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Home , Bivinia, Flacourtia, Lunania, Oncoba spinosa, Populus fremontii, Samyda

Nordic Journal of Botany 000: 001–007, 2017 doi: 10.1111/njb.01363, ISSN 1756-1051 © 2017 Th e Authors. Nordic Journal of Botany © 2017 Nordic Society Oikos Subject Editor: Simone Fior. Editor-in-Chief: Torbj ö rn Tyler. Accepted 30 December 2016

Dianyuea gen. nov. (: Scyphostegioideae) from southwestern China

Ce Shang* , Shuai Liao * , Yong-Jie Guo and Zhi-Xiang Zhang

C. Shang (http://orcid.org/0000-0003-1425-9403) and Z.-X. Zhang (http://orcid.org/0000-0002-1161-5065) ([email protected]), Laboratory of Systematic Evolution and Biogeography of Woody , College of Nature Conservation, Beijing Forestry Univ., Beijing, China. – S. Liao (http://orcid.org/0000-0002-3876-8002), Shanghai Chenshan Science Research Center, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, Shanghai, China. – Y.-J. Guo, Germplasm Bank of Wild Species in Southwest of China, Kunming Inst. of Botany, Chinese Academy of Sciences, Kunming, China.

Dianyuea C. Shang, S. Liao & Z. X. Zhang, a new monotypic of Salicaceae based on Flacourtia turbinata H. J. Dong & H. Peng, is described and illustrated. Morphologically, Dianyuea diff ers from Flacourtia Comm. ex L ’ H é r. by having six connate stamens, basal placentation and lobed seed appendages. All those features indicate that Dianyuea is allied with Scyphostegia Stapf. A molecular phylogenetic analysis using plastid trn L-F, matK, and rbc L sequence data for representatives of 16 genera in Salicaceae s.l. shows that Dianyuea is sister to Scyphostegia . Th e new combination Dianyuea turbinata (H. J. Dong & H. Peng) C. Shang, S. Liao & Z. X. Zhang is proposed.

An expanded Salicaceae s.l. has been proposed based on veins, campanulate , and solitary female fl owers, is molecular phylogenetic reconstructions (Chase et al. 2002), greatly dissimilar to the other species of Flacourtia Comm. consisting of Salicaceae s.s. (Salix L. and L.), ex L ’ H é r., but the basal placentation is reminiscent of Scyphostegiaceae, and the majority of species of . S. borneensis . In addition, the original material only con- More recently, based on morphological characteristics and tains female fl owers and , while male fl owers are molecular sequences, Alford (2005) highlighted the unique absent. Due to the lack of male fl owers and description of phylogenetic position of Scyphostegia borneensis Stapf (1894, their specifi c traits, the systematic position of this species p. 218) and off ered a new classifi cation for Salicaceae, from has remained unclear. which Samydaceae and Scyphostegiaceae were separated. Th e To solve this problem, we visited the type locality in April APG system (2016) circumscribed Salicaceae broadly, such 2015, and male fl owers were found. Th e connate stamens that it comprises approximately 981 species in 56 genera. were signifi cantly diff erent from Flacourtia , but more similar Within this enlarged Salicaceae, Stevens (2001 onwards) rec- to S. borneensis . To further evaluate the systematic position ognizes three subfamilies: Samydoideae, Scyphostegioideae of F. turbinata , we performed a phylogenetic analysis and Salicoideae. using sequences of the chloroplast trn L-F, rbcL, and mat K Scyphostegia borneensis, which is endemic to Borneo regions. (Steenis 1957), comprises the monotypic Scyphostegioideae. Th is species is characterized by a series of telescoping bracts, three stamens with their fi laments connate into a column Material and methods and a distinctive urceolate ovary with basal placentation. Although few morphological synapomorphies are shared Plant material among Scyphostegia and other genera of Salicaceae s.l. (Chase et al. 2002), the phylogenetic position of S. borneensis has Herbarium specimens of F. turbinata (maintained at KUN) been quite clear within a well-supported Salicaceae s.l. were studied, including female fl owers and fruits. Th is clade based on analyses of extensive DNA datasets (Xi et al. study was supplemented by fi eldwork at the type locality. 2012). Specimens with male fl owers and samples for molecular Flacourtia turbinata H. J. Dong & H. Peng (2013, experiments were collected at the same time. Voucher speci- p. 56) was described and collected from western Yunnan mens (Ce Shang 415) for DNA samples were deposited at Province, China. Its morphology, such as tripli-nerved BJFC. Pollen grains obtained from specimens were coated with a layer of gold, and then observed and photographed * Th ese authors contributed equally to this work. using a Hitachi S-520 scanning electron microscope.

Early View (EV): 1-EV A total of 24 species of Salicaceae s.l. were included in (MP) and MrBayes (Ronquist and Huelsenbeck 2003) for the molecular phylogenetic analysis, representing 11 genera Bayesian inference (BI), respectively. Both the MP and of Salicoideae, 4 genera of Samydoideae, and the only genus Bayesian were generated for the concatenated data of of Scyphostegioideae. Lacistema aggregatum (P. J. Bergius) trn L-F, rbcL, and mat K sequences, and gaps were treated as Rusby was used as an outgroup, based on the result of a pre- missing data. A total of 10 000 heuristic search replications vious phylogenetic study (Alford 2005). were used for the MP analysis, and node support was assessed using 1000 MP bootstrap (BS) replicates. Prior to Bayesian DNA extraction and PCR amplifi cation analysis, the dataset was tested for molecular evolution using Modeltest 3.7 (Posada and Crandall 1998), as implemented Samples for DNA extraction were dried with silica gel. in MrMTgui, based on the Akaike information criterion Genomic DNA was isolated using the Plant Kit following (Posada and Buckley 2004). TVM ϩ G was chosen as the the manufacturer' s protocol. Th ree chloroplast DNA regions, best model. Markov chain Monte Carlo searches were run trn L-F, rbc L, and mat K, were amplifi ed and sequenced using for 2 000 000 generations. All Bayesian analyses produced primers described by Taberlet et al. (1991: primers e and f), split frequencies of less than 0.01, showing convergence Olmstead et al. (1992), and Yu et al. (2011), respectively. between the paired runs. Of the 30 000 trees produced, the Amplifi ed products were purifi ed with the Plant Kit and then fi rst 2000 before stationarity was discarded as burn-in, and used for direct sequencing (performed by SinoGenoMax, the remaining trees were used to construct the majority-rule Beijing, China). consensus trees.

Sequence alignment and phylogenetic analyses Results and discussion In this study, 77 sequences, including three newly generated < and 74 retrieved from GenBank ( http://www.ncbi.nlm. Th e aligned matrix consisted of 1374 characters, of which > nih.gov/ ), were used for phylogenetic analyses (Table 1). 184 (13.4%) were parsimony-informative. Th e parsimony Th ree sets of sequences were independently aligned with analysis yielded 189 parsimonious trees of 455 steps length MAFFT (Katoh et al. 2005) and manually optimized in Bio- with a consistency index of 0.8088 and retention index of Edit (Hall 1999). We conducted preliminary separate phy- 0.8604. Bayesian analysis produced trees without signifi cant logenetic analyses using the parsimony criterion for each of diff erences from the MP topology (Fig. 1). Both the MP and the three markers, fi nding no incongruent grouping among Bayesian trees are largely consistent with the trees obtained any of the comparisons. Th us, the data were combined to from molecular and morphological data in previous system- form a three-gene matrix. Th e matrix was then analyzed atic studies (Alford 2005, Xi et al. 2012). Th us, Salicaceae * using PAUP (Swoff ord 2002) for maximum parsimony s.l. was divided into three clades. Flacourtia turbinata and

Table 1. Taxa analyzed and GenBank accession numbers for sequences used in this study. Accessions numbers in boldface indicate newly generated sequences.

Taxon trn L-F mat Krbc L Flacourtia turbinata H. J. Dong & H. Peng KU058673 KU058674 KU058675 Populus trichocarpa Torr. & A. Gray NC_009143 NC_009143 NC_009143 Populus fremontii S. Watson NC_024734 NC_024734 NC_024734 Salix purpurea L. NC_026722 NC_026722 NC_026722 Salix suchowensis W.C. Cheng ex G.H. Zhu NC_026462 NC_026462 NC_026462 Scyphostegia borneensis Stapf AY757073 EF135594 JX664072 (Lour.) Raeusch. AY757021 EF135541 AF206768 (Burm. f.) Merr. – AB233829 AB233933 Hasseltia fl oribunda Kunth AY757023 EF135546 GQ981759 Samyda dodecandra Jacq. AY757066 HM446743 HM446868 Homalium racemosum Jacq. AY757029 HM446700 AJ418822 Poliothyrsis sinensis Oliv. AY757052 EF135586 AF499236 spinosa Forssk. AY757047 JF270877 AJ418823 Lunania parvifl ora Spruce ex Benth. AY757037 AB233832 AB233936 Xylosma congesta (Lour.) Merr. JF950896 AB233834 AB233938 Xylosma chlorantha Donn. Sm. JF950897 JQ589122 JQ593990 mundii Warb. AY757071 X517610 AM235122 Scolopia spinosa Warb. AY757072 AB233833 AJ418833 Laetia procera (Poepp.) Eichler AY757033 JQ626344 JQ625734 Bivinia jalbertii Tul. AY757004 JX517831 JX572333 Trimeria grandifolia (Hochst.) Warb. AY757077 JF270978 AJ418835 Casearia commersoniana Cambess. AY757014 GQ981952 GQ981687 Casearia javitensis Kunth AY757015 AY935927 JQ626018 Casearia gladiiformis Mast. AY757008 JX517926 JX572383 Casearia praecox Griseb. AY757016 JQ589113 JQ593983 Lacistema aggregatum (P. J. Bergius) Rusby AY935787 AB233790 AB233894

2-EV Flacourtia jangomas 100/1.00 Flacourtia indica

Oncoba spinosa 84/1.00 Scolopia mundii

51/0.97 Scolopia spinosa

Xylosma congesta

Trimeria grandifolia Salicoideae 58/1.00

Xylosma chlorantha

79/0.97 Bivinia jalbertii 99/1.00 Homalium racemosum

Hasseltia floribunda 100/1.00 Salix purpurea 100/1.00 Salix suchowensis 100/1.00 Populus trichocarpa 100/1.00 65/1.00 100/1.00 Populus fremontii

Poliothyrsis sinensis Dianyuea turbinata 100/1.00 (Flacourtia turbinata) Scyphostegia borneensis

Samyda dodecandra Samydoideae 96/1.00 Casearia gladiiformis

100/1.00 Laetia procera

94/1.00 Casearia praecox

Casearia commersoniana 100/1.00 100/1.00 Casearia javitensis

Lunania parviflora

Lacistema aggregatum Outgroup

0.005

Figure 1. Majority consensus tree inferred from Bayesian analysis based on the trn L-F, mat K and rbc L sequences. Lacistema aggregatum (P. J. Bergius) Rusby (Lacistemataceae) was used as outgroup. Numbers at the nodes are maximum parsimony bootstrap percentages ( Ͼ 50%) and Bayesian posterior probabilities (Ͼ 0.90), respectively. Th e Scyphostegioideae clade, composed of Dianyuea turbinata (H. J. Dong & H. Peng) C. Shang, S. Liao & Z. X. Zhang (Flacourtia turbinata H. J. Dong & H. Peng) and Scyphostegia borneensis Stapf, is marked by a hollow rectangle.

S. borneensis were resolved as sisters with high branch support were strongly supported (BS ϭ 100%; PP ϭ 1.00). Th ere was (bootstrap, BS ϭ 100%; posterior probability, PP ϭ 1.00), only moderate support for the sister relationship between and the monophyly of both Salicoideae and Samydoideae Salicoideae and Scyphostegioideae (BS ϭ 65%, PP ϭ 1.00).

3-EV Th e genus Casearia Jacq. was not monophyletic, as also Etymology revealed in a previous comprehensive analysis (Alford 2005). Dianyuea is named after Dian-Yue ( 滇 越), an ancient king- Flacourtia turbinata diff ers from other species of dom recorded in Sima Qian ' s chronicles and located on Flacourtia in the following ways: it contains curved spines the trade route from China to India, since the type local- derived from peduncles (spines of Flacourtia species are ity of Dianyuea turbinata is also on the famous ‘ South Silk absent or erect, but never derived from peduncles), male Route ’ , adjoining a port on the boundary between Burma fl owers have six stamens connate to form a column (polys- and China. temonous and free in Flacourtia ), presence of a basal pla- centation (parietal in Flacourtia ), and fl eshy, pubescent, Description and lobed appendages around seeds (absent in Flacourtia ). Deciduous, diclinous , dioecious. Branches spread- Flacourtia turbinata resembles Scyphostegia with its connate ing, usually armed with curved spines at nodes; twigs terete, stamens and basal placentation, and is thereby separated pithy. simple, alternate, distichous, shortly petioled; from Salicoideae and Samydoideae, which usually have blade narrowly ovate to ovate-oblong, tri-nerved; veins pin- free stamens and parietal placentation. Th e seed appendage nipalmate, impressed adaxially and raised abaxially; stipules of F. turbinata is 5 – 6-parted at apex and connate into an small, linear, deciduous. Male infl orescences cymose, axil- inclined cupule surrounding the seed at base. Meanwhile, lary, pendulous; peduncles curved, persistent, transformed Heel (1967) revealed the arilloid formation of S. borneensis ; into spines after fl owers fading. Female fl owers solitary, a 5-lobed ring was observed during the early stages and then axillary; bracts linear, pubescent, deciduous; calyx petaloid, the ring became irregular branching arilloid enveloping the coalescent, campanulate; petals absent. Male fl owers with basal half of the seed. Th us, we inferred that the lobed seed calyx lobes 6– 8; stamens 6, connate in a column; fi laments appendage of F. turbinata and the arilloid of S. borneensis shorter than anthers; anthers 2-celled, dehiscing lengthwise, are homologous, which suggests a close affi nity between extrorse; pollen ellipsoid, tri-colporate. Female fl owers with these two species. However, several features of F. turbinata , calyx lobes 8– 11; ovary superior, ovoid to conical, 1-loculed; such as curved spines, linear bracts, absence of glands on styles 4 – 6, connate into a distinct column, slightly free at api- the disks, indehiscent fruits, and appendages surrounding ces; stigmas 2-lobed, dilated, recurved; placenta basal, fl eshy; the whole seed, are signifi cantly diff erent from those of ovules numerous, enveloped by fl eshy, pubescent, 5– 6-lobed S. borneensis (Table 2). appendages. ovoid to ellipsoidal, indehiscent, charac- Considering the morphological characters and the phylo- teristically longitudinally angled especially when dried. genetic position of F. turbinata , we here propose Dianyuea as a new genus of Scyphostegioideae to accommodate the new Habitat and distribution combination: Dianyuea turbinata (H. J. Dong & H. Peng) Th is genus is endemic to the Tongbiguan nature reserve in C. Shang, S. Liao & Z. X. Zhang. western Yunnan Province, China. Mountain rainforests; 1000 – 1300 m a.s.l. Dianyuea C. Shang, S. Liao & Z. X. Zhang gen. nov. Dianyuea turbinata (H. J. Dong & H. Peng) C. Shang, A genus related to Scyphostegia Stapf, but diff ering by hav- S. Liao & Z. X. Zhang comb. nov. (Fig. 2– 3) ing terete twigs (not 4-angular), curved spines at nodes, small and linear bracts (not tubular), absence of the three Basionym : Flacourtia turbinata H. J. Dong & H. Peng, glands on the disk of male fl owers, indehiscent fruits (not Phytotaxa 94 (2013, p. 56). 9 – 12-valved), ovoid ovary (not urceolate) and 5 – 6-lobed Type : China. Yunnan Province: Yingjiang County, Tong- appendages enveloping the whole seed (not surrounding biguan, elevation ca 1100 m a.s.l., 7 Jul 2012, Hua Peng only the basal half of the seed). and Hong-Jin Dong 2813 (holotype: KUN barcode Type : Dianyuea turbinata (H. J. Dong & H. Peng) C. Shang, 1217652!, isotypes: KUN barcode 1217651!, KUN barcode S. Liao & Z. X. Zhang comb. nov. 1217650!).

Table 2. Comparison of characters among Flacourtia Comm. ex L ’ H é r., F. turbinata H. J. Dong & H. Peng and Scyphostegia Stapf.

Flacourtia F. turbinata Scyphostegia Twigs round round 4-angular Spines straight or absent curved absent Infl orescence form racemes male cymes, female fl owers solitary panicles, consisting of racemosely-arranged racemes Bracts variable, not tubular linear tubular Filaments numerous, free 6, connate 3, connate Disc entire or broken to glands absent, without glands with 3 glands Placentation parietal basal basal Fruits indehiscent indehiscent 9– 12-valved Seed appendages/arilloid absent 5 – 6-lobed, enveloping the whole seed with irregular branching lobes, surrounding the basal half

4-EV Figure 2. Photographs of Dianyuea turbinata comb. nov. (A) branch, (B) male infl orescences, (C) SEM photograph of pollen, (D) seeds and appendages, (E) branch showing abaxial leaves and curved spines (photographed by Hong-Jin Dong), (F) fruit, (G) male fl ower with three sepals removed, (H) female fl owers (photographed by Yun-Fei Deng), (I) longitudinal section of fruit.

Description Infl orescences axillary; peduncles 1 – 2 cm long, pubescent, Deciduous shrub, 1– 2 m tall; bark yellow-brown; young curved, persistent, transformed into spines after fl owers branches green, pubescent, and later glabrescent, more fading; bracts linear, ca 1 ϫ 5 mm. Flowers appearing with or less zigzag. Petiole 2– 8 mm long, pubescent; blade young leaves, white to fl avescent; calyx coalescent, cam- chartaceous to thinly coriaceous, narrowly ovate or ovate- panulate, ca 1 ϫ 1 cm. Male fl owers 3 – 7, forming a cyme; oblong, 6– 12 ϫ 3 – 6 cm, acute or rounded at base, with calyx 6– 8-lobed; stamens 6, rarely 7 or 8, ca 2 mm long, margin serrate, long-acuminate at apex, tripli-nerved. connate in a column, pubescent. Female fl owers solitary;

5-EV Figure 3. Illustration of Dianyuea turbinata comb. nov. (A) branch with mature fruit, (B) abaxial leaf surface showing the abaxially raised veins, (C) longitudinal section of fruit, (D)– (E) seeds with appendages, (F) male infl orescence, (G) cut male fl ower with three sepals removed showing connate stamens, (H) and (I) female fl owers. Drawn by You-Zhi Han from Yong-Jie Guo 13CS8005 and En-De Liu 1897 in KUN, and Ce Shang 415 in BJFC. calyx 8 – 11-lobed; ovary ovoid to conical, 4 ϫ 3 mm; crowded in the fruit, spindly, blunt at apex, ca 1 cm long, styles 4– 6, united into a distinct column, ca 2 mm long. pubescent over the whole surface; appendage yellowish Fruit green, ovoid, 2.0– 2.5 cm long, characteristically lon- white, 12– 15 mm long, connate at base, deeply 5– 6-lobed gitudinally angled especially when dried. Seeds 14– 16, at apex.

6-EV Additional specimens examined Heel, W. A. van 1967. Anatomical and ontogenetic investiga- China. Yunnan Province, Yingjiang County, Tongbiguan: tions on the morphology of the fl owers and the fruit of 18 Apr 2007, En-De Liu 1897 (♀ , KUN); 23 Apr 2013, Scyphostegia borneensis Stapf (Scyphostegiaceae). – Blumea Yong-Jie Guo 13CS7442 (♂ , KUN); 26 Aug 2013, Yong-Jie 15: 107 – 125. Katoh, K. et al. 2005. MAFFT ver. 5: improvement in accuracy of Guo 13CS8005 ( ♀, KUN); 2 Apr 2015, Ce Shang 415 (♂ , ♂ multiple sequence alignment. – Nucl. Acids Res. 33: BJFC); 21 Apr 2016, Yi-Fei Xie XZ532 ( , BJFC). 511 – 518. Olmstead, R. G. et al. 1992. Monophyly of the Asteridae and identifi cation of their major lineages inferred from Acknowledgements – Th e authors thank Mac H. Alford (Univ. of DNA sequences of rbc L. – Ann. Miss. Bot. Gard. 79: Southern Mississippi) for providing us the electronic copy of his 249 – 65. doctoral dissertation, and Yun-Fei Deng (CAS) for his photographs. Posada, D. and Buckley, T. R. 2004. Model selection and model We are indebted to the help and advice from Hong-Jin Dong, Hua averaging in phylogenetics: advantages of Akaike information Peng (CAS), Qin-Wen Lin (CAS), Su Liu (Shanghai Chenshan criterion and Bayesian approaches over likelihood ratio tests. Botanical Garden), Yan-Feng Zhang (Beijing Forestry Univ.) and – Syst. Biol. 53: 793 – 808. Sue Zmarzty (Royal Botanic Gardens, Kew), and the illustration Posada, D. and Crandall, K. A. 1998. Modeltest: testing the model drawn by You-Zhi Han. Th is study was funded by Science and of DNA substitution. – Bioinformatics 14: 817 – 818. Technology Basic Work (grant no. 2013FY112100), and the Ronquist, F. and Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian National Natural Science Foundation of China (grant no. phylogenetic inference under mixed models. – Bioinformatics 31110103911, and J1310002). 19: 1572 ‒ 1574. Swoff ord, D. L. 2002. PAUP* : Phylogenetic Analysis Using Parsimony (* and other methods), ver. 4.0b10. – Sinauer References Associates. Stapf, O. 1894. On the fl ora of mount Kinabalu, in north Borneo. Alford, M. H. 2005. Systematic studies in Flacourtiaceae. – PhD – Trans. Linn. Soc. Lond. Bot. 4: 69– 263. thesis, Cornell Univ., USA. Steenis, C. G. G. J. van 1957. Scyphostegiaceae. – Flora Malesiana. Angiosperm Phylogeny Group 2016. An update of the Angiosperm Ser. I. Vol. 5. Noordhoff -Kolff N. V., pp. 297 – 299. Phylogeny Group classifi cation for the orders and families of Stevens, P. F. 2001 onwards. Angiosperm Phylogeny Website, ver. fl owering plants: APG IV. – Bot. J. Linn. Soc. 181: 1 – 20. 13. – < http://www.mobot.org/MOBOT/research/APweb/ > , Chase, M. W. et al. 2002. When in doubt, put it in Flacourtiaceae: accessed 27 Jul 2016. a molecular phylogenetic analysis based on plastid rbc L DNA Taberlet, P. et al. 1991. Universal primers for amplifi cation of three sequences. – Kew Bull. 57: 141 – 181. non-coding regions of chloroplast DNA. – Plant Mol. Biol. Dong, H. J. and Peng, H. 2013. Flacourtia turbinata (Salicaceae: 17: 1105 – 1109. Flacourtieae), a new species from Yunnan, China. – Phytotaxa Xi, Z. X. et al. 2012. Phylogenomics and a posteriori data partitioning 94: 56 – 60. resolve the Cretaceous angiosperm radiation . Hall, T. A. 1999. BioEdit: a user-friendly biological sequence – Proc. Natl Acad. Sci. USA 109: 17519 – 17524. alignment editor and analysis program for Windows 95/98/ Yu, J. et al. 2011. New universal mat K primers for DNA barcoding NT. – Nucl. Acids Symp. Ser. 41: 95 – 98. angiosperms. – J. Syst. Evol. 49: 176 – 181.

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