JSE
Journal of Systematics and Evolution
doi: 10.1111/jse.12295
Research Article
Phylogeny and a new tribal classification of Opiliaceae (Santalales) based on molecular and morphological evidence Chi-Toan Le1,2,3,4†, Bing Liu1,4†, Russell L. Barrett5,6, Li-Min Lu
1,4*
, Jun Wen7, and Zhi-Duan Chen1,4
1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China University of the Chinese Academy of Sciences, Beijing 100049, China 3 Hanoi Pedagogical University No. 2, Phucyen, Vinhphuc, Vietnam 4 Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China 5 National Herbarium of New South Wales, Royal Botanic Gardens and Domain Trust Sydney, Sydney, NSW 2000, Australia 6 Australian National Herbarium, Centre for Australian National Biodiversity Research, Canberra, ACT 2601, Australia 7 Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012, USA † These authors contributed equally to this work. *Author for correspondence. E-mail:
[email protected]. Tel.: 86-10-62836114. Fax: 86-10-62590843. Received 7 September 2017; Accepted 7 November 2017; Article first published online 28 December 2018 2
Abstract Opiliaceae are a pantropical family of the Santalales mainly distributed in the Old World with only one genus in the neotropics. Opiliaceae have remained taxonomically unresolved and the generic relationships within the family have been disputed. Here we present molecular phylogenetic analyses of the family and its close relatives using a combined dataset of the nuclear ribosomal (small subunit rDNA and large subunit rDNA) and the chloroplast rbcL, matK, and trnL-F regions. We also carried out a morphological phylogenetic analysis using 24 characters for all the species of Opiliaceae and three species in Santalaceae s.l. and Strombosiaceae as outgroups. Molecular analyses strongly supported the monophyly of Opiliaceae. Agonandra Miers ex Benth. & Hook. f. is sister to all the other genera of Opiliaceae. The remaining genera form two major clades: the Opilia clade (including Cansjera Juss., Lepionurus Blume, Opilia Roxb., Pentarhopalopilia (Engl.) Hiepko, Rhopalopilia Pierre, and Urobotrya Stapf.), and the other consisting of Anthobolus plus the Champereia clade (including Champereia Griff., Melientha Pierre, and Yunnanopilia C. Y. Wu & D. Z. Li). We propose a new classification of Opiliaceae, recognizing 12 genera and four tribes, with the description of a new tribe, Champereieae Bing Liu, C. T. Le, L. M. Lu & Z. D. Chen. Key words: Champereieae, classification, parasitic plants, phylogeny, Yunnanopilia.
1 Introduction The sandalwood order Santalales, containing 18 families, 160 genera, and over 2200 species, is the largest group of parasitic plants in the angiosperms (Nickrent et al., 2010). This order is distributed in tropical and temperate regions, but shows greatest diversity in the tropics (Kuijt, 2015). The systematic position of Santalales within angiosperms has been uncertain for decades. APG II (2003) placed Santalales in core eudicots, but did not identify its close relative. Both APG III (2009) and APG IV (2016) supported Santalales as sister to a clade of asterids and its close relatives (e.g., Caryophyllales and Berberidopsidales) based on Moore et al. (2010) and Soltis et al. (2011). A recent eudicot phylogeny based on hundreds of nuclear genes strongly supported rosids and Saxifragales as sisters, with Vitales and Santalales being successive next sister clades (Zhang et al., 2016 on shared morphology between Vitales and Santalales; Zeng et al., 2017). © 2017 Institute of Botany, Chinese Academy of Sciences
Opiliaceae are a small family of the Santalales with approximately 36 species (Hiepko, 2008; Nickrent et al., 2010). All species of Opiliaceae are root-parasites and the family has a pantropical distribution in tropical Africa, Asia, and Australia with only one genus, Agonandra Miers ex Benth. & Hook. f., in the neotropics (Hiepko, 2008; Nickrent et al., 2010; Kuijt, 2015). Compared with other members of the Santalales, Opiliaceae are characterized by their alternate leaves bearing cystoliths, erect and fleshy floral discs, distinct stamens (rarely forming a connate short tube), and superior ovaries (Kuijt, 2015). Some species of Opiliaceae are of cultural importance such as Melientha suavis Pierre and Champereia manillana (Blume) Merr., which have been cultivated and consumed as vegetables in South to Southeast Asia (Thailand, Vietnam, Laos, and Cambodia) for hundreds of years (Cruz-Garcia & Price, 2011; Abdul Wahab et al., 2015). Urobotrya siamensis Hiepko and Lepionurus sylvestris Blume are used as local medicines in Thailand and Malaysia (Hiepko, 2008). January 2018 | Volume 56 | Issue 1 | 56–66
Phylogeny and classification of Opiliaceae Opiliaceae have a complex taxonomic history with the currently included genera being treated in several families by early authors in the 19th century (Hiepko, 2008). For instance, Cansjera Juss., Agonandra, Lepionurus Blume, and Opilia Roxb. were placed in Olacaceae, and Champereia Griff. was included in Santalaceae (Bentham & Hooker, 1862, 1883; Engler, 1889), whereas Baillon (1892) included Opilia, Lepionurus, Champereia, Melientha Pierre, Agonandra, and Cansjera in a broadly circumscribed Loranthaceae. Valeton (1886) established Opiliaceae as a distinct family including five genera and two tribes: Opilieae (Cansjera, Champereia, Lepionurus, and Opilia) and Agonandreae (Agonandra). The tribes Opilieae and Agonandreae have been generally accepted, but the circumscription of each tribe has varied among authors (e.g., Qiu & cot et al., 2004; Hiepko, 2008). Sleumer Hiepko, 2003; Male (1935) was the first to include the genus Gjellerupia Lauterb. in Opiliaceae, specifically in the tribe Agonandreae. KoekNoorman & Rijckevorsel (1983) and Hiepko (1984) recognized nine genera in Opiliaceae, namely, Cansjera, Lepionurus, Champereia, Opilia, Agonandra, Gjellerupia, Melientha, Rhopalopilia Pierre, and Urobotrya Stapf. However, Koek-Noorman & Rijckevorsel (1983) and Hiepko (1984) provided different infrafamilial classifications. Koek-Noorman & Rijckevorsel (1983) divided the family into two groups: one including Champereia, Opilia, Rhopalopilia, and Melientha, and the other consisting of Gjellerupia, Lepionurus, and Urobotrya, with Cansjera and Agonandra having uncertain affinities. Hiepko (1984) classified the family into two tribes, the monogeneric Agonandreae and the Opilieae with the latter including all the other genera. Hiepko (1987) established a new genus Pentarhopalopilia (Engl.) Hiepko, which was formerly recognized as a section in Rhopalopilia. Wu & Li (2000) published the Chinese endemic genus Yunnanopilia C. Y. Wu & D. Z. Li with a single species Y. longistaminea (W. Z. Li) C. Y. Wu & D. Z. Li, which was previously placed either in Melientha or Champereia. Yang et al. (2017) provide support for its generic status. Nickrent et al. (2010) suggested that the santalaceous member Anthobolus R. Br. should also be placed in Opiliaceae. Most recently, Kuijt (2015) suggested four informal groups in Opiliaceae based on morphological characters, but Anthobolus was excluded from Opiliaceae and placed in Santalaceae. Therefore, the infrafamilial taxon circumscriptions within Opiliaceae need to be further explored. Several molecular phylogenetic studies of Santalales have been undertaken in the past 16 years and Opiliaceae were strongly supported as a clade sister to Santalaceae s.l. (including Comandraceae, Thesiaceae, Cervantesiaceae, Nanodeaceae, Santalaceae s.s., Amphorogynaceae, and cot, 2001; Der & Nickrent, 2008; Viscaceae) (Nickrent & Male cot & Nickrent, 2008; Vidal-Russell & Nickrent, 2008a, Male 2008b; Su et al., 2015; Chen et al., 2016). Although these studies have been carried out at the family level, some also discussed relationships within Opiliaceae. For instance, Der & Nickrent (2008) showed strong support for the placement of cot & Nickrent (2008) Anthobolus within Opiliaceae. Male resolved Lepionurus as sister to their other sampled genera of Opiliaceae, and strongly supported Cansjera, Pentarhopalopilia, and Opilia as close relatives. In a revised classification of the Santalales based on morphological, anatomical, and molecular data, Nickrent et al. (2010) supported Opiliaceae as an independent family with 11 genera sister to Santalaceae s.l., www.jse.ac.cn
57
although they did not discuss the generic relationships within Opiliaceae, nor provide a classification of the family. Opiliaceae thus merit further morphological and molecular analyses, emphasizing the intergeneric relationships using a broad taxon sampling. The present study aims to: (i) infer the phylogenetic relationships within Opiliaceae; and (ii) provide a phylogenetically based classification, integrating evidence from both molecular and morphological data. The phylogenetic framework established in this study should facilitate or stimulate further collections-based integrative studies with morphological, biogeographic, as well as phylogenomic analyses of Opiliaceae and the close allies (Wen et al., 2015, 2017).
2 Material and Methods 2.1 Taxon sampling, DNA extraction, amplification, and sequencing We sampled 17 species (21 individuals) for five molecular markers including the nuclear small subunit and large subunit ribosomal DNA, and the chloroplast rbcL, matK, and trnL-F regions. Fourteen of the 17 species (18 individuals, including the genera Rhopalopilia, Melientha, and Yunnanopilia) have not been sampled in previous molecular studies. The outgroups selected comprised the genera Strombosia Blume, Pyrularia Michx., and Viscum L. Forty sequences were downloaded from GenBank, representing 11 individuals of the above-mentioned ingroups and outgroups (Table S1). Genomic DNA was extracted from silica gel-dried tissues or herbarium material using the CTAB procedure (Doyle & Doyle, 1987). Polymerase chain reactions and sequencing were carried out using the primers designed by Vidal-Russell & Nickrent (2008a, 2008b) and Taberlet et al. (1991). We completed bidirectional sequencing using an ABI 3730 DNA Sequencer (Applied Biosystems, Carlsbad, CA, USA), and performed quality estimation and assembly for the newly generated sequences with Geneious version 8.0.5 (Kearse et al., 2012). The sequences were aligned in MUSCLE version 3.8.31 (Edgar, 2004) and then adjusted manually in Geneious. 2.2 Molecular phylogenetic analyses The combined dataset was partitioned into five subsets corresponding to the five DNA regions with each partition using a respective substitution model based on the results of jModelTest version 2.1.6 (Darriba et al., 2012). The combined dataset was analyzed with the maximum likelihood (ML) and Bayesian inference (BI) methods to establish the phylogenetic relationships of Opiliaceae. The ML analysis was carried out using the program RAxML version 8.2.8 (Stamatakis, 2006; Stamatakis et al., 2008) for the combined dataset applying 1000 bootstrap replicates with the default settings on the CIPRES Science Gateway Portal (Miller et al., 2010). Bayesian inference was conducted in MrBayses version 3.1.2 (Ronquist & Huelsenbeck, 2003) for the the combined dataset. The Markov chain Monte Carlo algorithm was run for 5 000 000 generations with four Markov chain Monte Carlo, and trees were sampled every 1000 generations. The program Tracer version 1.6 (Rambaut & Drummond, 2007) was used to check that effective sample sizes for all relevant parameters were well above 200 and that stationarity had probably been J. Syst. Evol. 56 (1): 56–66, 2018
58
Le et al.
reached. With the first 25% of sampled generations discarded as burn-in, a 50% majority-rule consensus tree and posterior probabilities (PP) were obtained by using the remaining trees. 2.3 Morphological analysis For the morphological analyses, we examined specimens from the Institute of Botany, Chinese Academy of Sciences, Beijing, China (PE) and the Institute of Ecology and Biological Resources, Hanoi, Vietnam (HN). The voucher information of the herbarium specimens used for the morphological analyses are presented in Table S2. We chose 24 vegetative, floral, and palynological characters, in which 15 were binary and nine were multistate (Table 1). Morphological characters and the coding of character states are detailed in Table S3. We corroborated each character state by examining specimens cot et al., 2004; and consulting published works (e.g., Male Hiepko, 2008; Kuijt, 2015). The morphological dataset covered all genera and species of Opiliaceae and three outgroups from Santalaceae s.l. and Strombosiaceae (Pyrularia, Viscum, and Strombosia) (Table 2). Additionally, we reduced the taxa sampling in the morphological dataset as in the molecular dataset, but included Gjellerupia papuana Lauterb. to construct a morphological simplified tree for a comparison between the molecular and the morphological results. Phylogenetic analyses of the morphological dataset were undertaken in PAUP version 4.0b10 (Swofford, 2003) using the maximum parsimony method with a heuristic search with 100 random taxa addition replicates, tree bisection–reconnection branch-swapping and MulTrees selected. All characters and character states were equally weighted and the transformations of binary and multistate characters were set as unordered. Bootstrap support values were calculated with 1000 heuristic-search replicates.
3 Results 3.1 Molecular phylogeny Our study generated 45 new sequences and produced a combined molecular dataset with 6778 aligned positions across all taxa. Of the 105 cells (21 individuals for five DNA regions), 85 cells had sequence data, thus the matrix was 81% filled (Table S1). Phylogenetic trees from individual nuclear and chloroplast partitions resulted in lower resolution of relationships within Opiliaceae than the combined dataset. Comparisons between the nuclear and chloroplast phylogenetic results are presented in Fig. S1. Opiliaceae were well supported to be monophyletic and the phylogenetic relationships among the sampled genera of Opiliaceae are shown in Fig. 1. Agonandra was supported as sister to the rest of Opiliaceae with weak support. The remaining genera formed two major clades: the Opilia clade (Cansjera, Lepionurus, Opilia, Pentarhopalopilia, Rhopalopilia, and Urobotrya) and a clade consisting of Anthobolus plus the Champereia clade (Champereia, Melientha, and Yunnanopilia) with a low bootstrap support (BS) value (Figs. 1, 2). 3.2 Parsimony analysis of morphological data Phylogenetic analysis of the 24 morphological characters resulted in six most parsimonious trees with a length of 74 steps, consistency index ¼ 0.472, and retention index ¼ 0.835. The strict consensus tree of the morphological analysis is depicted in Fig. S2. The genus Anthobolus was supported as sister to the remaining genera (BS ¼ 62%) that form a polytomy composed of the Agonandra clade (BS ¼ 76%) (including Gjellerupia and Agonandra), Lepionurus, the Opilia clade (BS ¼ 56%), the members of Urobotrya and the Champereia clade (BS ¼ 90%) (including Champereia,
Table 1 List of the 24 morphological characters and character states scored for the phylogenetic analysis of Opiliaceae 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Plant habit: mostly small trees and shrubs (0); mostly lianas and climbing shrubs (1). Twig tomentum: glabrous (0); sparsely to densely hairy (1). Leaf cystoliths: absent (0); present (1). Leaf tomentum: glabrous (0); sparsely to densely hairy (1). Inflorescence type: cyme (0); umbel (1); raceme (2); panicle (3); spike (4). Inflorescence position: mostly on young branches (0); mostly on older branches and trunks (1). Inflorescence length: >2 cm (0); ⩽2 cm (1). Inflorescence rachis tomentum: glabrous (0); sparsely to densely hairy (1). Bract length: 1 cm (0); ⩽1 cm (1). Pollen shape: equiaxial (0); breviaxial (1). Endoaperture granules: present and endoaperture smooth (0); none and endoaperture smooth (1); none and endoaperture endosculpture (2). 24. Exine in mesocolpium: smooth or microperforate tectum (0); reticulate exine (1); echinulate tectum (2). J. Syst. Evol. 56 (1): 56–66, 2018
www.jse.ac.cn
Phylogeny and classification of Opiliaceae
59
Table 2 Morphological matrix of Opiliaceae and outgroups Species
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Agonandra silvatica Ducke A. brasiliensis Benth. & Hook. f. A. fluminensis Rizzini & Occhioni A. goldbergiana Hiepko A. peruviana Hiepko A. excelsa Griseb. A. racemosa Standl. A. macrocarpa L. O. Williams A. obtusifolia Standl. A. ovatifolia Miranda Gjellerupia papuana Lauterb. Anthobolus leptomerioides F. Muell. Anthobolus filifolius R. Br. Anthobolus foveolatus F. Muell. Lepionurus sylvestris Blume Cansjera leptostachya Benth. Cansjera rheedei J. F. Gmel. Urobotrya congolana (Baill.) Hiepko U. sparsiflora (Engl.) Hiepko U. latisquama (Gagnep.) Hiepko U. longipes (Gagnep.) Hiepko U. siamensis Hiepko U. floresensis Hiepko U. parviflora Hiepko Opilia amentacea Roxb. Opilia campestris Engl. Pentarhopalopilia umbellulata (Baill.) Hiepko P. marquesii (Engl.) Hiepko P. madagascariensis (Cavaco & Keraudren) Hiepko P. perrieri (Cavaco & Keraudren) Hiepko Rhopalopilia hallei Villiers Rhopalopilia pallens Pierre Rhopalopilia altescandens Mildbr. Melientha suavis Pierre Champereia manillana (Blume) Merr. Yunnanopilia longistaminea (W. Z. Li) C. Y. Wu & D. Z. Li Viscum articulatum Burm. f.† Pyrularia edulis (Wall.) A. DC.† Strombosia grandifolia Hook. f. ex Benth.†
0 0
0 1
1 1
0 0
2 2
0 0
0 0
0 0
1 1
1 1
0 0
2 2
2 2
1 1
1 1
1 1
0 0
1 1
1 1
1 1
0 0
0 0
2 2
0 0
0
1
1
0
2
0
0
0
1
1
0
2
2
1
1
1
0
1
1
1
0
0
2
0
0 0 0 0 0 0 0 0 0
1 0 0 0 1 1 1 1 0
1 1 1 1 1 1 1 1 0
0 0 0 0 0 0 0 0 0
2 2 2 2 2 2 2 2 2
0 0 0 0 0 0 0 0 0
0 0 0 0 1 1 1 1 1
0 0 0 0 0 0 0 0 0
1 1 0 1 1 1 1 0 1
1 0 0 0 1 1 0 1 0
0 0 0 0 0 0 0 0 0
2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
1 1 1 1 1 1 1 1 0
1 1 1 1 1 1 1 1 0
1 1 1 1 1 1 1 1 0
0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 0
1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 0
0 0 0 0 0 0 ? 0 1
0 0 0 0 0 0 0 ? ?
2 2 2 2 2 2 2 ? ?
0 0 0 0 0 0 0 1 ?
0 0
0 0
0 0
0 0
2 2
0 0
1 1
0 0
1 1
0 0
0 0
2 2
2 2
0 0
0 0
0 0
0 0
0 0
1 1
0 0
1 1
? ?
? ?
? ?
1 1 1 0
1 1 1 0
1 1 1 1
0 1 1 0
2 4 4 2
0 0 0 0
0 1 1 0
0 1 1 1
1 1 1 1
1 0 0 1
0 2 2 0
0 0 0 0
2 1 1 0
0 0 0 0
0 1 1 1
0 1 1 1
1 1 1 0
0 0 0 1
1 1 1 1
0 0 0 0
0 0 0 0
? 0 0 1
? ? ? 1
1 ? ? 1
0 0
1 0
1 1
0 0
2 2
0 0
0 0
1 0
1 1
1 1
0 0 0 0
0 1
0 0
1 1
1 1
0 0
1 1
1 1
0 0
0 0
1 1
1 1
1 1
0 0 0 0 1 0 1
0 0 1 1 1 1 0
1 1 1 1 1 1 1
0 0 0 0 1 1 0
2 2 2 2 2 2 1
0 0 0 0 0 0 0
0 0 0 0 1 1 1
0 0 0 1 1 1 1
1 1 1 1 0 0 1
1 1 1 1 1 1 0
0 0 0 0 1 1 1
0 0 0 0 0 0 0
1 1 2 2 2 2 0
0 0 0 0 0 0 0
1 1 1 1 1 1 1
1 1 1 1 1 1 1
0 0 0 0 0 0 0
1 1 1 1 1 1 1
1 1 1 1 1 1 1
0 0 0 0 0 0 0
1 1 0 0 0 1 0
1 1 1 1 0 0 0
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1
1 1
1 1
1 1
1 1
0 0
1 1
1 1
1 0
0 0
1 1
0 0
0 0
0 0
1 1
1 1
0 0
1 1
1 1
0 0
0 0
0 0
1 1
1 1
1
1
1
1
1
0
1
1
1
0
1
0
0
0
1
1
0
1
1
0
0
0
1
1
0 0 1
1 1 0
1 1 1
1 1 1
2 2 2
0 0 0
1 1 1
1 1 0
1 1 1
0 0 0
1 1 1
0 0 0
1 1 1
0 0 0
1 1 1
1 1 1
0 0 0
0 0 0
1 1 1
0 0 0
? 0 0
0 1 0
1 2 1
1 1 1
0 0
0 0
1 1
0 0
3 3
1 1
0 0
0 0
1 1
0 0
0 0
2 1
2 0
0 0
0 0
1 1
0 0
2 2
2 2
2 2
0 0
0 0
1 1
1 1
0
0
1
0
3
1
0
0
1
0
0 0
1
0
0
1
0
2
2
2
0
0
1
1
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1 1
0 0
2 0
2 2
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1 0
0 0
1 1
2 0
0
0
0
0
0
0
0
0
0
0
0 0
0
0
0
0
0
0
0
0
0
0
0
0
†Outgroup; ?, Missing data.
www.jse.ac.cn
J. Syst. Evol. 56 (1): 56–66, 2018
60
Le et al.
Fig. 1. The 50% majority rule consensus tree of Opiliaceae from the Bayesian inference of the combined dataset of five DNA regions (large subunit ribosomal DNA, small subunit ribosomal DNA, matK, rbcL, and trnL-F). Values of maximum likelihood bootstrap and Bayesian inference posterior probabilities are presented above or below the branches.
Melientha, and Yunnanopilia). The Opilia clade included four genera: Cansjera, Opilia, Pentarhopalopilia, and Rhopalopilia. However, the generic relationships within the Opilia clade were not well resolved. Agonandra and Gjellerupia formed the Agonandra clade with strong support (BS ¼ 76%). Lepionurus was shown to be a member of the Opilia clade based on the molecular data, however, its position was not well resolved according to the morphological analysis. Three species of Urobotrya (U. latisquama (Gagnep.) Hiepko, U. longipes (Gagnep.) Hiepko, and U. siamensis) formed a clade (BS ¼ 62%), however, the systematic position of other species (U. congolana (Baill.) Hiepko, U. sparsiflora (Engl.) Hiepko, U. floresensis Hiepko, and U. parviflora Hiepko) was not well resolved. The Champereia clade was moderately supported by both the molecular and morphological data. The morphological data included the genus Gjellerupia that was not sampled in the molecular analyses, and revealed that Gjellerupia was sister to Agonandra (Figs. 3B, S2). The topology of the morphological tree including all 36 species of Opiliaceae (Fig. S2) was congruent with that of the simplified morphological tree including 15 species (Fig. 3B). The comparison between the molecular and morphological simplified trees in Fig. 3 also showed the distribution of morphological characters. The Champereia J. Syst. Evol. 56 (1): 56–66, 2018
clade is monophyletic and characterized by a paniculate inflorescence (5–3), inflorescences borne on older branches and trunk (6–1), stamens equal to petals in length (18–2), globose to ovoid ovaries (19–2), and styles absent (20–2). The Opilia clade usually has a densely hairy inflorescence rachis (8–1). Gjellerupia and Agonandra are characterized by the absence of petals in female flowers (14–1) and aborted styles (20–1) (Fig. 3B).
4 Discussion 4.1 Phylogeny and classification of Opiliaceae 4.1.1 Genus Agonandra Our molecular data weakly supported Agonandra as sister to the other genera of Opiliaceae in the absence of Gjellerupia. However, our morphological analysis supported Gjellerupia as sister to Agonandra (MP BS ¼ 76%). Agonandra has approximately 10 species endemic to the neotropics from Mexico to South America. Some taxonomists have treated Agonandra as constituting a distinct tribe Agonandreae (Valeton, 1886; Engler, 1897; Hiepko, 1984) or a separate group (Kuijt, 2015) because it is morphologically distinct from other genera of Opiliaceae with its dioecy, apetalous female flowers, dentate www.jse.ac.cn
Phylogeny and classification of Opiliaceae
61
Fig. 2. Summary topology of the molecular tree indicating inflorescence and flower character states in the four tribes of Opiliaceae. Values of maximum likelihood bootstrap and Bayesian inference posterior probabilities are presented above the branches. corolla lobes on male flowers, barrel-shaped ovaries, and pollen with an echinulate exine. The genus Gjellerupia contains only one species endemic to New Guinea (Hiepko, 2008; Kuijt, 2015). Sleumer (1935) suggested that Gjellerupia is closely related to Agonandra and should be included in the tribe Agonandreae. The two genera share morphological characters such as unisexual flowers, male flowers having four stamens opposite to each other with long filaments, apetalous female flowers, and aborted styles (Kuijt, 2015) (Fig. 3B; 14–1 and 20–1). Thus, in agreement with Sleumer (1935), we herein tentatively place Gjellerupia as belonging to the tribe Agonandreae. 4.1.2 Genus Anthobolus Anthobolus is endemic to central and northern Australia and shares relatively few morphological characters with other genera of Opiliaceae. It is the only genus of Opiliaceae that does not possess cystoliths in the foliar mesophyll. The placement of the genus in Opiliaceae has been disputed. Our phylogenetic analyses strongly support its inclusion in Opiliaceae, but it constitutes a distinct clade of its own (Figs. 1, 2, S1, S2), which is in agreement with Su et al. (2015). Anthobolus and the tribe Agonandreae share unisexual www.jse.ac.cn
flowers (Fig. 3B; 12–2). The inflorescences of Anthobolus are racemose and mostly borne on young branches, a character also found in Agonandra, Gjellerupia, and some genera of the Opilia clade. Our molecular data weakly supported Anthobolus as sister to the Champereia clade (BS ¼ 42%, PP ¼ 0.72; Fig. 1). Anthobolus is thus herein treated within the monogeneric tribe Anthoboleae. 4.1.3 Champereia clade Our molecular phylogeny supported the monophyly of Champereia and the sister relationship between Yunnanopilia and Melientha (BS ¼ 73%, PP ¼ 0.93; Fig. 1). Champereia and Melientha are widespread in SE Asia, whereas Yunnanopilia is endemic to the tropical area of SW China (i.e., Yunnan and Guangxi provinces), northern Vietnam, and northern Laos. Champereia was initially placed in Santalaceae by Engler (1889), but it was later treated as a member of Opiliaceae (Engler, 1897; Hiepko, 2008). Members of the Champereia clade can be distinguished by the following morphological characters: (i) inflorescence a panicle; (ii) inflorescence often borne on older branches or on the main trunk; and (iii) styles absent (Hiepko, 2008) (Figs. 2, 3B). The genus Yunnanopilia was erected by Wu & Li (2000) based on Melientha longistaminea W. Z. Li, J. Syst. Evol. 56 (1): 56–66, 2018
62
Le et al.
Fig. 3. Comparison between molecular and simplified morphological trees of Opiliaceae. A, A 50% majority-rule consensus tree from the Bayesian inference of the combined dataset of five DNA regions. Maximum likelihood bootstrap values and posterior probabilities of the Bayesian inference analysis are presented above the branches. B, Strict consensus tree of the maximum parsimony analysis of 24 morphological characters and 15 species of Opiliaceae. The distribution of morphological characters is represented by boxes: non-homoplasious characters are indicated with solid boxes; homoplasious characters are indicated with open boxes. Character number is shown above boxes and character state is shown below boxes. Parsimony support is given above the branches. but its generic status has been controversial. For instance, Tao (1993) transferred Melientha longistaminea to Champereia as C. longistaminea (W. Z. Li) D. D. Tao. More recently, Qiu & Chen (1997) reduced C. longistaminea to a variety of the only other species of Champereia, C. manillana, as C. manillana var. longistaminea (W. Z. Li) H. S. Kiu (also see Qiu & Hiepko, 2003). Wu & Li (2000) argued that Yunnanopilia merited the generic status due to its unique reproductive morphology within Opiliaceae. Yunnanopilia can be further distinguished from Champereia and Melientha by having bisexual, 4-merous, and sessile flowers, whereas Melientha has unisexual flowers and Champereia has 4 or 5-merous unisexual or stalked bisexual flowers (Wu & Li, 2000; Hiepko, 2008) (Fig. 4). Therefore, as reported in Yang et al. (2017), our results also support the generic status of Yunnanopilia because the inflorescence and floral morphology is sufficiently distinct from its sister taxon Melientha. We herein recognize the new tribe Champereieae as including the above three genera. 4.1.4 Opilia clade The circumscription of tribe Opilieae has been debated since its establishment by Valeton (1886). Our molecular analyses J. Syst. Evol. 56 (1): 56–66, 2018
revealed that the Opilia clade (BS ¼ 98%, PP ¼ 1.0) consists of six genera: Cansjera and Lepionurus from Asia–Australia, Pentarhopalopilia and Rhopalopilia from Africa, and Urobotrya and Opilia with disjunct distributions in both Asia–Australia and Africa. Inflorescence and stamen morphology are highly polymorphic in the Opilia clade (Figs. 2, 3). Four of the six genera of the Opilia clade have racemose inflorescences, whereas Pentarhopalopilia and Cansjera are described as being umbellate and spicate, respectively (Reed, 1955; Hiepko, 2008) (Fig. 2). Within this clade, two subclades were recognized. One subclade was formed by Cansjera, Lepionurus, and Urobotrya with strong support (BS ¼ 85%, PP ¼ 1.0). The other subclade included Opilia, Rhopalopilia, and Pentarhopalopilia with moderate support (BS ¼ 83%, PP ¼ 1.0). Pentarhopalopilia was not monophyletic with P. umbellulata (Baill.) Hiepko (the type species) and P. perrieri (Cavaco & Keraudren) Hiepko close to Opilia, and another speices P. marquesii (Engl.) Hiepko clustered with Rhopalopilia (Fig. 1). The relationship between Pentarhopalopilia and its relatives is unclear due to insufficient sampling and needs further study. The tribe Opilieae is herein circumscribed as including six genera. www.jse.ac.cn
Phylogeny and classification of Opiliaceae
63
Fig. 4. Floral structures of Champereia manillana, Melientha suavis, and Yunnanopilia longistaminea from left to right. A–C, Inflorescence. D–F, Bract. G–I, Flower structures. G1, Bisexual flower; G2, Female flower; H1, Female flower; H2, Male flower; a, Petal; b, Stamen; c, Pistil; d, Staminodes. Scale bars: A ¼ 5 mm; B ¼ 3 mm; C, G, H ¼ 2 mm; D ¼ 0.5 mm; E, F, I ¼ 1 mm. The illustrations B, E, G, H are reproduced from Hiepko (2008) with permission by Conservatoire et Jardin botaniques de la Ville de Gen eve.
4.2 Taxonomic treatment Opiliaceae Valeton, Crit. Overz. Olacin. 136. 1886. Type: Opilia Griff. (1) Agonandreae Engl. in Engl. & Prantl, Nat. Pflanzenfam. III, 1: 233. 1889. Type: Agonandra Miers ex Benth. & Hook. f. Included genera: Agonandra with 10 species from Mexico to South America; and Gjellerupia Lauterb. (tentatively placed here) with one species from New Guinea. g. 10: 461. 1841. (2) Anthoboleae Bartl. ex Spach, Hist. Nat. Ve Type: Anthobolus R. Br. Included genus: Anthobolus with three species endemic to central and northern Australia. (3) Champereieae Bing Liu, C. T. Le, L. M. Lu & Z. D. Chen, trib. nov. Type: Champereia Griff. Diagnosis: Tribus haec, inter tribus familiae Opiliaceae, ramulis et foliis glabris, inflorescentibus paniculatis magnis et saepe ramis senioribus vel truncis ortis, ovariis globosis vel ovatis, stigmatibus absentibus differt. www.jse.ac.cn
This tribe can be distinguished from other tribes of Opiliaceae by the glabrous branchlets and leaves; large, paniculate inflorescences that are often borne on older branches or on the main trunk; globose ovaries; and stigmas absent. Included genera: Champereia with one species endemic to SE Asia; Melientha Pierre with one species endemic to SE Asia; and Yunnanopilia C. Y. Wu & D. Z. Li with one species endemic to SW China. (4) Opilieae Benth., Ann. Mag. Nat. Hist., ser. 1, 7: 215. 1841. Type: Opilia Roxb. Included genera: Cansjera Juss. with three species from India and Sri Lanka to southern China, New Guinea, and northern Australia; Lepionurus Blume with one species from Nepal and Assam to western Malesia; Opilia Roxb. with two species from tropical Africa and one species from tropical Asia, the Solomon Islands, and northern Australia; Pentarhopalopilia (Engl.) Hiepko with four species from Africa and Madagascar; Rhopalopilia Pierre J. Syst. Evol. 56 (1): 56–66, 2018
64
Le et al. with three species from Central Africa; and Urobotrya Stapf. with two species from West and Central Africa and five species from tropical Asia.
Key to the tribes and genera of Opiliaceae 1a. Xerophytic, much branched shrubs; leaves linear to lanceolate, without cystoliths, often caducous ........................................trib. Anthoboleae —Anthobolus 1b. Shrubs or lianas; leaves usually broad, with cystoliths, often persistent.................................................................2 2a. Flowers unisexual; inflorescence a raceme; female flowers without petals.....................3. trib. Agonandreae 2b. Flowers bisexual; if flowers unisexual, then inflorescence a panicle; flowers always with petals..............................4 3a. Floral discs distinctly lobed; neotropical plants.......... ............................................................................Agonandra 3b. Floral discs weakly lobed; plants of New Guinea.................................................................Gjellerupia 4a. Inflorescence a panicle, often on older branches and trunks; twigs, leaves and pedicels usually glabrous; ovaries globose to ovoid, styles absent...................................................5. trib. Champereieae 4b. Inflorescence a raceme or spike, axillary; twigs, leaves, and pedicels sparsely to densely hairy (except Lepionurus); ovaries conical to cylindrical, styles distinct ....................................................................7. trib. Opilieae 5a. Flowers bisexual throughout, 4-merous, sessile................ .........................................................................Yunnanopilia 5b. Flowers unisexual or bisexual, 4 or 5-merous, bisexual flowers stalked..................................................................6 6a. Plants gynodioecious with both bisexual and female flowers; drupes 10–16 mm long.......................Champereia 6b. Plants dioecious with unisexual flowers; drupes 23– 40 mm long..........................................................Melientha 7a. Inflorescence a spike; bracts small and persistent; petals united into a tube..................................................Cansjera 7b. Inflorescence a raceme; bracts usually caducous; petals free or united at base........................................................8 8a. Inflorescence 5–15 mm long; flowers solitary in small bracts.................................................................................9 8b. Inflorescence mostly longer than 15 mm; 3 or more flowers clustered in broad bracts....................................10 9a. Stamens shorter than petals; petals 4..........Rhopalopilia 9b. Stamens longer than petals; petals 5.....Pentarhopalopilia 10a. Petals united at base; stamens shorter than petals....... ............................................................................Lepionurus 10b. Petals free; stamens longer than petals.........................11 11a. Bracts peltate; floral disc lobed..................................Opilia 11b. Bracts not peltate; floral disc annular...............Urobotrya
Acknowledgements We thank Professor Paul Hiepko of Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freie Universit€at, Germany for his critical reading of the manuscript and for his permission to use his illustrations. This study was supported by the Sino-African Joint Research Center, Chinese Academy of Sciences, CAS International Research and Education Development Program (SAJC201613), the CAS/SAFEA International J. Syst. Evol. 56 (1): 56–66, 2018
Partnership Program for Creative Research Teams, the National Natural Science Foundation of China (NNSF 31590822 and NNSF 31500179), and the CAS-TWAS President’s Fellowship for International Ph.D. Students. Fieldwork in Vietnam was partially supported by the External Cooperation Program of BIC, Chinese Academy of Sciences (GJHZ 201321), and Science and Technology Basic Work (2013FY112100). R.B. was supported by an Australian Biological Resources Study Applied Taxonomy Grant (ATC214-10).
References Abdul Wahab N, Ahdan R, Ahmad Aufa Z, Kong KW, Johar MH, Shariff Mohd Z, Ismail A. 2015. Nutritional values and bioactive components of under-utilised vegetables consumed by indigenous people in Malaysia. Journal of the Science of Food and Agriculture 95: 2704–2711. APG II. 2003. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: 399–436. APG III. 2009. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161: 105–121. APG IV. 2016. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society 181: 1–20. Baillon HE. 1892. Histoire des plantes, vol. 11. New York: Columbia University Press. Bentham G. 1841. Account of two new genera of plants, allied to Olacineae. Annals and Magazine of Natural History, including Zoology, Botany, and Geology ser. 1, 7: 214–217. Bentham G, Hooker JD. 1862. Dicotyledonum polypetalarum ordines LXXXIII: Ranunculaceas–Cornaceas. In: Bentham G, Hooker JD eds. Genera plantarum ad exemplaria imprimis in herbariis Kewensibus servata definita. London: Reeve. 1: 349. Bentham G, Hooker JD. 1883. Monochlamydearum, Gymnospermearum et Monocotyledonum ordines LXXIII. In: Bentham G, Hooker JD eds. Genera plantarum ad exemplaria imprimis in herbariis kewensibus servata definita. London: Reeve. 3: 231. Chen ZD, Yang T, Lin L, Lu LM, Li HL, Sun M, Liu B, Chen M, Niu YT, Ye JF, Cao ZY, Liu HM, Wang XM, Wang W, Zhang JB, Meng Z, Cao W, Li JH, Wu SD, Zhao HL, Liu ZJ, Du ZY, Wang QF, Guo J, Tan XX, Su JX, Zhang LJ, Yang LL, Liao YY, Li MH, Zhang GQ, Chung SW, Zhang J, Xiang KL, Li RQ, Soltis DE, Soltis PS, Zhou SL, Ran JH, Wang XQ, Jin XH, Chen YS, Gao TG, Li JH, Zhang SZ, Lu AM, China phylogeny consortium. 2016. Tree of life for the genera of Chinese vascular plants. Journal of Systematics and Evolution 54: 277–306. Cruz-Garcia GS, Price LL. 2011. Ethnobotanical investigation of ‘wild’ food plants used by rice farmers in Kalasin, Northeast Thailand. Journal of Ethnobiology and Ethnomedicine 7: 33. Darriba D, Taboada GL, Doallo R, Posada D. 2012. JModelTest 2: More models, new heuristics and high-performance computing. Nature Methods 9: 772. Der JP, Nickrent DL. 2008. A molecular phylogeny of Santalaceae (Santalales). Systematic Botany 331: 107–116. Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15. Edgar RC. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797.
www.jse.ac.cn
Phylogeny and classification of Opiliaceae
65
Engler A. 1889. Olacaceae trib. Opilieae and agonandreae. In: Engler A, Prantl K eds. Die nat€ urlichen Planzenfamilien. Leipzig: Engelmann. 3: 240–241.
Sanderson MJ, Beaman RS, Olmstead RG, Judd WS, Donoghue MJ, Soltis PS. 2011. Angiosperm phylogeny: 17 genes, 640 taxa. American Journal of Botany 98: 704–730.
Engler A. 1897. Olacaceae. In: Engler A, Prantl K eds. Die nat€ urlichen Planzenfamilien. Leipzig: Engelmann. 1: 144–149.
Spach E. 1841. Histoire naturelle des vegetaux. Phanerogames. Paris: Librairie encyclop edique de Roret. 10: 1–572.
Hiepko P. 1984. Opiliaceae. In: van Steenis CGGJ ed. Flora Malesiana. London: Martinus Nijhoff Garden. 31–52.
Su HJ, Hu JM, Anderson FE, Der JP, Nickrent DL. 2015. Phylogenetic relationships of Santalales with insights into the origins of holoparasitic Balanophoraceae. Taxon 64: 491–506.
Hiepko P. 1987. A revision of Opiliaceae, IV. Rhopalopilia Pierre and Pentarhopalopilia (Engler) Hiepko gen. nov. Botanische Jahrb€ ucher fur Systematik, Pflanzengeschichte und Pflanzengeographie 108: 280–288.
Stamatakis A. 2006. RAxML-VI-HPC, maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2221: 2688–2690.
Hiepko P. 2008. Opiliaceae. In: Anderson CE, Marhold K eds. Species Plantarum: Flora of the World. Gen eve: Conservatoire et Jardin botaniques de la Ville de Gen eve. 12: 1–71.
Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxMLWeb servers. Systematic Biology 575: 758–771.
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A. 2012. Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649.
Swofford DL. 2003. PAUP : Phylogenetic analysis using parsimony ( and other methods), version 4.0 Beta10. Sunderland: Sinauer Associates.
Koek-Noorman J, van Rijckevorsel P. 1983. Wood and leaf anatomy of Opiliaceae. Willdenowia 13: 147–174. Kuijt J. 2015. Santalales. In: Kuijt J, Hansen B eds. The families and genera of vascular plants. Flowering plants: Eudicots; Santalales, Balanophorales. Cham: Springer. 12: 2–189. Mal ecot V, Nickrent DL. 2008. Molecular phylogenetic relationships of Olacaceae and related Santalales. Systematic Botany 33: 97–106. Mal ecot V, Nickrent DL, Baas P, van den Oever L, Lobreau Callen D. 2004. A morphological cladistic analysis of Olacaceae. Systematic Botany 29: 569–586. Moore MJ, Soltis PS, Bell CD, Burleigh JG, Soltis DE. 2010. Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots. Proceedings of the National Academy of Sciences USA 107: 4623–4628. Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, LA, 14 November, 2010. 1–8. Nickrent DL, Mal ecot V. 2001. A molecular phylogeny of Santalales. In: Fer A, Thalouarn P, Joel DM, Musselman LJ, Parker C, Verkleij JAC eds. 7th International Parasitic Weed Symposium: Proceedings. Nantes: Facult e des Sciences, Universit e de Nantes. 69–74. Nickrent DL, Mal ecot V, Vidal-Russell R, Der JP. 2010. A revised classification of Santalales. Taxon 59: 538–558. Qiu HX, Chen BH. 1997. Noteworthy taxa from Southern China. Journal of Tropical and Subtropical Botany 5: 3–5. Qiu HX, Hiepko P. 2003. Opiliaceae. In: Wu ZY, Raven PH, Hong DY eds. Flora of China. Beijing: Science Press; St. Louis: Missouri Botanical Garden. 5: 205–207. Rambaut A, Drummond AJ. 2007. Tracer. Version 1.4 [online]. Available from http://beast.bio.ed.ac.uk/Tracer. Reed CF. 1955. The comparative morphology of the Olacaceae, Opiliaceae and Octoknemaceae. Memorias da sociedade broteriana. Coimbra 10: 29–79. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Sleumer HO. 1935. Olacaceae. In: Engler A, Prantl K eds. Die nat€ urlichen Planzenfamilien. Berlin: Borntraeger. 1–32. Soltis DE, Smith AS, Cellinese N, Wurdack KJ, Tank DC, Brockington SF, Redulio-Rodriguez NF, Walker JB, Moore MJ, Carlsward BS, Bell CD, Latvis M, Crawley S, Black C, Diouf D, Xi Z, Rushworth CA, Gitzendanner MA, Sytsma KJ, Qiu YL, Hilu KW, Davis CC,
www.jse.ac.cn
Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109. Tao DD. 1993. A new combination of the genus Champereia (Opiliaceae). Guihaia 1993: 8–11. Valeton T. 1886. Critisch overzicht der Olacineae B. et H. Groningen: P. Noordhoff. 136–161. Vidal-Russell R, Nickrent DL. 2008a. Evolutionary relationship in the showy mistletoe family (Loranthaceae). American Journal of Botany 95: 1015–1029. Vidal-Russell R, Nickrent DL. 2008b. The first mistletoes, origins of aerial parasitism in Santalales. Molecular Phylogenetics and Evolution 47: 523–537. Wen J, Harris AJ, Ickert-Bond SM, Dikow R, Wurdack K, Zimmer EA. 2017. Developing integrative systematics in the informatics and genomic era, and calling for a global Biodiversity Cyberbank. Journal of Systematics and Evolution 55: 308–321. Wen J, Ickert-Bond SM, Appelhans MS, Dorr LJ, Funk VA. 2015. Collections-based systematics: Opportunities and outlook for 2050. Journal of Systematics and Evolution 53: 477–488. Wu ZY, Li DZ. 2000. Yunnanopilia – A primitive new genus of Opiliaceae from Yunnan Plateau, China and its biogeographic significance. Acta Botanica Yunnanica 22: 248–250. Yang L, Yang GS, Ma HY, Wang YH, Shen SK. 2017. Phylogenetic placement of Yunnanopilia (Opiliaceae) inferred from molecular and morphological data. Journal of Systematics and Evolution. doi: 10.1111/jse.12285. Zeng LP, Zhang N, Zhang Q, Endress PK, Huang J, Ma H. 2017. Resolution of deep eudicot phylogeny and their temporal diversification using nuclear genes from transcriptomic and genomic datasets. New Phytologist 214: 1338–1354. Zhang N, Wen J, Zimmer EA. 2016. Another look at the phylogenetic position of the grape order Vitales: Chloroplast phylogenomics with an expanded sampling of key lineages. Molecular Phylogenetics and Evolution 101: 216–223.
Supplementary Material The following supplementary material is available online for this article at http://onlinelibrary.wiley.com/doi/10.1111/jse. 12295/suppinfo: Fig. S1. Comparisons of topologies between chloroplast (A) and nuclear (B) datasets of Opiliaceae. Maximum likelihood J. Syst. Evol. 56 (1): 56–66, 2018
66
Le et al.
bootstrap values and posterior probabilities of the Bayesian inference analysis are presented above the branches. , Support values
