 4 or fewer. In B. aromatica and B. loricata (Fig. 3B), tangential bands are also present at the beginning of the growth ring. There is no particular vessel pattern, except for in B. loricata (Fig. 3B) where a tendency toward diagonal arrangement is seen in latewood. Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 9

Vessel elements are short (average length mostly 230–330 μm, up to 351 μm in B. auriculata (KF003), 372 μm in B. pulchella, and 396 μm in B. dysophylla (KF009)). Length-on-age curves were prepared for all specimens except four (KF019–KF022 as samples were very narrow); in B. dysophylla (KF009) the curve is markedly ascending, in B. saligna (KF002) it is moderately ascending, and in the remaining species they are either flat or descending (Fig. 4). Perforation plates are exclusively simple (Fig. 3H). Intervessel pitting is alternate and with rounded pits in most species; in B. dysophylla and B. forrestii intervessel pits are rarely polygonal, and in B. aromatica they are oval. Pits are small to medium and of similar size in horizontal and vertical diameters (horizontal diameter means: 4.1–8.0 μm; vertical diameter means: 3.1–8.2 μm). Apertures are either lens-like (B. coriacea, B. forrestii, B. longiflora and B. virgata), slit-like (B. auriculata, very small; B. aromatica, B. dysophylla, B. glomerata and B. lindleyana), or round (B. loricata). In most species, vessel–ray pits are similar to intervessel pits in size and shape, occasionally co-occurring with scalariform (B. virgata) or lens-shaped (B. forrestii) pits. Vessel–ray pits have clearly reduced borders (except for in B. dysophylla where regular borders are present). In B. coriacea and B. loricata, vessel–ray pitting could not be clearly observed. Helical thickenings are found in all species (Fig. 3F,H) except B. virgata (Fig. 3E), and are very fine (B. aromatica) or more coarse. Usually, helical thickenings occur in all vessels throughout their length (Fig. 3F), but in B. dysophylla they are confined only to narrow vessels, and in B. aromatica they were not observed in some vessels irrespective of their diameter.Vascular tracheids were not found; however, fibriform vessels (sensu Carlquist 2001) were observed adjacent to the regular vessels in both samples of B. auriculata. Fibres are libriform, mostly non-septate (septate fibres occur rarely in B. coriacea, B.lindleyana, B.longiflora and B. virgata), and mostly thin- to thick-walled (Fig. 3D) or rarely very thick-walled (B. aromatica). Exclusively thick- to very thick-walled fibres are present in B. auriculata (Fig. 3C), B. dysophylla, B. lindleyana and B. longiflora. Pits are simple to minutely bordered and in selected species were observed mostly or exclusively in radial walls(B. aromatica, B. auriculata, B. dysophylla, B. glomerata, B. longiflora and B. virgata). Mean fibre lengths vary mostly between 400–650 μm, with 371 μm in B. loricata and 975 μm in B. pulchella. Fibre wall thickness ranges from 1.1 (B. virgata (KF010)) to 8.5 μm (B. saligna (KF002)), but in most cases it is 5μm. In all species, axial parenchyma proved difficult to observe. It is most likely completely absent (B. auriculata, B. coriacea, B. glomerata, B. lindleyana, B. longiflora and B. loricata; Fig. 3C), occasionally scanty paratracheal in solitary strands near the vessels (B. aromatica, B. forrestii), or diffuse (B. dysophylla and B. virgata). Rays are uni- and bi- to tetraseriate (Fig. 3E–F). Mean number of uniseriate rays ranges mostly between 0.4–5.6 mm−1, though it is up to 8.2 mm−1 in B. glomerata (Fig. 3F) and up to 14.75 in B. virgata (KF004, KF010; Fig. 3E); number of multiseriate rays spans 1.8–7.0 mm−1. Uniseriate rays are composed of upright cells sometimes intermixed with square ones. Multiseriate rays consist of procumbent body cells with few (1–3 in B. glomerata and B. longiflora, and 1–5 in B. dysophylla) or numerous (up to: 6 in B. auriculata,8inB. virgata, and 11 in B. lindleyana) marginal rows of upright and square cells, mostly of upright and square cells with few procumbent cells mixed throughout rays (B. aromatica, B. coriacea and B. forrestii), or exclusively of upright and square cells (B. loricata, though this is likely an effect of the sampled stem being only ca. 1 cm wide). Prismatic crystals were found in upright and square ray cells (occasionally also in procumbent ones) in B. aromatica, B. coriacea and B. longiflora (Fig. 3H). Droplets of tannin were observed in ray cells of B. coriacea, B. forrestii and B. lindleyana (Fig. 3G). All studied species tested positive for tannin.

Wood anatomy of Freylinia Wood descriptions of Freylinia (Fig. 5) are based on F. lanceolata and F. tropica. Growth ring boundaries are indistinctly to distinctly marked by 2–3 (up to 10) tangential rows of radially flattened fibres (F. lanceolata) or present and indistinctly marked with 1–2 tangential rows of fibres with thicker walls (F. tropica, Fig. 5B). Wood is diffuse-porous in both species (Fig. 5A–B). Vessels are circular to oval, very angular (F. lanceolata; Fig. 5A) to slightly angular (F. tropica; Fig. 5B), narrow to very narrow (mean tangential diameter: 33 μm and 18 μm in F. lanceolata and F. tropica, respectively; https://doi.org/10.6084/m9.figshare.12066717.v3), and numerous (87 mm−2 in F. lanceolata and 139 mm−2 in F. tropica). Vessels are partly solitary (46 and 39% in F. lanceolata and F. tropica, respectively) or disposed in small clusters of ca. 2–6 vessels. Mean vessel wall thickness is ca. 3.2 μm. Vessel element length averages 577 μm in F. lanceolata and 430 μm in F. tropica. Length-on-age curve is decreasing in both species (Fig. 4). Perforation plates are exclusively simple (Fig. 5C,E–F). Intervessel pitting is alternate and minute (1.7–3.6 μm in vertical size) in F. tropica or small to medium (4.2–8.2 μm in vertical size) in F.lanceolata.InF.lanceolata, pit borders are closely spaced, rounded, and sometimes polygonal. Apertures are slit-like. Vessel–ray pits are similar to intervessel pits in size and shape (Fig. 5E–F). Helical thickenings are very fine in F. tropica and coarser in F. lanceolata (Fig. 5E–F), and are present in all vessel elements throughout their length. Vascular tracheids were not found, but fibriform vessels adjoining regular vessels were observed. Fibres are libriform, non-septate, thin- to thick-walled (F. lanceolata; Fig. 5A) or very thick-walled (F. tropica; Fig. 5B), and slightly longer in F.Downloaded lanceolata from (meanBrill.com10/06/2021 length: 12:43:59AM 853 μm, via free access 10 IAWA Journal 42 (1), 2021

Figure 4. Linear models of vessel element length-on-age with 95% confidence intervals (grey areas) for specimens of species in sections of Buddleja and Freylinia. Colours represent different specimens in each graph. vs. 661 μm in F. tropica; Fig. 5E–F). Mean wall thickness is 4.5 μm and 5.6 μm in F. lanceolata and F. tropica, respectively. Pits are simple to minutely bordered, and present only in radial walls (F. lanceolata) or in radial and tangential walls (F. tropica). Axial parenchyma is absent (Fig. 5A–B). Rays are uni- and bi- to triseriate (Fig. 5C), and slightly more numerous in F. lanceolata than in F. tropica (uniseriate rays are 3.6 and 1.8 mm−1 respectively, and multiseriate ones are 4.2 and 3.6 mm−1, respectively). Uniseriate rays are composed of square and upright cells (Fig. 5D). Multiseriate rays have middle portions composed of slightly procumbent and square cells, with 1–3 marginal rows made mostly of upright cells (Fig. 5E). No crystals were found in ray cells. Droplets of tannins were observed in ray cells in F. lanceolata (Fig. 5E). Both species tested positive for tannin presence.

Bark anatomy of Buddleja virgata The epidermis on young stem parts is composed of a single layer of isodiametric dome- or bottle-like cells (9−)18(−35) μm in tangential size with thin radial and inner tangential walls and thicker outer tangential walls covered with prominent cuticle (Fig. 6A). Dark content is present in some epidermal cells. Trichomes were not found. The cortex is composed of up to 10 layers of rounded isodiametric parenchyma cells (12−)18(−28) μm in diameter, with an outermost layer of radially elongated palisade cells with large intercellular spaces in-betweenDownloaded (Fig. from 6A), Brill.com10/06/2021 followed by a12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 11

Figure 5. Wood anatomy of Freylinia. (A) F. lanceolata; deposits in fibres and vessel elements (TS); (B) F. tropica; weakly marked growth ring (TS); (C) F. lanceolata; multiseriate and uniseriate rays (TLS). (D): F. tropica; ray composed of square and slightly procumbent cells flanked by rows of upright cells (RLS). (E) F. lanceolata; ray composed mostly of square cells with few rows of procumbent cells (RLS). (F) F. lanceolata; helical thickenings in vessel elements and vessel–ray pits (RLS). layer of isodiametric, closely spaced cells (inset in Fig. 6A). The pericycle has a ring of discrete fibre strands embedded in parenchymatous ground tissue (Fig. 6A,C). The pericyclic fibres are moderately thick-walled, solitary, and in small groups of 2–5. No crystals or deposits were found in cortical parenchyma cells. Dilatation of cortex is affected by tangential stretching of cortical cells (Fig. 6D) and by anticlinal divisions of parenchyma cells with formation of tangential strands of 2–5 cells. Mature bark is partly peeling with shallow fissures. The first-formed periderm is initiated in the outer cortex; sequent periderms were not found (Fig. 6B–D). Phellem is not stratified and consists of 10–15 layers of isodiametric to tangentially elongated phelloid cells (Fig. 6B&D) — these cells do not stain with Sudan III nor phloroglucinol. Phelloderm is uni- to triseriate and difficult to distinguish in mature periderm (Fig. 6D). A lenticel with nonstratified (homogenous) filling tissue was observed in one sample (KF004; Fig. 6C). Conducting secondary phloem (Fig. 6C–D) is very homogeneous and consists of sieve tubes with companion cells (one per sieve tube as seen in TS), axial parenchyma, and rays. Sieve tube members are solitary and in small groups, rarely in rows of 3, (7−)13(−18) μm wide in tangential diameter, and ca. 130–200 μm long. Sieve plates could not be observed. Axial parenchyma forms the ground tissue (Fig. 6D) and consists of fusiform cells and strands of 2–7 cells. No crystals were found in axial parenchyma cells. Transition from conducting to nonconducting secondary phloemDownloaded is indistinguishable. from Brill.com10/06/2021 12:43:59AM via free access 12 IAWA Journal 42 (1), 2021

Figure 6. Bark anatomy of Buddleja virgata (sect. Gomphostigma). (A) Young stem with epidermis covered with prominent cuticle; inset shows epidermis, and a part of outer cortex under higher magnification (TS); (B) mature stem with fully developed periderm (RLS); (C) mature stem with periderm and pericyclic fibres in discrete strands in dilated cortex (TS); (D) close-up of phelloid cells and layers of phelloderm marked with arrow (TS); (E) secondary phloem rays and axial parenchyma (TLS).

Secondary phloem rays are uni- and bi- to tetraseriate (average 2.8 cells wide; Fig. 6E). They are composed of square and procumbent cells forming up to 15 rows, with upright cells in single marginal rows or all cell types intermixed throughout the ray body. No crystals or tannin deposits were observed in ray cells. No secretory structures were observed. Dilatation of secondary phloem is affected by tangential stretching of sieve tube members, axial parenchyma elements, and ray cells without anticlinal divisions. No sclerification of any secondary phloem elements was observed (Fig. 6C–D).

Bark anatomy of other Buddleja species Anatomical details of bark (Figs 7–8) from young stems are based on B. auriculata, B. cordata, B. dysophylla, B. glomerata, B. saligna and B. salviifolia, and bark anatomy of older stems was observed in the previous species, as well as B. aromatica, B. forrestii, B. lindleyana, B. longiflora, B. loricata and B. pulchella. In Buddleja glomerata, periderm initiation starts very early, so neither epidermis nor primary cortex were available even from the youngest stems (Fig. 7D). In the remaining species (Fig. 7A–B), the epidermis on young stem parts is composed of a single layer of tangentially elongated cells (except in B. saligna where cells are isodiametric). The epidermal cells are ca. 18(−28) μm in tangential diameter (smaller in B. dysophylla where width is 11 μm on average andDownloaded up to 19from μm) Brill.com10/06/2021 and have all 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 13

Figure 7. Bark anatomy of exemplar Buddleja species (TS). (A) B. auriculata; young stem with dense indumentum of trichomes on epidermis; (B) B. cordata; young stem, epidermis without trichomes, pericyclic fibres in discrete strands; (C) B. auriculata; solid sclerification of nonconducting secondary phloem, and multiple periderms, each including one layer of phelloid cells; (D) B.glomerata; multiple periderms, each including 2–4 layers of phelloid cells; (E) B. dysophylla; conducting non-sclerified secondary phloem and nonconducting phloem of which the outer part is sclerified; multiple periderms, each including 1–4 layers of phelloid cells; (F) B. salviifolia; conducting non-sclerified secondary phloem; multiple periderms, each including 1–3 layers of phelloid cells; cells with inulin crystals are seen as dark occlusions in conducting secondary phloem. walls thin (except in B. saligna where outer tangential walls are thicker; additionally epidermal cells have dark content in this species). Cuticle is not prominent (except in B. saligna and B. salviifolia where it is markedly thicker). Branched trichomes are commonly present — in B. cordata their number differs from stem to stem (from absent to very numerous; Fig. 7B), in B. salviifolia they are very numerous, and in B. auriculata (Fig. 7A) and B. dysophylla they form dense indumentum together with glandular trichomes. In B. saligna only trichome stalks were observed. The cortex consists of parenchymatic cells (1–3 layers in B. saligna and B. salviifolia,upto10inB. auriculata and B. dysophylla,upto14inB. cordata; Fig. 7A–B). The cells are isodiametric to tangentially stretched, in B. dysophylla rounded (sometimes oval), and in older stems of B. auriculata they might contain brownish deposits. The parenchymatic cells are ca. 30(−50) μm wide in tangential diameter. The pericycle has a ring of discrete fibre strands embedded in parenchymatous ground tissue (Fig. 7B) — the pericyclic fibres form bands (tri- to tetraseriate and very-thick walled in B. saligna) or clusters (10–20 thin-walled fibres in younger stems of B. auriculata to moderately thick-walledDownloaded fibres from in olderBrill.com10/06/2021 stems, and 12:43:59AM 20–40 via free access 14 IAWA Journal 42 (1), 2021 moderately thick-walled fibres in B. cordata). In B. dysophylla, the pericycle remains parenchymatous, i.e., pericyclic fibres are absent. Mature bark is macroscopically brittle, smooth, and partly peeling in B. auriculata and B. salviifolia; brittle, peeling, and with deep fissures in B. cordata, B. dysophylla, and B. glomerata (in the two latter species, fissures are also long); non-peeling with very shallow fissures in B. saligna; brittle and partly peeling with very shallow fissures in B. aromatica, B. coriacea, B. forrestii, and B. lindleyana. The first-formed periderm is initiated in the outermost layer of secondary phloem with concentric to reticulate arrangement of sequent periderms in deeper layers of secondary phloem (Fig. 7C–F). Periderm consists of phellem and phellogen, whereas phelloderm is absent (Fig. 8H). Phellem consists of isodiametric to radially elongated phelloid cells, which do not stain with Sudan III nor phloroglucinol, forming a single layer(B. salviifolia; Fig. 7F), single or occasionally two layers (B. auriculata and B. cordata; Fig. 7C, 8A), 1–3 layers (B. saligna, rarely 4 layers in B. dysophylla; Fig. 7E, 8C), or 2–4 layers (B. glomerata; Fig. 7D, 8B). Conducting secondary phloem is very homogenous and consists of sieve tubes with companion cells (one per sieve tube as seen in TS), axial parenchyma and rays. Sieve tube members are solitary and in small groups, and in B. dysophylla also in tangential bands. Sieve tube members are usually 10–20 μm wide and ca. 130–200 μm long. Sieve plates are compound (except in B. glomerata where they are simple), and sieve areas are located on oblique end walls (3–5; 4–6 in B. auriculata, 3–7 in B. cordata, 9–12 in B. dysophylla, 3–6 in B. saligna, up to 8 sieve areas in B. salviifolia; Fig. 8E). Axial parenchyma forms the ground tissue and consists of fusiform cells and strands of 2 (B. dysophylla), 2–3 (B. salviifolia), 2–4 (B. glomerata), 2–5 (B. auriculata and B. saligna), or 4–7 cells (B. cordata). No crystals were found in axial parenchyma cells except for inulin crystals in B. salviifolia (Fig. 7F). Transition from conducting to nonconducting secondary phloem is abrupt (Fig. 7E). Secondary phloem rays are uni- to tetraseriate (usually 2–3 cells wide; Fig. 8D). Ray bodies are composed of up to 6 rows of solely upright cells in B. glomerata (and in some rays in B. saligna) or of intermixed square, upright, and not markedly elongated procumbent cells forming multiple rows in the remaining species (up to: 9 in B. auriculata and B. cordata,11in B. saligna,15inB. dysophylla and B. salviifolia). They are flanked (in B. dysophylla not always) by marginal rows of upright cells (1 in B. auriculata, 1–2 in B. dysophylla and B. salviifolia,upto3inB. cordata and B. saligna). In B. cordata, most rays are composed of thin-walled cells, but some rays consist exclusively of very thick-walled cells. No crystals or secretory structures were observed in ray cells. Dark content (most likely tannins) commonly occurred in ray cells (except in B. salviifolia). Storied arrangement of sieve tube members, axial parenchyma elements, and low rays occasionally occurs in B. saligna. Dilatation of cortex is affected by tangential cells stretching without anticlinal cell divisions in B. cordata. Tangential stretching of cells of secondary phloem without anticlinal cell divisions was observed in B. dysophylla and B. salviifolia. In the remaining species, no dilatation of cortex or secondary phloem was observed. In all cases, solid sclerification (Fig. 8G–H) of conductive cells, axial parenchyma, and rays occurs in portions of the layers of secondary phloem separated by periderms (1–7 cell layers in B. auriculata, 1–10 in B. cordata, 2–5 in B. salviifolia, 2–8 in B. glomerata, 3–10 in B. saligna, and 3–16 in B. dysophylla; Fig. 7C–F, 8A–C,F). Solid sclerification was occasionally also observed in phloem prior to cut-off by periderm (Fig. 8G). Phloem cells which underwent solid sclerification do not stain with Sudan III, but develop intensive red colour when stained with phloroglucinol.

Bark anatomy of Freylinia Bark anatomy descriptions of Freylinia (Fig. 9) are based on F. lanceolata and F. tropica. The epidermis on young stem parts is composed of a single layer of rectangular, tangentially elongated, or bottle-like cells, ca. 25 μm in tangential diameter (F. lanceolata); or a single layer of dome-like cells, ca. 18 μm in tangential diameter (F. tropica), with thin walls covered with prominent cuticle (Fig. 9A). Dark content is present in some epidermal cells. No trichomes were observed. The cortex is composed of up to 10 layers of rounded to tangentially elongated parenchyma cells (Fig. 9A), usually 20 μm wide in F. tropica and ca. 40 μm wide in F. lanceolata.InF. lanceolata, the outermost layer of cortex cells is radially elongated. The pericycle remains parenchymatous in F. tropica, while in F. lanceolata it consists of a ring of discrete fibre strands embedded in parenchymatous ground tissue. The pericyclic fibres are thick-walled and in groups of 3–30 in F. lanceolata and completely lacking in F. tropica. No crystals were observed in cortical cells. Dilatation of cortex is affected by tangential stretching of cortical cells in both species (Fig. 9B). Mature bark is non-peeling. In F. lanceolata, it has long, longitudinal wrinkles but no fissures, and in F. tropica, it has short, shallow fissures. The first-formed periderm is initiated in the outer cortex, probably in its subepidermal layer (Fig. 9B), with reticulate arrangement of sequent periderms (Fig. 9C). Phellem consists of up to 10 layers of isodiametric to tangentially elongated phelloid cells in F. tropica (Fig. 9C) and in F. lanceolata phellem cells are distorted andDownloaded cannot be from precisely Brill.com10/06/2021 counted 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 15

Figure 8. Bark anatomy of exemplar Buddleja species. (A) B.auriculata; solid sclerification of conductive cells and ray cells in nonconducting secondary phloem; uniseriate phellem consisting of phelloid cells (RLS); (B) B. glomerata; five periderms, each including 2–4 layers of phelloid cells (RLS); (C) B. dysophylla; three fully developed periderms and a fourth in the process of formation (RLS); (D) B. cordata; secondary phloem with mostly biseriate rays and axial parenchyma cells (TLS); (E) B. saligna; compound sieve plate (RLS); (F) B. dysophylla; portion of sclerified secondary phloem surrounded by a uniseriate phellem composed of phelloid cells (TLS); (G) B. cordata; nonconducting phloem, in outer part of which certain cells underwent solid sclerification; early stages of phellem initiation can be observed at the top; (H) B. cordata; (from the bottom upwards) nonconducting secondary phloem, 2–3 layers of phellogen cells, 1 layer of phellem composed of phelloid cells, and a cut-off phloem which underwent solid sclerification.

(ca. 5; Fig. 9B). Phelloid cells do not stain with Sudan III nor with phloroglucinol. Phellem is nonstratified (insets in Fig. 9B–C). Phelloderm is 3–5-seriate and found only in the first-formed periderm. Conducting secondary phloem consists of sieve tubes with companion cells (one per sieve tube as seen in TS), axial parenchyma, rays, and sclereids in groups of 3 to 12 (F. lanceolata; Fig. 9B) or 17 (F. tropica). Sieve tube members are solitary and in small groups, and in F. lanceolata also in radial rows. They are wide in F. lanceolata (ca. 30 μm wide) and narrow in F. tropica (ca. 13 μm wide), and ca. 110 or 150 μm long (in F. lanceolata and F. tropica, respectively). Sieve plates are compound, with up to 3–5 sieve areas on oblique end walls. Axial parenchyma forms the ground tissue and consists of fusiform cells and strands of 2–4 (F. tropica) or 2–5 (F. lanceolata) cells. No crystals were found in axial parenchyma cells except for inulin crystals in F. tropica, and dark content (most likely tannins) is common in F. lanceolata (Fig. 9E). Transition from conducting to nonconducting secondary phloem is gradual. Nonconducting phloem is marked by theDownloaded presence from of Brill.com10/06/2021 thick-walled sclereids12:43:59AM via free access 16 IAWA Journal 42 (1), 2021

Figure 9. Bark anatomy of Freylinia. (A) F. tropica; young stem with epidermis covered with prominent cuticle (TS); (B) F. lanceolata; mature bark with one periderm, dilated cortex, and clusters of sclereids in nonconducting secondary phloem; inset shows periderm under higher magnification (TS); (C) F. tropica; discontinuous tangential bands of sclereids in nonconducting secondary phloem; 3–5-seriate sequent periderms composed of phelloid cells arranged in reticulate pattern; inset shows fragment of periderms under higher magnification (TS); (D) F. lanceolata; sclerification of secondary phloem elements at early and final stages (TS); (E) F. lanceolata; multiple narrow multiseriate secondary phloem rays, and tannins in cells (dark occlusions in cells; TLS); (F) F. lanceolata; first-formed periderm with 3–5-seriate phelloderm, dilated cortex, sclereids in nonconducting secondary phloem (RLS).

(Fig. 9D), 30–70 μm wide and 220–450 μm long, arranged in clusters of 3–12 in F. lanceolata (Fig. 9B&D) and in discontinuous tangential bands in F. tropica (Fig. 9C). Secondary phloem rays are uni- to biseriate (Fig. 9E). In F. lanceolata they are composed of square and procumbent cells forming up to 9 rows with upright cells flanking rays, and in F. tropica they are composed of intermixed square, markedly elongated procumbent, and upright cells forming up to 6 rows. No crystals were observed in ray cells, but in F. lanceolata they commonly contain dark content (most likely tannins; Fig. 9E). Dilatation of cortex and secondary phloem is affected by tangential cells stretching without anticlinal cell divisions (Fig. 9B). In secondary phloem, dilatation is combined with partial sclerification, which is more prominent in F.tropica (Fig. 9B–D). Secretory structures were not observed. Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 17

Figure 10. Scatterplot of fibre wall thickness versus vessel element length in species of Buddleja (black dots) and Freylinia (red dots).

Statistical analyses Discrimination of taxa based on wood anatomy — Freylinia differs from Buddleja in having a combination of relatively long vessel elements (>400 μm) and thicker fibre walls (average thickness 4.5 μm; Fig. 10). We did not find any pairs of traits that were suitable for distinguishing among sections within Buddleja (not shown). Correlations between wood traits and climatic variables and plant height — The first factors from principal component analyses of bioclimatic variables (PC1 and pPC1) accounted for 36% and 40% of variance in PCA and pPCA, respectively. The second factors (PC2 and pPC2) explained 25% and 29% of variance, respectively. The results of PCA and pPCA (top plot in Fig. 11) were congruent — first factors were affected by similar bioclimatic variables: PC1 increased with precipitation of driest month (bio14) and quarter (bio17) and decreased with mean diurnal range in temperature (bio2). When including a phylogenetic correction, the first factor (pPC1) positively corresponded with precipitation of driest month (bio14) and quarter (bio17) and with minimal (bio6) and mean (bio11) temperature of coldest month, and negatively corresponded with mean diurnal range in temperature (bio2) and precipitation seasonality (bio15). Therefore, going from lower to higher values of the first factor represents a shift from seasonably dry climates with greater temperature variance to wetter climates with more stable temperatures and milder winters. The second factors, PC2 and pPC2, increased with maximal (bio5) and mean (bio10) temperature of warmest month and quarter and decreased with isothermality (bio3); PC2 decreased also with altitude, but this effect disappeared after the inclusion of phylogeny in the inference. Going from lower to higher values of second factors represents a shift from climates with stable, milder temperatures throughout the year to climates that are seasonally hot. Calculations of Pagel’s lambda and Blomberg’s K indicated that phylogenetic signal was either low or non-existent for all quantitative wood traits and maximal plant height. Although a few traits had significant p-values (<0.05), lambda was below 0.46 and K was below 0.4 for those traits, which is interpreted to mean there is little correlation between related species (Table A2 in the Appendix). Several wood traits were significantly correlated with bioclimatic factors (Table 2, Fig. 11, Table A3 in the Appendix). Vessel frequency was negatively correlated and fibre length was positively correlated with PC1 and pPC1. Weakly significant correlations were retrieved for vessel element length (positive) and number of vessels per group (negative) with PC1 and pPC1. Intervessel pit diameter, vessel element length, and fibre length were negatively correlated with PC2. Except for vessel element length, these traits were also negatively correlated with pPC2. Weakly significant correlations were found for vessel frequency (positive) and vessel lumen diameter (negative) with PC2; and vessel frequency (positive), vessel lumen diameter (negative), and vessel element length (negative) with pPC2 (Table A3 in the Appendix). Some wood traits had weakly significant correlations with maximal plant height. Vessel frequency correlated negatively, while vessel element length, vessel lumen diameter, intervessel pit diameter, fibre length, and fibre wall thickness correlated positively (Fig. 12). Maximal plant height itself was significantly negatively correlated withDownloaded PC2 and from pPC2 Brill.com10/06/2021 (Fig. 11). 12:43:59AM via free access 18 IAWA Journal 42 (1), 2021 2 R -value p + 0.004* 0.2 + 0.03* 0.1 ––- Slope direction 2 R -value 0.002* 0.2* p 0.0002* 0.3* 0.0002* 0.3* ith –, and intervals spanning across 0 are marked with +/–. 95% confidence interval excluding 0, i.e., strongly statistically – + 0.009* 0.2 – 0.04* 0.1 – 2 R Slope direction 2 R -value <0.05 and p 0.2 0 -value 0.001* 0.2* p 0.0004* 0.3* + + 0.03* 0.1 – 0.01* 0.1 + 0.02* 0.1 +/– +/– 0.3 0 – Slope direction s for correlations with both a 2 R -value 0.002* 0.2* p 0.0002* 0.3* 0.0002* 0.3* 0.0003* 0.3* ly positive intervals are marked with +, exclusively negative intervals w its and bioclimatic principal components or maximal plant height. + 0.009* 0.2 – – essel element length ratio. Slope direction 2 R -value 0.001* 0.2* p 0.0004* 0.3* PC1 PC2 pPC1 pPC2 Maximal plant height + 0.03* 0.1 – – 0.005* 0.2 +/– 0.6 0 – 0.005* 0.2 +/– 0.6 0 +/– 0.4 0 +/– 0.6+/– 0 1 +/– 0 0.7 +/– 0 +/– 0.3 0 0.6 +/– 0 +/– 1 0 0.7 0 +/– +/– 0.3 0 0.7 0 +/– 0.2 0.1 +/– 0.1 0.1 – 0.01* 0.1 +/– 0.1 0.1 – 0.01* 0.1 + 0.02* 0.1 +/– 0.3 0 – Slope direction 95% confidence intervals excluding 0 are marked with asterisks, and field 2 R Direction of slope was assessed based on 95% confidence limits. Exclusive height per group width diameter length diameter -values <0.05 and Multiseriate rays Number of vessels Vessel wall thicknessMultiseriate rays F/V ratio +/–Maximal plant height 0.5 +/– 0 +/– 0.2 +/– 0 0.1 0.1 1 – +/_ 0 +/– 0.6 0 0.5 +/– 0 +/– 0.1 0.1 1 +/– 0 0.6 +/– 0 0.4 +/– 0 1 0 Fibre wall thicknessFibre length +/– 0.6 + 0 +/– 0.1 0 +/– 0.6 0 +/– 0.1 0 + 0.04* 0.1 Vessel lumen Vessel element Vessel frequency – Intervessel pit significant, are highlighted in italics. F/V ratio is the fibre length to v P Table 2. Summary statistics for linear regressions between quantitative wood tra Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 19

Figure 11. Principal component analysis of bioclimatic variables, and regressions between bioclimatic principal components and wood traits. (Top plot) Bioclimatic variables from WorldClim and their effect on principal component values. Variables with the greatest effect are shown in colours. (Other plots) Regression line plots and 95% confidence limits for selected strongly significant correlations between bioclimatic principal components and wood traits or maximal plant height. Colours follow the scheme from the top plot, and function formulas and coefficients of determination (R2) are provided.

DISCUSSION

Systematic patterns in wood traits The species of Buddleja and Freylinia examined in the present study are fairly uniform in their wood structure and differ between genera only in the combination of relatively long vessel elements and thicker fibre walls in Freylinia (Fig. 10). This needs to be taken with caution, however, as only two samples of Freylinia were studied. The seven sections of Buddleja recognised by Chau et al. (2017) showed only weak quantitative differences in their wood anatomy. No reliable diagnostic wood characters could be identified. Very high vessel frequency (average 511 vessels mm−2) in combination with lack of helical thickenings could possibly be diagnostic for section Gomphostigma, but it was represented by only one (B. virgata)of two species in our study. Moreover, similarly high vessel frequency and lack of helical thickeningsDownloaded infrom selected Brill.com10/06/2021 vessel elements 12:43:59AM via free access 20 IAWA Journal 42 (1), 2021

Figure 12. Regression line plots and 95% confidence limits for significant correlations between wood traits and maximal plant height. Function formulas and coefficients of determination (R2) are provided.

were reported in B. utahensis (Carlquist 1997). Since quantitative wood traits are apparently not related to phylogeny, they may be more influenced by climatic factors and species habit. One of the qualitative traits suggested by Carlquist (1997) as possibly being useful for taxonomic discrimination in Buddleja is the presence of prismatic crystals in ray cells (Fig. 3H). Reported data and our observations show that prismatic crystals occur only in sections Buddleja and Chilianthus (Carlquist 1997; Aguilar-Rodríguez & Terrazas 2001). However, contrary to previous suggestions, this character is not suitable for delimitation of these two lineages because many of their members lack prismatic crystals in their ray cells.

Ecological patterns in wood traits We found that four quantitative wood traits strongly correlate with climatic factors (Fig. 11) and six traits weakly correspond with maximal plant height (Fig. 12). Four traits overlap in both categories (vessel frequency, vessel element and fibre lengths, intervessel pit size), while vessel diameter and fibre wall thickness are weakly correlated with plant size. Plant di- mension, in turn, decreases with higher temperature in the species range (Fig. 11, Table A2 in the Appendix). Our study shows that Buddleja species with higher vessel frequency, shorter and narrower vessel elements, and smaller intervessel pits are found in areas with more extreme annual and diurnal temperature changes and lower precipitation (although the degree of correlation between these wood traits and climate varies). Some of these traits directly influence water conductance and xylem susceptibility to cavitation. Higher frequency of narrower vessels (and vice versa) is a case of the well-known trade-off between safety and efficiency of water conductance (Baas et al. 2004). Wider vessels allow for faster flow at the cost of higher embolism risk. That risk can be mitigated by more numerous vessels allowing water to bypass em- bolised vessel elements (Lens et al. 2013; Carlquist 2014). In our study, however, the response of vessel diameter to climate is only weakly significant (Table 2). Such a weak response could result from a considerable functional effect of even minor changes in this trait, as water conductivity increases to the fourth power of vessel diameter (Tyree & Zimmermann 2002). Additionally, the shortening of vessel elements and fibres in drier habitats has been reported as an environmental trend in several other genera (Baas 1973; Van der Graaff & Baas 1974; Van Den Oever et al. 1981; Baas 1986; Noshiro & Baas 2000; Lens et al. 2003; Kotina et al. 2013). The length of tracheary elements is linked to the size of cambial fusiform cells, but the adaptive or functional significance of these parameters remains obscure (Noshiro & Baas 2000). As for the size of intervessel pits, variation in this trait in different taxa can show opposite responses to drought and precipitation seasonality (Sonsin et al. 2012; Kotina et al. 2013). The functional and adaptive effects of this character cannot be assessed separatelyDownloaded from from the Brill.com10/06/2021 estimation of 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 21 other wood characters, such as the density of bordered pits on vessel walls, the area of pit membranes on vessels, the sectoriality of long-distance transport in the stem, and especially the thickness and porosity of pit membranes (Orians et al. 2004; Wheeler et al. 2005; Ellmore et al. 2006; Hacke et al. 2006; Tixier et al. 2014; Schenk et al. 2015; Li et al. 2016). Interestingly, all of the above traits and also fibre length and wall thickness weakly correlate (nevertheless with slope P-value <0.05) with maximal plant height. In previous studies, vessel diameter was shown to be affected by plant size (Fichtler &Worbes 2012; Olson etal. 2013; Olson & Rosell 2013; Olson 2014).Terrazas etal. (2008) showed that fibre length increases with plant height in Buddleja, and this trend, i.e., shorter libriform fibres in shrub species relative to congeneric tree species, has also been reported in other taxa (Cumbie & Mertz 1962). In our study, vessel element length and diameter and intervessel pit diameter were positively correlated with plant size, while vessel frequency decreased with it (Fig. 12). A negative correlation between vessel frequency and plant height may result from higher auxin concentrations in the distal stem areas (near the apical meristem) and lower in the proximal areas (Aloni 1987). In addition, fibre length and fibre wall thickness were shown to increase with maximal height (Fig. 12), which likely provides mechanical support in taller species. Why did we find only weak correlations between wood traits and plant height in Buddleja in the present study? Most likely our methods were not sensitive enough to detect trends. It is established that axial sampling height and individual plant height affect certain wood traits (Olson & Rosell 2013; Dória et al. 2019; Olson et al. 2020). However, since these data were not available, we used species maximal height as a rough proxy, unavoidably making the correlations weaker. Nevertheless, at least some influence of plant size on wood traits in Buddleja can be inferred. In our analyses, maximal plant size showed a significant negative trend with maximal temperature (Fig. 11), a climatic situation conducive to higher transpiration rates and therefore also to a higher likelihood of developing cavitation. Simul- taneously, Buddleja wood traits promoting protection against cavitation (more numerous and smaller vessels) are better expressed in smaller plants. Therefore, evolution in Buddleja in response to climate may not be acting directly on wood traits, but rather on plant size, which in turn impacts quantitative xylem features.

Systematic patterns in bark structure Unlike wood, bark structure varies considerably in Buddleja and Freylinia. All studied species of these genera share very regular arrangement of sieve tubes and axial parenchyma in conducting secondary phloem lacking idioblasts, as well as periderm composed of phelloid cells. However, the two Freylinia species and B. virgata, the only studied member of Bud- dleja section Gomphostigma, are very distinctive in their lack of trichomes, initiation of phellogen in the outer cortex, and only partial (Freylinia spp.) sclerification in dilated secondary phloem or complete absence (B. virgata) of its sclerification. Nonetheless, it must be noted that B. virgata stems were described as ‘glabrous or more often with a silvery indumentum of stellate scales’ (Leeuwenberg 1977); therefore, the lack of trichomes in our samples might be incidental. In contrast, the remaining Buddleja species usually have trichomes on the epidermis, phellogen initiated in secondary phloem, and dilated secondary phloem among periderms undergoing solid sclerification in their rhytidome. As genus Freylinia was an outgroup in our study and section Gomphostigma may be sister to the rest of the genus Buddleja (Chau et al. 2017), the latter suite of bark traits may be synapomorphic for the clade of Buddleja species excluding section Gomphostigma. More comprehensive examination of bark structure within sections of Buddleja is required, however, to confirm these results. The deep formation of narrow periderms with thin-walled phellem cells occurs in many plants with stringy or peeling bark, such as Clematis (Ranunculaceae), Lonicera (Caprifoliaceae), Melaleuca (Myrtaceae), Dodonaea (Sapindaceae), and Mahonia (Berberidaceae; Eryomin & Kopanina 2012; Crivellaro & Schweingruber 2013; Schweingruber et al. 2013). Such periderms function as separation layers for the outer portions of nonconducting secondary phloem shedding off in the course of bark dilatation. Those portions of secondary phloem usually contain numerous sclereids or fibres undertaking the protective role from phellem. Unlike in the genera mentioned above, some Buddleja species show ultimate manifestations of these phe- nomena.Their periderms are very homogenous in their structure, consisting only of phellogen and phellem without any trace of phelloderm (Fig. 8H). Moreover, the phellem in B. auriculata, B. cordata, B. dysophylla, B. saligna, and especially in B. salviifolia is often reduced to uniseriate layers of phelloid cells. This condition has not been reported elsewhere. At the same time, in most Buddleja species, the elements of nonconducting secondary phloem are almost totally involved in fast sclerification starting most likely simultaneously with their separation by newly-formed periderm (possibly also after the separation and occasionally just prior to it; Fig. 7G). In contrast, in other studied genera with such bark organisation (including those with solid sclerification of separated phloem portions, as in Clematis and Dodonaea), the sclereids occur in the secondary phloem before that part is cut off by new periderm (Crivellaro & Schweingruber 2013; Schweingruber et al. 2013). These observations suggest that the initiation of phellogen in Buddleja species may be involved in triggering the sclerification in adjacent regions of secondary phloem. Such a morphogenetic relationship between these two processes may be thought of as an evolutionary novelty in Buddleja, but this hypothesis needs additional testing. Downloaded from Brill.com10/06/2021 12:43:59AM via free access 22 IAWA Journal 42 (1), 2021

CONCLUSIONS Buddleja wood anatomy does not differ substantially among infrageneric sections. Wood traits providing higher embolism resistance are correlated with hotter, drier climates and with smaller-sized plants. Therefore hotter climates, which usually harbour smaller plants, will consequently see woods less susceptible to cavitation. Wood traits not directly affecting water conductance (fibre length and wall thickness) are also positively correlated with plant maximal height, providing more mechanical support. The patterns of periderm formation and secondary phloem sclerification found in Buddleja sect. Gomphostigma are similar to those in Freylinia and very unlike those in other Buddleja species, supporting its position as sister to the rest of the genus. Bark of the remaining Buddleja species is characterised by deep formation of very homogenous and often uniseriate phellem, which most likely is simultaneous with solid sclerification of nonconducting phloem, suggesting that it influences sclerification. The mechanism for this developmental connection deserves further investigation.

ACKNOWLEDGEMENTS

We thank the University of Washington Botanic Gardens, University of Washington Biology Greenhouse, Walter Sisulu National Botanic Garden, and Anthony R. Magee and John Manning at the Compton Herbarium (NBG) for facilitating sampling; Łukasz Banasiak (University of Warsaw) for suggestions on statistical analyses; and Krzysztof Spalik and Bożena Zakryś (University of Warsaw) for making sledge microtome available to us. The study was financially supported by the Polish Ministry of Science and Higher Education through the Faculty of Biology, University of Warsaw intramural grant (DSM 501-D114-01-1140300 to K.F.), University of Warsaw Integrated Development Programme (ZIP), co-funded by the European Social Fund (Operational Programme Knowledge Education Development 2014–2020, path 3.5 to K.F.), the National Research Foundation of South Africa (incentive grant No. 109531 to A.O.), the Russian Foundation for Basic research (grant no. 19-04-00714 to A.O.), the University of Johannesburg, and the Komarov Botanical Institute (institutional research Project No. AAAA–19-119030190018-1 to A.O.).

AUTHOR CONTRIBUTIONS

The study was designed by K.F. and elaborated by A.O. and J.H.C. Sample collection was done primarily by J.H.C., who also provided taxonomic ex- pertise. K.F. performed anatomical work under supervision by A.O.; J.H.C. obtained and checked bioclimatic data, and K.F. compiled the anatomical data matrix. Statistical work was performed by K.F. and A.O. The manuscript was written by K.F. and A.O. and edited by J.H.C.

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Edited by Marcelo R. Pace

Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 25 structure general general general, detailed general, detailed detailed - general general, detailed qualitative, quantitative qualitative, quantitative qualitative, quantitative qualitative, quantitative quantitative general, qualitative, quantitative quantitative qualitative, quantitative r detailed young and mature bark anatomy pecimen description Wood anatomy Bark mation, specimen collection information, specimen description, Shrub, ca. 0.25 m; sampled dead pruned stem Shrub, ca. 0.5 m; sampled main stem Shrub, ca. 1.5 m; older growth had burned in previous winter, new growth from base Shrub, ca. 2 m, sampled main stem Small tree, ca. 4 m; sampled main stem Shrub, ca. 0.5 m; sampled dead pruned stem Rambling shrub; flowers whiteScandent shrub, ca. 10 m; sampled qualitative, main stem North America, USA, Washington, University of Washington, Center for Urban Horticulture; partly shaded; Lat. 47.6581 Long. -122.2909; cultivated; 12.08.2019 North America, USA, Washington; University of Washington, Biology greenhouse; Lat. 47.7138 Long. -122.1295; cultivated; 08.08.2019 Africa, South Africa, Gauteng, Melville Koppies East; open grassland on low slopekoppie on (rocky hill); Lat. -26.1718 Long. 28.0047; Alt. 1710 m; 22.01.2019 Africa, South Africa, Western Cape, Kirstenbosch Botanic Garden; artificial side, next to a communal building; cultivated; 30.08.2019 Africa, South Africa, Gauteng, Melville Koppies West; open grassland on low slope on koppie (rocky hill); Lat. -26.1680 Long. 27.9981; Alt. 1635 m; 22.01.2019 North America, USA, Washington, University of Washington, Center for Urban Horticulture; partly shaded; Lat. 47.6581 Long. -122.2909; cultivated; 12.08.2019 Africa, South Africa, Vitval (Dundee) N.P.; among acacias; Alt. ca. 4000’ National Botanic Garden; south of concert stage, area with trees; Lat. -26.088 Long. 27.843; cultivated; 18.07.2019 of wood anatomy, and note on whether general structure of mature bark and/o used in study, including species, sample accession number, voucher infor Freylinia and (WTU)) (WTU)) (J)) (J)) (J)) (WTU)) NBG1472734.0 (NBG)) KF009 (John Chau 348 (J)) Africa, South Africa, Gauteng, Walter Sisulu Buddleja (Benth.)(Benth.) KF022 (Justus Thode, Benth. KF003 (KamilBenth. Frankiewicz 3 KF012 (Kamil Frankiewicz 12 J.Rémy KF014 (John Chau 359 J.Rémy KF016 (John Chau 361 Diels KF017 (John Chau 362 Kunth KF005 (Kamil Frankiewicz 5 Buddleja auriculata Buddleja auriculata Buddleja cordata Buddleja coriacea Buddleja dysophylla Buddleja dysophylla Radlk. Buddleja forrestii SpeciesBuddleja aromatica Sample accession ID (voucher) Specimen collection information S note on whether sample was used for qualitative and/or quantitative study Appendix Table A1. Sample information for specimens of was studied in sample. Downloaded from Brill.com10/06/2021 12:43:59AM via free access 26 IAWA Journal 42 (1), 2021 structure general, detailed general general general detailed detailed qualitative, quantitative qualitative, quantitative qualitative, quantitative qualitative, quantitative quantitative - quantitative general quantitative general, quantitative general, pecimen description Wood anatomy Bark Branch bearing inflorescence Shrub, ca. 1.5 m; sampled main stem Shrub, ca. 1.5 m; sampled main stem Shrub, ca. 2 m; sampled main stem Branch bearing inflorescence stalk sampled stalk sampled Specimen in flower at time of collection Small tree, ca. 3 m; sampled main stem Small tree, ca. 4 m; sampled main stem oo National Park, Rooivalle lookout point on Klipspringer Pass road; rocky, south-facing steep slope in karoo; Lat. -32.32561 Long. 22.45138; 20.04.2019 North America, USA, Washington; University of Washington, Chemistry Library; partly shaded; Lat. 47.6581 Long. -122.3097; cultivated; 09.08.2019 North America, USA, Washington, University of Washington, Center for Urban Horticulture; partly shaded; Lat. 47.6581 Long. -122.2909; cultivated; 12.08.2019 Africa, South Africa, Basutoland, Mamalapi; abundant on slopes in valley; Alt. 9000’ no collection information Africa, South Africa, KwaZulu-Natal, Mbona Estate, Karkloof, near house #41 (Holbeck); forest, forest/grassland, interface vegetation, moderate hill slope; well-drained, loamy soil, partial shade, no biotic effect seen; Lat. 29°18’S Long. 30°23’E; Alt. 1250 m; 02.08.2003 Africa, South Africa, Gauteng, Melville Koppies West; wooded east-facing slope on koppie (rocky hill); Lat. -26.1693 Long. 27.9977; Alt. 1635 m; 22.01.2019 Africa, South Africa, Gauteng, Melville Koppies West; wooded east-facing slope on koppie (rocky hill); Lat. -26.1693 Long. 27.9977; Alt. 1635 m; 22.01.2019 (WTU)) (WTU)) NBG14727154.0 (NBG)) KF020 (NBG1472745.0 (NBG)) NBG2930AC/NBG199486.0 (NBG)) (J)) (J)) Fortune KF015 (John Chau 360 H. Wendl. KF006 (John Chau 339 (J)) Africa, South Africa, Western Cape, Kar Brade KF018 (John Chau 363 (L.) Lam. KF001 (Kamil Frankiewicz 1 N.E.Br. KF019 (I. Nänni, Leeuwenberg KF021 (Compton, Willd. KF002 (Kamil Frankiewicz 2 SpeciesBuddleja glomerata Sample accession ID (voucher) Specimen collection information S Buddleja lindleyana Buddleja longiflora Buddleja loricata Buddleja madagascariensis Lam. Buddleja pulchella Buddleja saligna Buddleja salviifolia Table A1. (Continued.) Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 27 structure general, detailed general, detailed detailed general, detailed general, detailed general, detailed qualitative, quantitative qualitative, quantitative quantitative general, qualitative, quantitative qualitative, quantitative qualitative, quantitative of Washington. pecimen description Wood anatomy Bark Shrub, ca. 3 m; sampled main stem Shrub, ca. 4 m; sampled main stem Shrub, ca. 3 m; sampled main stem Dwarf shrub, ca. 50 cm; sampled main shoot with two branches/twigs Shrub, ca. 1.5 m; sampled main stem Shrub, ca. 2 m; sampled main stem u u sulu Sisulu National Botanic Garden; back of concert stage, area with many shrubs and someLat. trees; -26.088 Long. 27.843; cultivated; 18.07.2019 National Botanic Garden; back of concert stage, area with many shrubs and someLat. trees; -26.088 Long. 27.843; cultivated; 18.07.2019 Africa, South Africa, Western Cape, Kirstenbosch Botanic Garden; artificial side, next to the main entrance; cultivated; 30.08.2019 Africa, South Africa, Gauteng, Melville, 27 Boxes Mall; ornamental thicket growing along mall; Lat. -26.1756 Long. 28.0076; cultivated; 22.01.2019 National Botanic Garden; People’s Plants Garden, open area; Lat. -26.088 Long. 27.843; cultivated; 18.07.2019 National Botanic Garden; side of restaurant, on stream; Lat. -26.088 Long. 27.843; cultivated; 18.07.2019 ational Biodiversity Institute – The Compton Herbarium; WTU, University (J)) (J)) (L.) G.Don KF007 (John Chau 346 (J)) Africa, South Africa, Gauteng, Walter (L.) Lam. KF013 (Kamil Frankiewicz 13 L.f.L.f. KF004 (Kamil Frankiewicz 4 L.f. KF010 (John Chau 349 (J)) KF011 (John Chau Africa, 350 South (J)) Africa, Gauteng, Walter Sisul Africa, South Africa, Gauteng, Walter Sisul S.Moore KF008 (John Chau 347 (J)) Africa, South Africa, Gauteng, Walter Si Herbarium acronyms: J, University of Witwatersrand; NBG, South African N Freylinia tropica Buddleja virgata Buddleja virgata Buddleja virgata Freylinia lanceolata SpeciesBuddleja salviifolia Sample accession ID (voucher) Specimen collection information S Table A1. (Continued.) Downloaded from Brill.com10/06/2021 12:43:59AM via free access 28 IAWA Journal 42 (1), 2021

Table A2. Statistics for Pagel’s lambda and Blomberg’s K for 11 quantitative wood traits and maximal plant height.

Blomberg’s Kp-value Pagel’s lambda p-value

Vessel frequency 0.39011 0.046* 0.67088 0.112 Vessel lumen diameter 0.29532 0.047* 0.36802 0.012* Vessel element length 0.14142 0.648 0.29157 0.149 Intervessel pit diameter 0.22858 0.161 0.45675 0.043* Fibre wall thickness 0.20011 0.277 0.00006 1.000 Fibre length 0.17487 0.395 0.31239 0.191 Multiseriate rays height 0.13721 0.667 0.00005 1.000 Number of vessels per group 0.16135 0.762 0.00007 1.000 Vessel wall thickness 0.19674 0.565 0.00007 1.000 Multiseriate rays width 0.22867 0.419 0.00007 1.000 F/V ratio 0.33232 0.010* 0.36651 0.159 Maximal plant height 0.29981 0.053 0.40708 0.075

P-values <0.05 are marked with asterisks.

Downloaded from Brill.com10/06/2021 12:43:59AM via free access Frankiewicz et al. – Wood and bark anatomy of Buddleja 29 95% confidence limits 2 R Standard error ) 2 R determination ( 14 0.2 0.1 0.01 0.4 0.2 0.3 0.1 0.1 0.5 9.3 0.3 0.1 0.1 0.5 0.4 0.3 0.1 0.1 0.5 11.3 0.3 0.1 0.1 0.5 11.3 0.3 0.1 0.1 0.5 − − − − − − its and bioclimatic principal components or maximal plant height. 1.3 0.5 28.9 36.1 36.1 58.5 − − − − − − Lower Upper Lower Upper -value Slope 95% confidence limits Coefficent of P 0.001* 13.6 52.7 0.2 0.1 0.02 0.4 0.002* 0.001* 13.6 52.7 0.2 0.1 0.02 0.4 0.0002* 0.0003* 0.0004* 0.0004* 0.000197* Intervessel pit diameterFibre wall thicknessFibre length 0.3 0.6 −0.1 −0.1 0.3 0.1 0.0 0.0 0.0 0.0 0 0 0.11 0.0 Multiseriate rays heightNumber of vessels per groupMultiseriate rays widthF/V ratioMaximal plant height 0.005* 0.6 1 −0.2 −11.4 0.2 0.1 −0.04 −0.04 −0.1 20 −0.01 0.001 0.7 0.2 0.1 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.1 0.4 0.0 0.1 0.0 0.0 0.1 0.2 Intervessel pit diameter Intervessel pit diameterFibre wall thicknessFibre length 0.3 0.6 −0.1 −0.1 0.3 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 Fibre wall thicknessFibre length Multiseriate rays heightNumber of vessels per groupMultiseriate rays widthF/V ratioMaximal plant height 0.1 0.6 0.7 0.3 −0.1 −14.2 −0.2Multiseriate rays height −0.02 0.6 0.02 21.4 0.1 −0.03 0.6 0.1 −11.4 0.0 0.0 0.1 0.0 0.0 20 0.1 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.04 0.1 0.0 0.0 0.1 0.0 0.0 0.1 Vessel frequency Vessel lumen diameterVessel element length 0.1 0.03* −0.5 0.8 3.7 20 0.1 0.1 0.1 0.1 0.0 0.0 0.2 0.25 Vessel frequencyVessel lumen diameterVessel element length 0.01* 0.009* −5.3 5.9 −0.7 38.3 0.1 0.2 0.1 0.1 0.0 0.0 0.3 0.4 Vessel wall thickness 0.5 −0.1 0.1 0.0 0.0 0 0.1 Vessel frequency Vessel lumen diameterVessel element length 0.1 0.03* −0.5 0.8 3.7 20 0.1 0.1 0.1 0.1 0.0 0.0 0.2 0.2 Vessel wall thickness 1.0 −0.1 0.1 0.0 0.0 0.0 0.0 PC1 PC2 pPC1 Table A3. Summary statistics for linear regressions between quantitative wood tra Wood trait Slope Downloaded from Brill.com10/06/2021 12:43:59AM via free access 30 IAWA Journal 42 (1), 2021 95% confidence limits 2 R ficant, are highlighted in italics. F/V ratio is Standard error ) 2 R determination ( 95% confidence interval excluding 0, i.e., strongly statistically signi 2 R 14 0.2 0.1 0.01 0.4 0.2 0.3 0.1 0.1 0.5 0.4 0.3 0.1 0.1 0.5 − − − -value <0.05 and p 1.3 0.5 58.5 − − − Lower Upper Lower Upper -value Slope 95% confidence limits Coefficent of P 0.002* 0.0002* 0.0002* -values <0.05 are marked with asterisks, and correlations with both a P Number of vessels per groupMultiseriate rays widthF/V ratioMaximal plant height 0.005* 1Intervessel −0.2 pit diameter 0.2 0.1 −0.05 −0.04 −0.1 −0.01 0.05 0.7 0.2 0.1 0.0 0.1 0.0 0.1 0.0 0.0 0.1 0.1 0.0 0.4 0.0 0.0 0.0 0.1 0.2 Fibre wall thicknessFibre length Multiseriate rays heightNumber of vessels per groupMultiseriate rays widthF/V ratioMaximal plant height 0.1Maximal plant 0.6 height 0.7 0.3Intervessel pit diameter −0.1Fibre −14.2 wall −0.2 thicknessFibre lengthMultiseriate rays heightNumber of −0.02 vessels per 0.6 group 0.004*Multiseriate rays width 0.0 21.4F/V ratio 0.1 0.04* 0.4 −0.03 0.7 0.1 0.1 0.03* 0.001 0.2 −0.04 −12.9 0.0 0.0 0.1 0.0 1.2 0.3 −0.1 1 0.1 0.0 0.1 8.4 29.6 0.1 0.0 −0.03 0.0 0.01 0.0 0.2 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.3 0.1 0.0 0.0 0.1 0.1 0.2 0.0 0.1 0.1 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.2 0.1 0.0 0.3 0.0 0.2 0.0 Vessel wall thicknessVessel frequencyVessel lumen diameterVessel element 0.5 length −0.1 0.01* 0.009* 0.01* −5.3 5.9 −5.3 0.1 −0.7 38.3 −0.7 0.0 0.1 0.2 0.1 0.0 0.1 0.1 0.0 0.1 0.0 0.0 0.0 0.1 0.3 0.4 0.3 Vessel wall thicknessVessel frequencyVessel lumen diameterVessel element length 1 0.02* −0.1 0.04*Vessel wall thickness 0.02* 0.3 −21.3 1.2 0.1 0.4 −0.5 3.1 14.1 −0.04 0.0 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.3 0.3 0.3 0.0 0.1 pPC2 the fibre length to vessel element length ratio. Table A3. (Continued.) Wood trait Slope Downloaded from Brill.com10/06/2021 12:43:59AM via free access