bs_bs_banner Botanical Journal of the Linnean Society, 2014, 176, 452–468. With 2 figures Phylogenetic analysis based on structural and combined analyses of Rhus s.s. (Anacardiaceae) AGUSTINA ROSA ANDRÉS-HERNÁNDEZ1, TERESA TERRAZAS2*, GERARDO SALAZAR2 and HELGA OCHOTERENA2 1 Escuela de Biología, Benemérita Universidad Autónoma de Puebla, Puebla, México Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-233, 04510 México, D.F., México 2 Received 15 December 2012; revised 29 December 2013; accepted for publication 21 August 2014 Structural data were combined with trnL-F and internal transcribed spacer sequences from other studies and with new sequences representing ten additional species to clarify the phylogenetic relationships of Rhus s.s. These data indicate that Rhus s.s and both subgenera, Rhus and Lobadium, are monophyletic. The genus Rhus is supported as monophyletic by the presence of red glandular hairs on the berries and inflorescence axis, cilia on the sepals and glands on the leaf blades. Subgenus Rhus can be identified by the presence of more than seven resin channels in the petiole, weakly percurrent tertiary veins and a type I vascular system in the mid-vein. Subgenus Lobadium is characterized by the presence of short bracteoles and pedicels. This subgenus is divided into four sections, Lobadium, Rhoeidium, Styphonia and Terebinthifolia. Section Lobadium has trifoliate leaves; section Rhoeidium is monotypic, including only Rhus microphylla; section Styphonia is supported by five synapomorphies, including an incomplete marginal vein, fibres in the petiole, a thick cuticle, two layers of palisade parenchyma and prismatic crystals in the mesophyll; and section Terebinthifolia has gelatinous xylary fibres in the petiole. Hypotheses about the evolutionary changes of these characters are presented based on the cladograms. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468. ADDITIONAL KEYWORDS: leaf anatomy – Lobadium – molecular data – Rhoeidium – Styphonia – wood. INTRODUCTION Rhus L. has received widely varying circumscriptions over time, as outlined by Brizicky (1963) and Young (1975). Barkley (1937) recircumscribed the group by segregating many separate genera, and his circumscription of Rhus s.s. is generally followed today. Barkley (1940) further restricted Rhus by segregating a genus he called ‘Schmaltzia’ (an invalid name), but most authors in recent decades have followed Young (1975, 1978, 1979) in maintaining Barkley’s (1937) circumscription of the genus. Barkley (1937) and Young (1975) agreed that Rhus s.s. is morphologically characterized principally by the presence of red berries with red trichomes (Table 1). Barkley (1937) recognized two subgenera, subgenus Sumac (DC.) A.Gray and subgenus Schmaltzia Desv. ex Steud. *Corresponding author. E-mail: tterrazas@ib.unam.mx 452 Subgenus Sumac was characterized by, among other traits, thyrses appearing with or after the leaves, one bract per flower and short-pedicellate flowers. This subgenus was not divided into sections. Subgenus Schmaltzia was characterized by compound spikes appearing with or before the leaves, one bract and two bracteoles per flower, and usually sessile flowers, and was divided into five sections (Table 1). Brizicky (1963) noted that the correct names for Barkley’s subgenera are subgenus Rhus (for Barkley’s subgenus Sumac) and subgenus Lobadium (Raf.) A.Gray (for Barkley’s subgenus Schmaltzia), and all subsequent authors have followed Brizicky on this issue. Young (1975, 1978) added and/or refined data on secondary chemistry, morphology and wood anatomy to support Barkley’s two subgenera and partially amended Barkley’s (1937) sections (Table 1). Young’s modified classification of the genus has been the starting point for most subsequent work. In © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES 453 Table 1. Infrageneric categories of Rhus s.s. by different authors Author Subgenus Sections Barkley (1937) Sumac Schmaltzia Young (1975, 1978, 1979) Rhus (= Sumac) Lobadium (= Schmaltzia) None Lobadium Rhoeidium (= Rhus microphylla) Pseudosumac Styphonia Pseudoschmaltzia None Lobadium (Lobadium + Rhoeidium) Terebinthifolia (= Pseudosumac) Styphonia (Styphonia + Pseudoschmaltzia) subgenus Lobadium, Young recognized only three sections as opposed to Barkley’s five. In Young’s scheme, section Lobadium (Raf.) DC. was expanded to include R. microphylla Engelm. [the sole species of Barkley’s section Rhoeidium (Green) Barkley] and comprised deciduous shrubs or subshrubs with flowers opening before the leaves. The remaining two sections were characterized as evergreen trees or shrubs that flower with the leaves. Section Terebinthifolia D.A.Young shared the circumscription of Barkley’s section ‘Pseudosumac’ (an invalid name) and was defined by its thinner textured, pinnately compound leaves, looser inflorescence and glandular pubescence restricted to the fruits and flowers. Section Styphonia (Nutt.) Barkley was expanded to include Barkley’s section ‘Pseudoschmaltzia’ (another invalid name) and was defined by its more coriaceous leaves that range from simple or unifoliate to pinnately compound, contracted inflorescences and often glandular pubescent leaflets. Young divided this latter section into three subsections (Table 1) (Young, 1975, 1979), adding flavonoid and wood-anatomical data to support his infrageneric classification. The monophyly of Rhus s.s. has been supported recently by molecular-sequence data (Miller, Young & Wen, 2001; Pell, 2004; Yi, Miller & Wen, 2004, 2007; Pell et al., 2008). However, the relationships in the genus are complicated by inconsistencies between plastid and nuclear genes. For example, nuclear data support the monophyly of the two subgenera, but plastid data do not because R. microphylla (Yi et al., 2004) and R. rubifolia Turcz. (Yi et al., 2004, 2007) are nested in subgenus Rhus; these two species were assigned to subgenus Lobadium by Young (1975). Notably, Yi et al. (2004, 2007) recognized subgenera Rhus and Lobadium as monophyletic only if R. microphylla and R. rubifolia were excluded from their com- Subsections – Styphoniae Compositae (= Pseudoschmaltzia) Intermediae bined molecular analyses, and therefore no argument can be made about the status of these species. However, Yi et al. (2007: 32) argued that the varying positions of R. microphylla and R. rubifolia were ‘likely an indication of hybridization between members of subgenus Rhus and subgenus Lobadium’. These authors considered that reticulate evolution played an important role in the phylogeny of Rhus. However, they did not discuss the possibility that poor sampling, especially in subgenus Lobadium, might be a problem. At the section level and using Young’s (1975) circumscriptions, section Lobadium has been supported as monophyletic only if R. microphylla is omitted (Yi et al., 2004). Section Styphonia was not recovered as monophyletic when using plastid DNA; however, combined nuclear and plastid DNA data supported this group as monophyletic, except that R. kearneyi F.A.Barkley appeared to belong in subsection Compositae rather than in subsection Styphoniae (Yi et al., 2007). Subsection Intermediae was not included by these authors (Yi et al., 2004, 2007). Concerning the wood anatomy of Rhus, Heimsch (1940) noted resin canals in R. aromatica Ait. and R. trilobata Nutt., two species in section Lobadium, which have biseriate rays. Young (1974) found resin canals in the rays of R. microphylla and small vessels in a flame-like cluster in the latewood, similar to R. aromatica and R. trilobata, and no resin canals in five species of section Styphonia. Most genera of Anacardiaceae have wood with diffuse porosity. Although Rhus s.s. has ring-porous wood with libriform fibres, vessel elements with alternate pitting and helical thickenings, simple perforation plates, scanty paratracheal parenchyma (except for R. chinensis Mill. with diffuse apotracheal parenchyma and R. standleyi F.A.Barkley with diffuse-in-aggregates) and heteroge- © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 454 A. R. ANDRÉS-HERNÁNDEZ ET AL. neous rays (Young, 1978; Terrazas, 1994; AndrésHernández, 2006), the quantitative characters do not appear to be statistically significant (Terrazas, 1994; Andrés-Hernández, 2006), showing a continuous variation among species of Rhus s.s. Leaf architecture, including venation patterns and foliar and petiolar anatomy, suggests that some traits may be phylogenetically informative (Andrés-Hernández, 2006; Andrés-Hernández & Terrazas, 2006, 2009), but these characters have not been incorporated in recent phylogenetic analyses. Despite the advances in the taxonomy of Rhus s.s., no study has tested the morphological characters traditionally used for the infrageneric classification in this group, and no study has sampled most of the species of this genus. Therefore, the present study includes 31 species, ten more than previous studies have sampled (Miller et al., 2001; Yi et al., 2007), and analysed such important taxonomic characters as the leaves, inflorescences, flowers and fruits. We also include new morpho-anatomical characters for species of Rhus s.s. and test the congruence of the structural characters with molecular phylogenetic trees generated by us and other authors. MATERIAL AND METHODS TAXON SAMPLING Thirty-one species of Rhus s.s. were included in this study (Appendix 1). Seven species representing other genera of Anacardiaceae that have previously been included in Rhus s.l. [Actinocheita filicina (D.C.) Barkley, Malosma laurina (Nutt) Abrams, Searsia ciliata (Licht. ex Schult.) A.J.Mill., S. quartiniana (A.Rich.) A.J.Mill., Toxicodendron diversilobum (Torr. & A.Gray) Greene, T. radicans Kuntze, T. vernix Kuntze] were included to test the monophyly of Rhus s.s., and Schinus molle L. was used as the functional outgroup to root the tree because this genus has never been included in Rhus s.l. Sequences of trnL-F, ndhF, Nia-i3, trnC-D and internal transcribed spacer (ITS) were obtained from GenBank (Appendix 1) and trnL-F and ITS sequences were generated for R. allophylloides Standl., R. andrieuxii Engl., R. barclayi Standl., R. chondroloma Standl., R. hartmanii F.A.Barkley, R. muelleri F.A.Barkley, R. oaxacana Loes., R. schmidelioides Schtdl. (only trnL-F), R. standleyi and R. terebinthifolia Schltdl. & Cham. (Appendix 1). STRUCTURAL CHARACTERS We examined the collections of Rhus s.s. and related taxa in the following herbaria: ANSM, ARIZ, DUKE, GH, IBUG, IEB, MEXU, NCU, NY, TEX and US. Leaves (blade and petiole) were removed for leaf architectural and anatomical studies. The methods used for these studies are described in detail elsewhere (Andrés-Hernández & Terrazas, 2006, 2009). Wood samples (one to four mature-stem samples per species) were provided by CAFw, FHOw, MADw, RSA, SJRw, TWTw and USw or were personally collected in Mexico and deposited at MEXUw (Andrés-Hernández, 2006). Wood characters are commonly diagnostic at the generic level (Carlquist, 2001); therefore, few characters provided information at the hierarchical level of interest (in subgenera). Forty structural characters (25 morphological and 15 anatomical) were coded as primary homology hypotheses (De Pinna, 1991). Sixteen characters had multiple states, and all characters were treated as unordered and equally weighted (Fitch parsimony; Fitch, 1971). The coded structural-data matrix of Rhus s.s. and related taxa is given in Appendix 2 and Table 2. This partition was analysed independently and in combination with molecular data to allow any secondary signal to emerge (Nixon & Carpenter, 1996). DNA EXTRACTION, AMPLIFICATION AND SEQUENCING Total DNA was extracted from silica gel-dried or herbarium leaves with a modification of the 2× CTAB procedure of Doyle & Doyle (1987). DNA was cleaned directly with QIAquick silica columns (Qiagen) or precipitated with 100% ethanol at −20 °C and purified on a caesium chloride/ethidium bromide density gradient (1.55 g mL−1) with subsequent dialysis and removal of ethidium bromide with butanol. All DNA regions were amplified in 100 μL PCR reactions including 0.5 μL 5 u μL–1 Taq DNApolymerase (Promega), 10 μL 10× Mg-free DNA polymerase buffer (Promega), 12 μL 25 mmol L−1 MgCl2, 2 μL 10 mmol L−1 each dNTP, 1 μL 0.4% bovine serum albumin (BSA), 1 μL each primer (100 ng μL–1), 72.5 μL double-distilled H2O (ddH2O) and template DNA. Alternatively, 50-μL reactions were prepared using 45 μL 1.1× PCR Master Mix (Advanced Biotechnologies), including 1.25 μL Taq DNA polymerase, 75 mmol Tris-HCl (pH 8.8 at 25 °C), 20 mmol [NH4]2SO4, 1.5 (for ITS) or 2.5 mmol (for plastid DNA) MgCl2, 0.01% Tween 20 and 0.2 mmol each dNTP, to which were added 0.5 μL each primer (100 ng μL–1), 0.5 μL 0.4% BSA, 2 μL ddH2O and template DNA. The PCR mix used to amplify the ITS region included 2% dimethyl sulphoxide to reduce problems related to secondary structure and efficiency of PCR primer binding. The trnL-F region, including the trnL intron and the intergenic spacer, was amplified either as a single piece with primers c and f or as two non-overlapping fragments using primers c-d and e-f (all from Taberlet et al., 1991). The PCR profile consisted of an initial 2-min premelt at 94 °C; 28–30 cycles of 1 min © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES 455 Table 2. Matrix of morphological characters Characters Taxon 1. . . .5 6. . . .10 Actinocheita filicina 00100 11010 Malosma laurina 2–201 03020 Searsia ciliata 1-210 0T0?? Searsia quartiniana 1-012 02000 Schinus molle 01020 02002 Toxicodendron diversilobum 10002 10000 Toxicodendron radicans 1–002 10000 Toxicodendron vernix 00012 02000 Rhus subgenus Rhus Rhus chinensis 0??00 11001 Rhus copallina 01100 02101 Rhus coriaria 0?000 11001 Rhus glabra 00100 11011 Rhus lanceolata 01120 12001 Rhus michauxii 01101 11011 Rhus sandwicens 01100 11001 Rhus typhina 01100 11001 Rhus subgenus Lobadium (Section Lobadium) Rhus allophylloides 1-110 11010 Rhus aromatica 1-000 11000 Rhus schmidelioides 1-110 11000 Rhus trilobata 1-100 11000 Rhus microphylla 01110 0?110 (Section Terebinthifolia) Rhus barclayi 00002 02000 Rhus hartmanii 00110 02000 Rhus jaliscana 00111 02000 Rhus palmeri 00112 02010 Rhus rubifolia 00111 02000 Rhus terebinthifolia 00110 02100 (Section Styphonia) Rhus andrieuxii 00210 03002 Rhus chondroloma 00200 03022 Rhus choriophylla 00210 03022 Rhus integrifolia 2-201 03022 Rhus kearneyi 2-201 03022 Rhus muelleri 2-211 03012 Rhus oaxacana 00201 03022 Rhus ovata 2-211 03022 Rhus pachyrrhachis 00200 03122 Rhus schiedeana 00201 03122 Rhus standleyi 2-211 0T012 Rhus virens 00200 03102 11. . . .15 16. . . .20 21. . . .25 26. . . .30 31. . . .35 36. . . .40 00101 00001 ???0? 00000 01001 0010? 00101 00100 01101 02?10 ?1?01 01?01 00001 ????1 01001 0???1 00000 -0000 ?0000 ?0000 00000 ?0000 00000 ?0000 101?? 100?? ????? ????? 0000? 200?? 2100? 200?? ?0000 ?0011 ????? ????? 00000 ??0?0 ?0000 ??0?0 1000? 10113 ????? ????? 0000? ??0?? 0000? ??0?? ?0110 00110 00111 00010 00110 01110 00110 01110 01001 01001 0???1 01001 01001 01001 01001 01001 ?1101 01101 ??101 01101 01101 01101 01101 01101 201?? 20110 20110 20110 2?110 2?110 2?110 20110 ????? 00100 00100 00100 00100 00100 00100 00100 ????? 10000 10000 10000 10000 10000 10000 10000 00110 00111 00110 01111 00111 02211 02211 02211 02211 02211 11111 11111 11111 11111 11111 20100 20100 2?100 20100 20100 00100 00100 00100 00100 00100 10002 10002 10002 10002 10002 00111 00111 00111 00111 00111 00111 01100 01100 01100 01100 01100 01100 -1111 -1111 -1111 -1111 -1111 -1111 20100 20100 20100 2?100 2?100 20100 10010 10010 10010 10010 10010 10010 10002 10002 10002 10002 10002 10002 11112 10112 11112 11112 11112 10112 21112 10112 10112 11112 10112 11112 11000 12110 11000 02110 02110 02110 01000 02110 11000 11000 02110 11000 -1111 -1111 -1111 -1111 -1111 -1111 -1111 -1111 -1111 -1111 -1111 -1111 20101 20101 2?101 21101 20101 20101 20101 20101 2?101 20101 20101 20101 01011 01011 01?11 01012 01012 01012 01011 01013 01011 01011 ?1012 01011 01111 01111 01111 11113 11113 11113 01111 11113 01111 01111 11113 01111 denaturation at 94 °C, 30 s annealing at 48 °C and 1 min extension at 72 °C, and a final extension of 7 min at 72 °C. The entire ITS region was amplified using primers ITS4 and ITS5 (White et al., 1990) and in some cases using primers 17SE and 26SE (Sun et al., 1994). The PCR profile for ITS5–ITS4 included an initial 2-min premelt at 94 °C; 28–30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 52 °C and 2 min extension at 72 °C; and a final extension of 7 min at 72 °C. The PCR profile for 17SE–26SE differed only in using a lower annealing temperature of 50 °C. The PCR products were cleaned with QIAquick or CONCERT (Life Technologies) silica columns accord- © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 456 A. R. ANDRÉS-HERNÁNDEZ ET AL. ing to the manufacturers’ protocols and used in cycle sequencing reactions with the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (Applied Biosystems). The 10-μL cycle-sequencing reactions included 1 μL terminator mix, 3 μL 2.5× cycle-sequencing buffer (200 mmol L−1 trizma base, 5 mmol L−1 MgCl2, pH 9.0), 1 μL primer (5 ng μL–1) and 3–5 μL PCR product plus ddH2O as required. The cycle-sequencing products were cleaned by precipitation in 25 μL 100% ethanol with 1 μL 3 mol L−1 NaOAc (pH 4.6) on ice for 30 min, after which they were centrifuged at 12289 g for 25 min. The alcohol/ salt mix was discarded, and the pellet was subjected to two washes with 300 μL 70% ethanol, each followed by centrifugation at 13 000 r.p.m. for 15 min. The cleaned cycle-sequencing products were allowed to dry overnight at room temperature or oven-dried at 65–70 °C for 15 min and were then protected from light until they were analysed. The forward and reverse sequences were analysed on a PE 377 automated sequencer (Applied Biosystems), and the resulting electropherograms were edited and assembled with Sequencher versions 3.1 or 4.1 (Gene Codes). SEQUENCE ALIGNMENT AND INDEL CODING The 3′ portion of the trnL-F and ITS regions were aligned with Clustal W (Thompson, Higgins & Gibson, 1994) and visually adjusted as necessary, following the guidelines of Kelchner (2000). All nonautapomorphic indels were coded as binary (presence/ absence) characters using the simple indel-coding method used by Simmons & Ochoterena (2000) and appended to the sequence matrices. PHYLOGENETIC ANALYSES Parsimony analyses of three datasets (molecular data, including trnL-F, ndhF, Nia-i3, trnC-D and ITS; structural data; and combined data) were conducted separately with PAUP* version 4.0b10 (Swofford, 2002). All analyses consisted of 1000 replicates of random sequence addition with tree bisection-reconnection (TBR) branch swapping and the MULTREES option, saving all most- parsimonious trees. Individual gap positions were treated as missing data because the indel characters were appended to the molecular matrices. Internal clade support was evaluated using bootstrap resampling (Felsenstein, 1985), with 300 replicates using TBR branch swapping and saving up to 20 trees per replicate to reduce time spent swapping on large islands. Clade support in the combined analyses was evaluated by bootstrap and jackknife resampling (Farris et al., 1996), with 300 bootstrap and jackknife replicates using the searching strategy described above. To assess the level of congruence between the data sets, we employed the incongruence length difference (ILD) test or the partition-homogeneity test (Farris et al., 1995), implemented in PAUP*. One hundred heuristic-search replicates were performed, with all characters equally weighted and TBR branch swapping. RESULTS MOLECULAR ANALYSES The maximum-parsimony analysis of the sequence data yielded 4113 most-parsimonious trees with a length of 1328 steps [consistency index (CI) 0.79; retention index (RI) 0.80; homoplasy index (HI) 0.34, excluding uninformative characters]. The percentage of potentially informative characters was 6.8% (Table 3). Although 30% of the data were missing for three markers (ndhF, Nia i3 and trnC-D), the topology was well supported. The bootstrap analysis recovered Rhus s.s. as monophyletic with maximum (100%) bootstrap support (BP). Both subgenera were monophyletic with high support: 96% BP for subgenus Lobadium and 98% BP for subgenus Rhus. In subgenus Lobadium, two clades were also recovered, here named A and B. Moreover, in clade B, three lineages were recovered: R. microphylla was sister to subclade B1, and these lineages together were sister to subclade B2 (Fig. 1A). Clade A received moderate support, but the relationships in this clade were poorly resolved (Fig. 1A), except for the sister-taxa relationships of R. integrifolia Engl.–R. ovata S.Watson and R. muelleri–R. standleyi. Table 3. Character diagnostics and trees resulting from the analysis of the molecular, morphological and global datasets Data set No. of characters Potentially parsimonyinformative characters No. of optimal trees No. of steps CI RI HI Molecular (trnL-F, ndhF, Nia-i3, trnC-D, ITS) Structural Combined 6973 40 7013 448 40 488 4113 5380 56 1328 125 1476 0.79 0.45 0.75 0.80 0.84 0.80 0.34 0.54 0.25 © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES 457 Figure 1. Strict consensus of trees resulting from the phylogenetic analyses. A, cladogram derived from the combined molecular markers, trnL-F, ndhF, Nia-i3, trnC-D and ITS (4113 trees, 1328 steps; CI = 0.79, RI = 0.80; HI = 0.34). B, cladogram derived from the structural characters (5380 trees, 125 steps, CI = 0.45, RI = 0.84, HI = 0.54). Bootstrap support values (≥ 50%) are indicated above branches. Colours correspond to subgenera or sections in Figure 2. STRUCTURAL ANALYSIS A parsimony analysis based on 40 informative structural characters (Table 3) yielded 5380 trees of 125 steps. The bootstrap analysis (Fig. 1B) retrieved Rhus s.s. as monophyletic with strong support (99% BP), only if Malosma laurina is part of it. The characters supporting the monophyly of Rhus were red glandular hairs on the berries and inflorescence axis, cilia on the sepals, glands on the leaf blades and a striate cuticle on the lamina. These characters were absent in M. laurina. Both subgenera were recovered as monophyletic, with moderate support, 75% BP for subgenus Rhus and 72% BP for subgenus Lobadium. In subgenus Lobadium, clade A had the strongest support values, with M. laurina as its sister taxon and R. microphylla as sister to both plus subclade B1. However, clade B2 was not recovered; only R. allophylloides and R. schmidelioides were resolved as sister taxa. Several leaf character states are similar between M. laurina and members of clade A. COMBINED ANALYSIS The 40 morphological characters plus the combined molecular data contained a total of 488 potentially informative characters. The total-evidence approach yielded 56 minimum-length trees of 1476 steps (CI 0.75; RI = 0.80; HI = 0.25; 6.9% informative characters, Table 3) with a topology similar to that recovered in the analysis of the molecular data alone. In the strict-consensus tree, Rhus s.s. was recovered with maximum support (100% BP), including 31 species (Fig. 2), and its structural synapomorphies were the same as those found in the structural analysis, except for the striate cuticle on the leaf blade. Both subgenera were also recovered with high support. The © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 458 A. R. ANDRÉS-HERNÁNDEZ ET AL. Figure 2. Strict consensus of the simultaneous analysis of structural and molecular characters (56 trees, 1476 steps, CI = 0.75, RI = 0.8, HI = 0.25). Bootstrap/jackknife support values (≥ 50%) are indicated above branches. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES 459 relationships in subgenus Lobadium were similar to those found in the analysis of the sequence data alone but with stronger support (Fig. 2). indumenta on the fruit surface, but these hairs differ from the glandular hairs of Rhus s.s. DISCUSSION Subgenus Rhus is monophyletic and is supported by several molecular and three structural synapomorphies: the occurrence of weakly percurrent tertiary veins (10), more than seven resin canals in the petiole (29) and type I vascular bundles in the midvein (40) (Fig. 2). These synapomorphies are conserved in the subgenus, which has a wider geographical distribution than subgenus Lobadium. Barkley (1937) proposed imparipinnate leaves and thyrses with pedicellate flowers as the diagnostic features for this subgenus, but these characters were not revealed as evolutionary novelties in our analyses. The sistertaxa relationship of R. copallinum L. and R. lanceolata A.Gray ex Engl. is supported by their eucamptodromous venation; other members of this clade have craspedodromous venation (7). Moreover, the R. michauxii Sarg.–R. typhina L.–R. glabra L. clade is supported by the presence of incomplete areoles (12), with a transformation to imperfect areoles in R. glabra. We found other informative structural characters to support the subgroups in subgenus Rhus. The sister-group relationship of R. chinensis Mill. and R. sandwicensis A.Gray was supported exclusively by molecular characters. Some authors have suggested that these species are distinguished by seed size; however, we did not include this character due to the lack of seed-size information for most Rhus species. These species are geographically isolated, one in Eurasia and the other in Hawaii (Yi et al., 2004). RHUS Our supermatrix analysis not only supports previous findings about the circumscription of Rhus but also provides strong support for the subgenera and sections, delimitation of which has previously been uncertain. Both partitions (molecular and structural) supported the major lineages in Rhus. Therefore, combining the data did not introduce conflict but provided higher support and more resolution. Although the precise species relationships differed among analyses, the backbone relationships were supported by all partitions. Our analyses suggest that the five-sectional classification proposed by Barkley (1937) for subgenus Lobadium (as the incorrectly named Schmaltzia) is partly correct. Based on our results, we recognize Barkley’s sections Lobadium, Rhoeidium and Terebinthifolia (incorrectly named Pseudosumac by Barkley) and Young’s section Styphonia (Barkley’s Styphonia + Pseudoschmaltzia). Although our supermatrix had missing data for three genes (30%), the simultaneous inclusion of all information (additional taxa, previously available DNA sequences and structural characters) was critical in resolving the conflicting topologies generated in previous studies (Yi et al., 2004, 2007) especially within the genus, and revealed the phylogenetic signal of the microstructural characters. The simultaneous analysis presented here reinforces the utility of supermatrices with missing data (Pirie et al., 2008; Chatrou et al., 2012) and the value of morphological characters (Donoghue & Sanderson, 1992; Wiens, 2004). RHUS S.S. Total-evidence analysis yielded the strongest support to the monophyly of Rhus s.s. Our results confirm that the presence of red glandular hairs on the fruit surface (25) is a synapomorphy for this genus, as suggested by Barkley (1937). The occurrence of glands on the foliar blade (14), ciliate sepals (22) and red glandular hairs on the inflorescence axis and branches (23) are also synapomorphies of Rhus s.s. Gillis (1971) and Moffett (1993) considered Toxicodendron Mill. to be part of Rhus; however, our combined analysis does not support this circumscription. Moreover, Toxicodendron is recognized by its glabrous fruits and the occurrence of toxic resins (Aguilar-Ortigoza, Sosa & Aguilar-Ortigoza, 2003). Other members of Rhus s.l. such as Actinocheita F.A.Barkley and Malosma (Nutt.) Abrams show pilose RHUS SUBGENUS SUBGENUS RHUS LOBADIUM Subgenus Lobadium was recovered as monophyletic and supported by two structural synapomorphies: short (< 1 mm) pedicels (21) and the presence of bracteoles (24) (Fig. 2). Barkley (1937) suggested the presence of bracteoles as diagnostic features for this subgenus, and our results support this hypothesis. However, the remaining diagnostic features suggested by Barkley (1937) (inflorescence in spikes and simple, trifoliolate or imparipinnate leaves) were not found to be synapomorphies. Subgenus Lobadium encompasses the largest number of species, most of which are distributed in the Mexican Transition Zone (Andrés-Hernández et al., 2006). Our results suggest that speciation associated with several anatomical and morphological features has occurred in different eco-regions: sections Lobadium, Rhoeidium and Terebinthifolia in temperate warm environments and section Styphonia in dry environments. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 460 A. R. ANDRÉS-HERNÁNDEZ ET AL. The combined analysis supports the recognition of four previously proposed sections (Barkley, 1937; Young, 1975, 1978), as discussed below. CLADE B The clade here named Clade B encompasses all recognized species of the previous sections Lobadium (B2), Rhoeidium and Terebinthifolia (B1) and shares type III vascular bundles (40) in the midrib (Fig. 2). Subclade B2 is monophyletic and corresponds to Barkley’s section Lobadium, with the combination of trifoliolate leaves (1) with serrate margins (6) being unique in the genus Rhus. This subclade does not correspond to Young’s (1975) Lobadium, which also included R. microphylla (see below). The trifoliolate leaf character is acquired independently in Searsia F.A.Barkley and Toxicodendron, whereas the serrate margin represents a reversal. Moreover, serrate margins are common in species inhabiting temperate forests (Baker-Brosh & Peet, 1997), like most species of subgenus Rhus with imparipinnate leaves. The species of subclade B1, section Terebinthifolia, share the unique combination of the following characters: three-branch inflorescences (17); a distance of 3–5 mm from the base of the second branch to the flower (18); a distance of 3–5 mm between flowers (19); the absence of a pedicel (20); a smooth cuticle on the leaf lamina (33); a papillose foliar epidermis (34); and the presence of gelatinous xylary fibres in the petiole (31) (Fig. 2). These characters were acquired independently in this clade and are not combined in other clades in Rhus. Barkley (1937) characterized this section as containing evergreen trees or shrubs with three to 15 leaflets and inflorescences in lax spikes. None of these characters was recovered in our analysis, although the inflorescences were represented by four informative characters rather than simple, lax spikes (see below). STATUS OF R. MICROPHYLLA Our results support Barkley’s (1937) recognition of the monotypic Rhoeidium (R. microphylla) as separate from section Lobadium. Young (1975, 1978) placed R. microphylla in section Lobadium based on its chemical characters and the occurrence of radial canals in its wood. However, similar resin canals are present in the wood of R. integrifolia of clade A, section Styphonia and R. aromatica and R. trilobata of clade B2, section Lobadium. Our molecular, morphological and combined analyses all place R. microphylla as sister to section Terebinthifolia. These lineages share eucamptodromous venation. Yi et al. (2004, 2007) suggested that R. microphylla is a hybrid between R. copallinum or R. lanceolata (subgenus Rhus) and R. rubifolia (subgenus Lobadium, section Terebinthifolia), but our results do not support this hypothesis. Conflicting sister-taxa relationships are common in phylogenetic analyses of genes such as ITS, trnL-F and rbcL (Bradford & Barnes, 2001; Arias, Terrazas & Cameron, 2003; Muellner et al., 2003). We suggest that poor sampling in sections Lobadium and Terebinthifolia resulted in misleading signals for R. microphylla and R. rubifolia in the analyses of Yi et al. (2004, 2007). CLADE A As traditionally defined by Young (1978), members of section Styphonia have evergreen, coriaceous and simple or compound leaves. Our results provide strong support for this clade, which exhibits several evolutionary novelties, including incomplete marginal vein (11), xylary fibres in the petiole (30), a thick foliar cuticle (> 6 μm) (32), two equal rows of palisadeparenchyma cells (35) and prismatic crystals in the mesophyll (37). In addition to these synapomorphies, the coriaceous leaves noted by Barkley (1937) and Young (1978) constitute a unique character combination for Styphonia. Other characters such as cladodromous venation (7), transversely ramified tertiary veins (10), type II veinlets (15), a papillose epidermis on the midvein (38) and xylary fibres in the midvein (39) are homoplasious; these traits were acquired independently in other genera, such as Malosma, Searsia and Toxicodendron, and in members of other tribes of Anacardiaceae (Smith & Stern, 1962; Wilkinson, 1983; Dickison, 1989; Buijsen, 1995; Fariña et al., 2003; Martínez-Millán & CevallosFerriz, 2005). Styphonia is well supported by anatomical characters that represent adaptations to xeric environments. Young’s (1975, 1978) subsections Compositae, Intermediae and Styphoniae were not recovered in molecular studies (Yi et al., 2004, 2007) and are not supported by our combined analysis, even though all recognized species have been included here. However, R. ovata and R. integrifolia (Nutt.) Engl. are sister taxa (100% BP), sharing wood with septate fibres, and R. muelleri and R. standleyi (97% BP) are sister taxa, sharing sinuous secondary veins. Moreover, the five species previously classified in Styphoniae are the earliest-diverging species in clade A and have a disjunct distribution pattern. Rhus kearneyi F.A.Barkley occurs with R. ovata and R. integrifolia in xerophytic scrublands in California and Baja California provinces, whereas R. standleyi occurs in the eastern Trans-Mexican Volcanic Belt and the Sierra Madre del Sur; R. muelleri occurs in the Sierra Madre Oriental and on the Mexican Plateau in xerophytic scrub or xerophytic scrub/temperate forest ecotones © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES (Andrés-Hernández et al., 2006). Based on this distribution pattern, we hypothesize that the foliar features shared by these five species and the other members of section Styphonia were acquired after subgenus Lobadium diverged from subgenus Rhus > 30 million years ago, as suggested by Yi et al. (2004). PLANT-ORGAN EVOLUTION Leaves Eucamptodromous venation is common in Anacardiaceae (Hickey & Wolfe, 1975; Martínez-Millán & Cevallos-Ferriz, 2005). Craspedodromous and eucamptodromous venation occur in both subgenera, while cladodromous venation is unique to section Styphonia. The tertiary veins show a randomly reticulate condition in Rhus, changing to weakly percurrent in subgenus Rhus and transversely ramified in section Styphonia. Both transitions represent evolutionary novelties. Type I idioblast veinlets appear several times in Rhus s.s. However, type III idioblast veinlets occur only in species of Styphonia. One layer of palisade-parenchyma cells is the plesiomorphic condition in Rhus, and two or three layers is the derived condition. The larger number of palisade-parenchyma cells is a unique feature of Styphonia, although two layers appear independently in Malosma laurina. The changes associated with the number of palisade-parenchyma layers, such as the cuticle thickness and the lignification of the midvein and petiole (Andrés-Hernández & Terrazas, 2006), may explain the maintenance of a unique type of wood porosity. Stem Semi-ring wood porosity is the plesiomorphic condition present in Schinus L.; ring porosity and diffuse porosity are derived conditions. Ring porosity occurs in all species of Rhus s.s. and Toxicodendron, whereas diffuse porosity appears in Malosma laurina and Actinocheita filicina. Ring porosity is present in all species of Rhus s.s. independent of their evergreen or deciduous character, even though these species are widely distributed in northern Eurasia, the United States and southward into the drier and warmer regions of southern Mexico. In the eudicotyledons with wide latitudinal distributions, wood porosity typically varies from ring to diffuse-porous as latitude decreases (Noshiro & Baas, 1998; Aguilar-Rodríguez, Terrazas & López-Mata, 2006). Contrary to expectation, the retention of ring porosity across the latitudinal range of Rhus does not affect conductivity due to the acquisition of several leaf-structural evolutionary novelties that favour evergreen leaves in the warmer 461 and more xeric environments of the Mexican Sierras (Andrés-Hernández et al., 2006). Inflorescences The inflorescences of Anacardiaceae are described as thyrses and panicles. Thyrses are a distinctive trait of tribe Rhoeae, except for Toxicodendron species, which have panicles (Barfod, 1988). In Rhus s.s., Barkley (1937) recognized thyrses in Rhus subgenus Rhus and compound spikes in Rhus subgenus Lobadium. However, the diversity of the spikes in subgenus Lobadium is not adequately represented by these typological terms. Therefore, we coded the inflorescences using five characters (17–21). Rhus s.s. shows a reduction in the number of inflorescence branches. Subgenus Lobadium shows a reduction in the flower position along the second branch (18) and in the distance between flowers (19), whereas sections Terebinthifolia and Styphonia independently acquired the absence of pedicels. CONCLUSIONS This study demonstrates that more complete taxon sampling can resolve the conflicting phylogenetic relationships of certain taxa. In addition, wellunderstood morphological characters can be consistent with molecular phylogenetic analyses and can add support and resolution when combined with molecular data. The potential ‘noise’ of morphological characters due to their supposed high levels of homoplasy is not a problem when the appropriate phylogenetic levels are addressed. Rhus s.s. contains two well-supported subgenera: Lobadium and Rhus. In subgenus Lobadium, four sections are recognized: Lobadium, Styphonia, Terebinthifolia and the resurrected Rhoeidium. A key to these subgeneric categories is given below. Adaptive leaf features have buffered wood evolution in Rhus s.s., all species of which have ring-porous wood. Thus, xerophytic leaf characters evolved once in the Styphonia lineage, originating in the drier environments of the Mexican provinces of California and Baja California and the scrubs of the Trans-Mexican Volcanic Belt, Sierra Madre del Sur, Sierra Madre Occidental and the Mexican Plateau. ACKNOWLEDGEMENTS We are grateful to the curators of ANSM, ARIZ, DUKE, GH, IBUG, IEB, MEXU, NCU, NY, TEX and US for allowing us to remove material for this study. The first author thanks the Consejo Nacional de Ciencia y Tecnología (National Council for Science and Technology) for providing a scholarship (169599) to support her doctoral studies at the Colegio de © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 462 A. R. ANDRÉS-HERNÁNDEZ ET AL. KEY FOR RHUS S.S. AND INFRAGENERIC CATEGORIES A. Species lacking glandular hairs on inflorescences axes and fruits; sepals with entire margin ........................ .......................................................................................................... Toxicodendron, Actinocheita AA. Species with glandular hairs on inflorescences axes and fruits; sepals with ciliate margin ................... Rhus B. More than seven resin canals in the petiole, weakly percurrent tertiary veins, pedicel 1.5–2.5 mm, lacking bracteoles ........................................................................................................ Subgenus Rhus BB. Less than seven resin canals in the petiole, reticulate or transversely ramified tertiary veins, pedicel < 1 mm, bracteolate ............................................................................................... Subgenus Lobadium C. Leaflets chartaceous with serrate margins; type III vascular bundles in the midrib; cuticle < 6 μm; prismatic crystals lacking in mesophyll ...................................................... Section Lobadium D. Rachis winged; fibres lacking in the petiole ......................................... Section Rhoeidium DD. Rachis non-winged; gelatinous fibres in the petiole ......................... Section Terebinthifolia CC. Leaflets coriaceous with entire margin; type IV vascular bundles in the midrib; cuticle > 6 μm; prismatic crystals present in the mesophyll .............................................................. Section Styphonia Postgraduados. We thank Tom Wendt for his assistance during herbarium work at TEX; the Molecular Systematic Laboratory of the Instituto de Biología (Institute of Biology), UNAM, for use of the sequencing facilities; Julio César Montero-Rojas for helping to prepare the figures; and two anonymous reviewers and the associate editor for their valuable comments on earlier versions of the manuscript. REFERENCES Aguilar-Ortigoza CJ, Sosa V, Aguilar-Ortigoza M. 2003. Toxic phenols in various Anacardiaceae species. Economic Botany 57: 354–364. Aguilar-Rodríguez S, Terrazas T, López-Mata L. 2006. Anatomical wood variation of Buddleja cordata (Buddlejaceae) along its natural range in Mexico. Trees 20: 253–261. Andrés-Hernández AR. 2006. 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Young DA. 1975. Systematics of Rhus subgenus Lobadium section Styphonia. PhD thesis, Claremont: Claremont Graduate School. Young DA. 1978. Reevaluation of the sections of Rhus L. subgenus Lobadium (Raf.) T. & G. (Anacardiaceae). Brittonia 30: 411–415. Young DA. 1979. Heartwood flavonoids and the infrageneric relationships of Rhus (Anacardiaceae). American Journal of Botany 66: 502–510. APPENDIX 1 Information for the samples of species of Rhus s.s. Voucher information is listed as follows: taxon, name, country, locality, collector name and number, (herbarium). For GenBank vouchers the accession numbers are given in parentheses for (ITS, trnL-F, ndh-F, Nia-i3, trnC-D). Abbreviations of herbaria according to Holmgren et al. (1981). *Blades that were cleared, **blades and petiole for anatomical study. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 464 A. R. ANDRÉS-HERNÁNDEZ ET AL. Rhus subgenus Rhus: R. copallina L. USA Southeast of Bamberg Dorchester, C. Ahles 31816*; Jackson Co. Illinois, L. Bastian s.n.*; Warren Co., W. Seaman 2944* (NCU); Sondy Bottom, Texas, F. Barkley 13597; Freestone, Texas, L. Do 226; Gregg Co., W. Holmes 5454; Henderson Co., A. Lundell 9565; Hardin Co., A. Lundell 11542; South of Jasper, C. Lundell 11818; Anderson Co., E. Morsh Jr. 242; Todville Road, F. Waller et al. 2806** (TEX); Winter State Park, R. Bochinski s.n**(US); Wake Co. N. Carolina, R. Currie 884; Hopkins Co. Texas, B. Jenning 31 (IBUG). Wen 7134, 7165, (AY641483, AY640437, AY643098, DQ382288, DQ400539). R. chinensis Mill. Wen 6389, 7086, 7310 (AY641480, AY640435, AY643095, DQ382286, DQ 400536). R. coriaria L. AFGHANISTAN Kabul, K. Rechinger 31176 (US). ESPAÑA Toledo, F. Meyer s.n. ISRAEL Jerusalem, D. Goldman 1080**, Amdursky 550* (NY). Wen 7150 (AY641485, AY640439, AY643099, DQ382291, DQ400540). R. glabra L. MEXICO. Chihuahua: Mpio. Madera, A. Benítez 1417* (ANSM); Mpio. Madera, S. Rodríguez 1511* (IEB). USA. Maple Creek Cleveland Co., H. Ahles 15244*; Stanly Co., J. Morgan 1551* (NCU); Texas: Hemphill Co., Chester & M. Rowell 4175**, 10255b; D. Correll & H. Correll 30047; Texas Co., H. Gentry 306; Palo Pinto Co., R. McVaugh 8347 (TEX). Wen 7171 (AY641486, AY640440, AY643100, DQ382293, DQ400541). R. lanceolata A.Gray ex Engl. MEXICO. Coahuila: Múzquiz-Boquillas, J. Villareal 6944* (IEB); Sierra la Babia, M. Carranza C-3061; Rancho los potros, D. Castillo 512; Rancho las pilas Múzquiz, Vázquez-Aldape s.n.*; La Babia, J. Villareal et al. 8876*(ANSM); Nuevo León: Mpio. Iturbide, Hinton et al. 21234; San Luís Potosí: Ciudad del maíz, F. Barkley 3470; Tamaulipas: La Purificación, F. González 17227 (ANSM); Puebla: Pahuatlán, F. Salazar s.n.* (US). U.S.A Texas: Guadalupe Mountains, A. Chose 5961; Real Co., D. Correll 38069**; Corner Co., L. Hinckley s.n.; Davis Mountains, B. Wornock 9303, 10967 (TEX). Wen 7277 (AY641487, AY640441, AY643101, DQ382299, DQ400544). R. michauxii Sarg. USA north of Efland Orange Co., H. Ahles 58816**(NCU); Florence Co., S. Carolina, H. Bartlett 2881; Hoke Co. N. Carolina, W. Fox & R. Godfrey 4230 (TEX). Hardin 13984 (AY641488, AY640442, AY643102, DQ382307, DQ400545). R. sandwincensis A.Gray. USA Hawaii: Maunakia, A. Hitchcook 14300* (NCU); Volcanoes Nacional Park, S. Darwin 1201**; Ola Puma, O. Deneger s.n., 9527**; Waichn Valley, H. Mann & W. Brigham 412; Galch Mahaeli, J. Rock 5837 (TEX); P. Palmer 30024 (NY). Wen 7052 (AY641491, AY640445, AY643105, DQ382316, DQ400553). R. typhina L. CANADA Quebec, S. Blake 1984 (TEX). USA. McDowell Co., H. Ahles 17709**; West of Tapoco, Gram. Co., A. Radford 16002**; Nilkes Co., H. Tottens s.n.* (NCU); Rolling Hills, G. Baker 555**; Stoughton N. Cork, S. Blake 10930; Franklin Co., Vermont, S. Blake 3146; Middlesex Co. Massachusetts, S. Blake 4234; Ashland Co. Ohio, J. Stevenson 8640; Sussex Co. New Jersey, H. Maldenke 25982 (TEX). Wen 7082 (AY641492, AY640446, AY643106, DQ382319, DQ400556). Rhus subgenus Lobadium section Lobadium: R. allophylloides Standl. MEXICO. Jalisco: Mpio. Mezquitic, C. Chávez 4999; Mazamitla, J. Machuca 7726; Los Espinos, Tapalpa, M. Macías & B. Arbayo 633; La Manzanilla, Mazamitla, R. Ornelas 1378; La Joyas, Autlán, A. Vázquez 3773; R. Vega 2509* (IBUG); Las Minas Zimapán, J. Cházaro et al. 911; Sierra de Manantlán, R. Guzmán 29089 (IEB); Real Alto, Hinton et al. 2317* (US); State of Mexico: Mina de Agua, Temascaltepec, Hinton et al. 2817**(US). Rzedowski 45608 (HE862264,HE862254---). R. aromatica Ait. CANADA Ontario: Manitoulin Strait, D. Soper 8950* (NCU). MEXICO Chihuahua: Casas Grandes, GómezDurán s.n**; Nuevo León: Cerro el Viejo Aramberri, Hinton et al. 25181 (IEB); Aramberri, Hinton et al. 22643, 24072 (ANSM). USA Lancaster County, H. Ahles 27466*; Riley C. Kansas, W. Baker 2565*; Bennington C., Vermont, E. Baufford 17799; Missouri, T. Croat 17119*; Pennsylvania, H. Wahlx 17406* (NCU); Texas Co., D. Corell 16214; Morion Co., D. Corell & C. Lundell 18796; Wood Co., C. Lundell & A. Lundell 9474; Red River, M. Nee 44041**; Freestone Co., B. Thorp 2908 (TEX). Wen 7086 (AY641496, AY640447, AY643107, DQ382285, DQ400535). R. microphylla Engelm. MEXICO. Coahuila: Rancho Morteros, Múzquiz, M. Carranza et al. 1341; Ramos Arizpe, J. Encina 839; Mpio. G. Cepada, Hinton et al. 16560; Guerrero Nuevo, R. Pérez 612; Ramos Arispe, A. Rodríguez & M. Carranza 874; Sierra Paila, J. Villareal 5218 & M. Carranza (ANSM); Carr. Múzquiz-Boquillas, R. Vázquez 259* (IEB). Guanajuato: Dolores Hidalgo, Rzedowski 41071*, 41085; San Miguel Allende, Rzedowski 43470; Mpio. San Luís la Paz, Rzedowski 47060; Mesa de Jesús, E. Ventura 90204*; Cerro Sta. Cruz, E. Ventura & E. López 6817, 6824 (IEB). Nuevo León: West of Linares, C. Anderson 4624*(DUKE); San Pablo Galeana, Hinton et al. 21896; Aramberri, Hinton et al. 21968*; La Carbonera Galeana, Hinton et al. 25346; Mpio Galeana, Hinton et al. 25712 (IEB). Sonora: Cañón de la Bellota, S. White 4699*(DUKE); Durango: Mpio. Guadalupe Victoria, Ochoa-Martínez 248; San Luis Potosí: Mpio. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES Charcas, J. Reyes 415; Querétaro: Vizarrón, S. Zamudio 11085 (IEB). H. Wornock s.n.**, (TEX). Wen 7288 (AY641495, AY640448, AY643108, DQ382305, DQ382305, DQ400546). R. schmidelioides Schltdl. MEXICO. Michoacán: San Andrés Ziracuáreriro, H. Díaz-Barriga 3516***; La Esperanza Morelia, V. Huerta 521; W. San Miguel, C. Medina 1652; Mil Cumbres, Rzedowski 45608*; Cerro Blanco Pátzcuaro, S. Zamudio 10642 (IEB). Querétaro: Tres Lagunas, H. Díaz-Barriga 3850*; El Madroño Mpio. Landa, E. González 408; Acatitlán de Zaragoza, E. González 634; La Yesca Mpio. Landa, H. Rubio 1554; Puerto Hondo, H. Rubio 609; La Parada Mpio. Jalpan, Sotero-Servín 56; Pinal de Amoles, S. Zamudio 2430 (IEB). Jalisco: Concepción de Buenos Aires, R. Ornelas & A. Flores Macías 1371, 1372; Manzanilla-Mazamitla, R. Ornelas & A. Flores Macías 1378; Ejutla, R. Ornelas & A. Flores Macías 1397 (IEB); Cerro Gordo, Arandas, R. Ramírez 1217*(IBUG). R. trilobata Nutt. MEXICO Chihuahua: Ignacio Zaragoza Mpio. Casas Grandes, P. Tenorio & C. Romero 6491**; Puerto Colorado, G. Rodríguez 261; Ejido El Largo Mpio. Madera, A. Benítez 1443, (IEB); Coahuila: Ejido Nurias, Cuatrocienegas, M. Carranza 1867; El Cedral, Sierra Paila, J. Villareal 3949 (IEB); Sierra Paila, D. Castillo & J. Aguilera 849; Saltillo, F. Encina et al. 562; General Cepeda, J. Marroquín 2271; Cañón de la Carbonera, Mpio. Arteaga, J. Villareal 3742; Saltillo, J. Villareal, 1537 (ANSM). Querétaro: 6.5 km to the SW of Vizarrón, Mpio. Cadereyta, S. Zamudio 2760, 8 km to the NE of Vizarrón, S. Zamudio 2854, Peña Azul, El Jabalí, S. Zamudio 3044* (IEB). Huapanguillo Mpio. Miquihuana, Tamaulipas, L. Hernández 2086* (IEB). U.S.A: Lawrence Co. C. Bennett s.n.*; Medora North Dakota D. Stevens 2413*; Henry Co. Illinois, Sears 1065* (NCU). Miller 21 (AY641497, AY640449, AY643109, DQ382317, DQ400555). Section Terebinthifolia: R. barclayi Standl. MEXICO Jalisco: Autlán, R. Ornelas et al. 1506, 1585, Lagunillas de Ayotitlán, F. Santana et al. 4307*; Autlán de Navarro, R. Delgadillo et al. 1109 (IEB); Autlán, R. Cuevas & M. Rosales 1823; Ayotitlán, S. Guerrero 247; Arroyo la Calera, A. Guzmán et al. 977; Ayotitlán, R. Ornelas 1604**; Puerto los Mazos Autlán, R. Ornelas 1614** (IBUG); Talpa, E. Palmer s.n.* (US). Cochrane 12157(HE862264, HE862253---). R. hartmanii F.A. Barkley. MEXICO Sonora: Las Chinazas, M. Fishbein et al. 102a; Barranca Huicochic, M. Fishbein et al. 121; Río Mayo, H. Gentry 3682; Santa Rosa, L. Toolin 310***; Yecora, L. Toolin 1376* (ARIZ); Nacore Chico, C. Muller 3689 (GH). Felger 95-181, (-HE862260---). R. jaliscana Standl. MEXICO Jalisco: Barranca Huentitlán, R. Acevedo et al. 1632; A. Flores & M. 465 Cházaro 2531; Ornelas 1429*; Barranca de Oblatos, M. Cházaro et al. 6743*; Cerro de Lima Mpio. Jocotepec, H. de Alba & M. Cházaro 10; Las Siete Cascadas, A. Flores 2422; 2 Km to the E. of Juanacatlán, R. Ornelas & J. García Castañeda 1456 (IEB); Cerro Amatitlán, E. Estrada, 8555; Siete Cascadas, Tonalá, M. Huerta & S. Guerrero 256**; La Primavera, Zapopan, O. Reyna 551; Laguna de Chapala, L. Villareal 3176, Las Tinajas Tonalá, L. Villareal 7223; Ixtlahuacán, L. Villareal 9401; Teocaltiche, F. Zapata 10 (IBUG); Guadalajara, C. Pringlei s.n.*** (US). R. palmeri Rose. MEXICO Sinaloa: Sierra Tacuichamona, H. Gentry 5672; Los Pucheros, Sierra Surotato, H. Gentry 7203; along hwy, R. Worthington 7939 (GH); Chihuahua: Canyon Tarahumara, H. Gentry 7296; Mina El Bravo, P. Martin et al. s.n. (GH); Sonora: Vicinity of Alamos, P. Standley et al. 1310*; La Huerta Sierra Alamos, J. Wiens et al. 93-121(GH). R. rubifolia Turcz. MEXICO Jalisco: Mpio. Hostotipaquillo, R. Ornelas 1545***; 10 km before Corcovado, R. Ornelas & J. García 1461 (ANSM); 3 km to the N of Tecolotlán, R. Ornelas & A. Flores 1388*; 8 km to the N. of Ayutla, 1391*; Unión de Tula 1392, Los Pilares, Mpio. Ameca, 1513* (IEB). Steinmann et al. 3146 (AY641508, AY640459, AY643119, DQ382315, DQ400552). R. terebinthifolia Schltdl. & Cham. MEXICO Chiapas: Amatenango del Valle, Shilom Tom 1845*; Tenejapa, Shilom Tom 4041* (DUKE); Tenejapa, Breedlove 12669; Lagos de Monte Bello, Breedlove 9676; Lagos de Montebello, Tziscao, M. Lavin et al. 4578; Amatenango del Valle, Shilom Tom 1128 (TEX); Oxchuc, F. Gómez 296 (IEB). Guerrero: Chilpancingo, W. Anderson 4338* ( DUKE). Oaxaca: Sola de Vega, Breedlove 12284 (ARIZ); Grutas de San Sebastian Sola de Vega, R. Cedillo 1745*(IEB). Querétaro: Las Mesitas to the SW of El Rincón, E. González 1401*; Tongojo El Rincón, A. Herrera 56; 1.5 km to the NW of La Mesa de Corozo, H. Rubio 1750 (IEB). GUAEMALA El Petén, E. Contreras 10464* (DUKE); Honduras N. of Teguzigalpa T. Croat 63933*(DUKE). Calzada 19102 (HE862259, HE862269---). Section Styphonia subsection Styphonia: R. integrifolia Engl. MEXICO Baja California: Bahía de Todos los Santos, Carter 3181; Ensenada, S. Stephenson 67-135* (DUKE); Punta Banda, M. Dillon et al. 1829; Rancho Morron, R. Moran 17211 (TEX). Punta Banda J. Elizondo 311 (ANSM). USA California: La Habra Heights, S. Myer s.n.; San Bernardino Valley, S. Parish 6890; Santa Barbara, H. Pollard s.n**; Santa Monica Mountains, P. Raven s.n.; San Luis Obispo Co., D. Keil 13688**; San Diego, Wallace & D. Thompson 108 (TEX). Millar, 28 (AY641499, AY640451, AY643111, DQ382294, DQ400542). © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 466 A. R. ANDRÉS-HERNÁNDEZ ET AL. R. kearneyi F.A.Barkley. MEXICO Sonora: Sierra Niña, R. Felger 89-47 (TEX). Baja California: San Pedro Martir, R. Moran 18308***; Canyon Diablito, G. Webster 18261 (TEX). Modson 6979* (NCU); Paniel 2312* (NCU). Ickert-Bond, 1298 (AY641500, AY640452, AY643112, DQ382295, DQ400543). R. muelleri F.A.Barkley. MEXICO Nuevo León: Galeana, K. Nixon 4008; San Isidro y Lirios, C. Peterson 1277; Montemorelos, Sierra Cebolla, T. Petterson et al. 7164**; El Fraile, R. Smith M657, M683 (TEX); El Sauce, Mpio. Galeana Hinton et al. 18082, 19208*, Los Lirios, Mpio. Santiago, Hinton et al. 25584 and 24937*; NW of Galeana, M. Poole et al. 2476***; between Rayones and Galeana, S. Zamudio et al. 6220 (IEB); on the road to Madero, J. Marroquín 3705; Villa Santiago, V. Valdez 790; J. Villa 4787 (ANSM). Coahuila: El Cedral, Sierra Paila, J. Villareal 3942*, (IEB); Sierra Paila, J. Villareal et al. 5263 (ANSM). Hinton 24937 (HE862265, HE862255---). R. ovata S.Watson. MEXICO Baja California: Sierra San Francisco, Mpio. Mulege, M. Domínguez 2236; 9 miles E of Mission Borja, K. Nixon 960*(IEB); Punta Prieta H. Gentry 8979*** (US). USA California: Azuza, K. Murata & E, Lee 20; San Bernardino, S. Parish 6802; Sta. Barbara, M. Pollard s.n.; Santa Monica, T. Ross & A. Ross 5989; Liebre Mountains, Ross et al. 4946; Riverside Co., D. Seigier et al. DS-2200; Orange Co. R. Thorne 32857; San Diego Co., Wallace & D. Thompson 111; Sentenac Canyon, T. Crovello 270** (TEX); S. Boyd 6744* (NY). Miller, 6, 22 (AY641501, AY640453, AY643114, DQ382308, DQ400547). R. standleyi F.A.Barkley. MEXICO Oaxaca: Estación Microondas, P. Guerrero 135; Santiago Teotongo, Mpio. Ayutla, Salinas Dorado s.n.*(IEB); Cuicatlán, Stone 2785*(DUKE), Tepelmeme, Tamazulapan, Breedlove & B. Bartholomew 60721; Nochixtlán, M. Luckow 2538**(TEX). Puebla: Santa Lucia Atlixco, J. Jiménez 1678; Tepoxtlan, Mpio. San Martín, P. Tenorio 4918 (IEB); Tehuacán, C. Anderson 5318* (DUKE). Salinas 8087 (HE862258, HE862268---). Section Styphonia subsection Compositae: R. andrieuxii Engl. MEXICO Coahuila: Sierra Rancho Nuevo, Mpio. Santiago, Carranza et al. 1802; Estación de Microondas Saltillo, E. Rodríguez & J. Villareal 1751; Rancho Demostrativo Saltillo, J. Valdés s.n.; 12 km to the S of Saltillo, J. Villareal et al. 2705 (ANSM). Oaxaca: Juxtlahuaca, Calzada 21794* (IBUG); detour toward Jaltepec, Nochixtlán, M. Cházaro et al. 7065*; before the Jaltepec detour, Nochixtlán, M. Negrete 7065* (IEB). Puebla: Nicolás Bravo Chapulco, M. Cházaro et al. 6090*; P. Tenorio 5137 (IEB). Medrano 4285 (-HE862262---) MEXU. R. choriophylla Wooton & Standl. MEXICO Sonora: Tepehuanes, E. Torrecillas 35 (IEB); Coa- huila: La paila, B. Hinton 16514**, L. Lundell 12477** (US). Miller 27 (AY641498, AY640450, AY643110, DQ382287, DQ400534). R. oaxacana Loes. MEXICO Oaxaca: San Carlos Yautepec, T. Croat 46237*** (DUKE); Topanala Mpio. Yautepec, S. Acosta 947; Santa María Albarrados, Ayutla, M. Cházaro et al. 6801; San Pedro Tabiche, R. Robles 84; Tapónala, Mpio. San Carlos Yautepec, A. Flores 1168* (IEB). Juárez 567 (HE862256, HE862266). R. pachyrrhachis Hemsl. MEXICO Coahuila: Mpio. De Candela M. Carranza 2727, Carranza et al. 2786 (IEB); W. of Palmilla, R. Moran 10021*(US). Guanajuato: Comonfort, A. Mora 913* (IEB). Nuevo León: Las Norias, Mpio. Arramberi, Hinton et al. 17473, 23602*; La Poza, Mpio. Galeana, Hinton et al. 22214 (IEB); 5 km to the south of Zaragoza, J. Villareal & M. Carranza 536; J. Villareal et al. 5154 (ANSM). Querétaro: 6 km to the NW of La Luz, Rzedowski 52479*, 54479; Bornalejo, Mpio. de la Paz, S. Zamudio et al. 11601 (IEB). Tamaulipas: Bustamante J. Henrickson 19091** (TEX). Steinmann et al. 3724 (AY641503, AY640455, AY643115, DQ38239, DQ4005448). R. schiedeana Schltdl. MEXICO Chiapas: Teopisca Mpio. Totolapa, Breedlove 26174*, 46237*; Amatenango del Valle, Shilom Tom 1844* (DUKE). Guanajuato: Chupaderos, Mpio. La Paz, Steinmann et al. 3696; Puerto Gallo, Mpio. Atarjea, E. Ventura 6493*(IEB); Atarjea, E. Ventura & E. López 9121 (ANSM). Querétaro: 4 km to the NE of Acatitlán de Zaragoza, E. Carranza 786; to the S of La Parada Jalpan, E. Carranza 820; Laguna Colorada, Mpio. Jalpan, M. Chávez 112**; 7 Km to the W of Tilaco, R. Fernández 3101, 3112; Acatitlán de Zaragoza, E. González 59*; Cerro Fresnos and La Barrada, C. Guzmán 64, 114*; Puerto Sabino, H. Rubio 320; El Rincon, Rzedowski 42980; El Lobo Landa, Rzedowski 43997; 3 km from La Parada, B. Servin 1863; S. Zamudio et al. 10480 (IEB). Steinmann et al. 3696 (AY641504, AY640456, AY643116, DQ382313, DQ400554). R. virens Lindh. ex A.Gray MEXICO Coahuila: Sierra La Encantada, M. Carranza et al. C- 682, C-865, C-2279, 2126; Ramos Arizpe, J. Encina 836*; Sierra Paila, J. Marroquín 1388; Muzquiz J. Villareal 3545, 16948*; Sierra de la Madera, J. Villareal et al. 7313, (ANSM); Limeston Canyon, D. Flyr 1149* (DUKE). Nuevo León: Mpio. Bustamante, M. Carranza C-3643; Santa Catarina, Hinton et al. 24984 (ANSM). Tamaulipas: Near Villagran, C. Lundell 12477* (US). Querétaro: Mpio. Tolimán, Rzedowski 50139*; Sierra Peña Azul Vizarrón, S. Zamudio 2762; Jabalí Mpio. Cadereyta S. Zamudio 3027 (IEB). Zacatecas: 14 mi W of Sombrerete, D. Ward 5786*, (DUKE). Wen 7282 (AY641505, AY640457, AY643117, DQ 382320, DQ 400557). © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 RHUS PHYLOGENY: STRUCTURE AND MOLECULES R. chondroloma Standl. MEXICO Oaxaca: Tamazulapan, Teposcolula, L. Rico et al. 329; Sn Pedro Nopala, T. Salinas et al. s.n., (ANSM). Huajuapan de León, F. McCarten 2976*** (US). Tehuacán, Rzedowski 33957 (IEB). Hunn 59 (HE862257, HE862267---). Actinocheita filicina (D.C) F.A.Barkley. MEXICO Guerrero: Iguala, C. Catalán et al. 783; Xochipala, E. Martínez 711, (IEB), Rzedowski 18652 (TEX). Oaxaca: Justlahuaca, S. Zamudio & G. Ocampo 11057 (IEB). Puebla: Izúcar de Matamoros, E. Guízar, 914, (IEB); Chapulco, J. Panero 7354; Tehuacán, Rzedowski 19130 (TEX). Panero s.n. (AY641509, AY640460, AY643120, DQ382321, DQ400558). Malosma laurina (Nutt) Abrams. USA California: Rancho Sta. Ana, Gillis 9009; P. C. Everet, 2163; San Gabriel Mountains, T. Ross 3698; La Jolla, San Diego, J. Thorner s.n. (TEX). Miller 34 (AY641510, AY640461, AY643121, DQ382322, DQ400559). Toxicodendron diversilobum Torr. & Gray. MEXICO Baja California: El Mandadero, Mpio. Ensenada, Tenorio & Romero 13397; El Observatorio, San Pedro Martir, Tenorio and Romero 13231* (IEB). Wen 6693 (AY677202, AY677208, AY677205, DQ382328, DQ4005689). Toxicodendron radicans (L.) Kuntze. MEXICO Baja California: Sierra de La Laguna, R. 944 (IEB). Chihuahua: Municipio de Madera, Bravo-Bolaños, 890 (ANSM). Jalisco: Zapopan, Santana F. et al. 3178 (IEB). Nuevo León Sierra de Anahuac, Sánchez Vega,640 (ANSM). Sonora: Mpio. Yecora, Tenorio L. 4566; Tamaulipas: Gómez Farias, Avendaño R. & Naruve F. 1701; Veracruz: Chicantepec, Duran E.C. et al. 290; Chiconquiaco, Gutíerrez C. 3232 (IEB). USA. River at Co. H. E. Ahles 59889*; Northeast of Pollocksville, Jones Co. M. N. Sears 6736 (NCU). Wen 6236 (AY677203, AY677207, AY677206, DQ382329, DQ400569). Toxicodendron vernix L. USA. Chowan river, Gates Co. H. E. Ahles 40371; Swampy Roads, road to Morston H.E. Ahles 24843; Swampy Hollow, Wake Co. W. B. Fox 3806 (NCU). Woodfort C. Illinois, H. Chase, 16013 (ANSM). Wen 7146 (AY541520, AY640471, AY643131, DQ382330, DQ400570). Searsia ciliata Miller 47 (AY641513, AY640464, AY643124--). Searsia quartiniana Miller 51 (AY641517, AY640468, AY643128, DQ382331, DQ400566). Shinus molle Wen 6686 (AY641512, AY640463, AY643123, DQ382333, DQ400565). APPENDIX 2 Structural characters and character states of Rhus s.s. analysed in this work. Morphological characters: 467 (1) Leaf organization: 0 = multifoliate, 1 = trifoliate, 2 = simple. (2) Rachis (midrib) winged: 0 = absent, 1 = present. (3) Texture: 0 = membranaceous, 1 = chartaceous, 2 = coriaceous. (4) Leaflets or leaf shape: 0 = ovate, 1 = elliptic, 2 = lanceolate. (5) Leaflets or leaf base: 0 = obtuse, 1 = rounded, 2 = acute. (6) Margin of leaflets: 0 = entire, 1 = serrate. (7) Venation pattern: 0 = brochidodromous, 1 = craspedodromous, 2 = eucamptodromous, 3 = cladodromous. (8) Course midvein: 0 = right, 1 = sinous. (9) Course secondary veins: 0 = recurved, 1 = sinous, 2 = zig-zag. (10) Tertiary veins pattern: 0 = irregular reticulate, 1 = weakly percurrent, 2 = transversely ramified. (11) Marginal ultimate venation: 0 = looped, 1 = incomplete, 2 = fimbriate. (12) Type of areole: 0 = imperfect, 1 = incomplete. (13) Pilose indument: 0 = absent, 1 = present. (14) Glands: 0 = absent, 1 = present. (15) Freely ending veinlets terminals: 0 = without idioblast, 1 = idioblast type I, (veinlets simple to widening in spherical form at the end), 2 = idioblast type II (widened veinlets form spherical group due to the association with brachysclereids). (16) Fiber in the freely ending veinlets: 0 = absent, 1 = present. (17) Number of branches in the inflorescences: In mature inflorescences, the number of inflorescence axes are considered branches: 0 = more than three branches, 1 = three branches, 2 = two branches. (18) Distance from base of the secondary branch to the first flower (measured with caliper at least three samples for species): 0 = 5–10 mm, 1 = 3– 5 mm, 2 = 0.5–1.5 mm. (19) Distance between flowers: 0 = 3–5 mm, 1 = smaller than 0.5 mm. (20) Pedicel: 0 = absent, 1 = present. (21) Pedicel length: 0 = 1.5–2.5 mm, 1 = smaller than 1mm. (22) Ciliate sepals: 0 = absent, 1 = present. (23) Red glandular hairs on principal axis of inflorescences and branches: 0 = absent, 1 = present. (24) Bracteoles: 0 = absent, 1 = present. (25) Red glandular hairs on the fruit surface: 0 = absent, 1 = present. Anatomical characters: Wood: (26) Porosity: 0 = semi-ring, 1 = diffuse, 2 = ringporous. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468 468 A. R. ANDRÉS-HERNÁNDEZ ET AL. (27) Septate fibers: 0 = absent, 1 = present. (28) Ray number of cells width: 0 = more than 4 cells wide, 1 = 1–3 cells wide. Petiole: (29) Number of canals in petiole: 0 = fewer of seven, 1 = more than seven. (30) Xylary fibres in petiole: 0 = absent, 1 = present. (31) Gelatinous fibre in xylem: 0 = absent, 1 = present. Lamina: (32) cuticle thickness: 0 = 6 μm or less, 1 = more than 6 μm. (33) Cuticle surface: 0 = smooth, 1 = striate. (34) Papillose epidermis : 0 = absent, 1 = present. (35) Number of layer of palisade parenchyma: 0 = one layer, 1 = two unequal layers, 2 = two equally long layers, 3 = three layers. (36) Presence of druses in mesophyll: 0 = absent, 1 = present. (37) Prismatic crystals in mesophyll: 0 = absent, 1 = present. Midvein: (38) Presence of papillose epidermis: 0 = absent, 1 = present. (39) Fibres in xylem: 0 = absent, 1 = present. (40) Arrangement of vascular tissue of midvein: 0 = type I (simple, open arch with one, additional vascular bundle in the adaxial region without fibre encircling the bundle), 1 = type II (simple, open arch with one additional vascular bundle in the adaxial region with fibres encircling the bundle), 2 = type III (simple, open arch with fibres encircling the vascular system), 3 = type IV (closed arch). © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 452–468