Plant Syst Evol (2016) 302:231–238 DOI 10.1007/s00606-015-1253-7 SHORT COMMUNICATION Phylogeny of Isolepis (Cyperaceae) revisited: non-monophyletic nature of I. fluitans sensu lato and resurrection of I. lenticularis 1,5 2 3 4 Yu Ito • Jan-Adriaan Viljoen • Norio Tanaka • Okihito Yano • A. Muthama Muasya2 Received: 18 March 2015 / Accepted: 19 September 2015 / Published online: 30 October 2015 Ó Springer-Verlag Wien 2015 Abstract The aquatic and wetland ephemeral genus Australasian species. The overall morphological evidence Isolepis (Cyperaceae) comprises 76 species mostly in the conflicts with the species recognition of I. fluitans sensu southern hemisphere, and especially Africa and Australa- lato and rather indicates the non-monophyly of I. fluitans, sia. The latest taxonomic revision recognizes three sub- which we tested in a phylogenetic framework. Sequence genera (Fluitantes, Isolepis and Micranthae) and three data from three plastid DNA regions and nuclear ITS were sections in subgen. Isolepis. Subgen. Fluitantes, mat- analyzed using maximum parsimony, maximum likelihood, forming perennial herbs typically bearing a single terminal and Bayesian inference. We obtained moderately resolved spikelet, comprises nine species with a nearly cosmopolitan phylogenies with the plastid DNA and ITS data sets. distribution except in Americas and Antarctica. Of these, Although partially conflicting, both phylogenies rejected I. fluitans includes infraspecific taxa from Africa–Europe the monophyly of I. fluitans and instead revealed inter- and Asia–Australasia that are distinguished by the length of continental pattern with infraspecific taxa showing close the involucral bract relative to the spikelet. This morpho- relationships with species in the subgenus within their logical character is also used in the key to subgen. geographic area. A revised key to species of subgenus Fluitantes that separates African–European and Asian– Fluitantes is provided with the Asian–Australasian I. flui- tans var. lenticularis resurrected to species rank as I. len- ticularis. The phylogeny reveals a single dispersal event from Africa to Australasia, or vice versa in subgen. Handling editor: Pablo Vargas. Fluitantes. Electronic supplementary material The online version of this article (doi:10.1007/s00606-015-1253-7) contains supplementary Keywords Aquatic plants Á Cyperaceae Á Isolepis Á ITS Á material, which is available to authorized users. Molecular phylogeny Á Plastid DNA & Yu Ito [email protected] Introduction 1 Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo 112-0001, Japan 2 Isolepis R.Br. (Cyperaceae) is an aquatic and wetland or Department of Biological Sciences, University of Cape ephemeral plant genus that comprises 76 species mostly in Town, Rondebosch 7701, South Africa 3 the southern hemisphere, and especially Africa and Aus- Tsukuba Botanical Garden, National Museum of Nature and tralasia (Muasya 1998;WCSP2015). This sedge genus, Science, Tsukuba 305-0005, Japan 4 defined as having ‘‘bisexual flowers with glumes spirally Faculty of Biosphere-Geosphere Science, Okayama (or occasionally distichously) arranged’’ (Muasya and University of Science, Okayama 700-0005, Japan Simpson 2002; Muasya et al. 2006, 2007), has been phy- 5 Present Address: Plant Phylogenetics & Conservation Group, logenetically studied based on morphological (Muasya and Centre for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Simpson 2002) and molecular data (Muasya et al. 2001, Kunming 650223, People’s Republic of China 2009; Muasya and de Lange 2010). Although the Ficinia- 123 232 Y. Ito et al. Isolepis clade was strongly supported ([90 % Bootstrap Materials and methods support; Muasya et al. 2009), none of these studies recovered Isolepis as monophyletic and instead, for Taxon sampling example, dispersed the genus at least into three weakly supported clades (Muasya et al. 2009). With a primal focus on Isolepis subgen. Fluitantes,we Muasya and Simpson (2002) revised the taxonomy of obtained a total of 43 samples (41 unique OTUs), which the genus Isolepis, recognizing three subgenera (Isolepis, were equivalent to 32 species from all subgenera and Fluitantes (C.B.Clarke) Muasya and Micranthae sections of Isolepis sensu Muasya and Simpson (2002) (C.B.Clarke) Muasya) and three sections in subgenus Iso- including five samples of each of the three varieties of lepis (Isolepis, Cernuae (C.B.Clarke) Muasya and Prolif- I. fluitans (Online Resource 1). The sample set contained erae Muasya). Subgen. Fluitantes is defined as being those used in the previous molecular phylogenetic studies ‘‘plants mat-forming, with above ground rhizomes; spikelet (Muasya et al. 2001; Hirahara et al. 2007a, 2007b; Muasya single and not proliferating’’ and has been previously et al. 2009; Muasya and de Lange 2010; Yano et al. 2012) classified as the segregate genus Eleogiton Link (Muasya and newly added 15 samples, including I. pottsii and and Simpson 2002). Isolepis fluitans is among the nine I. reticularis, species new to molecular phylogenetic species of subgen. Fluitantes and includes three varieties studies. that are in part distinguished by the length of involucral The outgroup included Dracoscirpoides falsa, Ficinia bract relative to the spikelet: Australasian var. lenticularis pinguior, and Hellmuthia membranacea, following Muasya (‘‘longer’’) and African–European var. fluitans and Ethio- et al. (2001, 2009) and Muasya and de Lange (2010). pian endemic var. nervosa (‘‘shorter’’) (Muasya and Simpson 2002). This morphological character is also found DNA extraction, amplification and sequencing in the key to species of subgen. Fluitans provided by Muasya and Simpson (2002; Table 1), where ‘‘longer’’ is Total genomic DNA was extracted from either silica gel- given for Australasian I. crassiuscula and I. producta, dried leaf tissues or herbarium specimens using the CTAB while ‘‘shorter’’ is for South African Cape endemic I. ru- method described in Ito et al. (2010). The targeted DNA bicunda and I. striata. Besides, the other African members regions, three from plastid DNA (ptDNA; rbcL, rps16, and of subgen. Fluitantes had involucral bracts ‘‘shorter’’ than trnL) and one from nuclear DNA (ITS), were amplified spikelets: I. graminoides; I. inyangensis; I. ludwigii using the following primers: rbcL-1F (Fay et al. 1997) and (Muasya and Simpson 2002). Meanwhile, the length of rbcL-729R (50-CTTCGCATGTACCTGCAGTAGC-30; glumes and the length of anthers divide I. fluitans, modified from Fay et al. 1997) plus rbcL-636F (Asmussen I. graminoides, I. inyangensis, plus I. ludwigii and I. cras- and Chase 2001) and rbcL-1379R (Little and Barrington siuscula, I. producta, I. rubicunda, plus I. striata (Muasya 2003) for rbcL, ‘‘c’’ and ‘‘d’’ for the trnL intron (Taberlet and Simpson 2002; Table 1). These characters, however, et al. 1991), rpsF and rpsR2 for rps16 (Oxelman et al. need to be reevaluated because, for example, in the Flora of 1997), and ITS-4 and ITS-5 for ITS (Baldwin 1992). PCR Victoria, Australia, I. crassiuscula, I. fluitans, and I. pro- amplification was performed following the procedure of Ito ducta are largely overlapped (Wilson 1994; Table 1). The et al. (2010). overall morphological evidence conflicts with the species Sequences of the rbcL, rps16, and trnL, and ITS were recognition of I. fluitans sensu Muasya and Simpson (2002) aligned using the software Mafft (ver. 7.058; Katoh and and rather indicates that the Australasian and African plus Standley 2013) and then edited manually. Gaps associated African–European varieties of I. fluitans have close rela- with mononucleotide repeats were not included in the tionships with the respective geographical relatives in phylogenetic analyses, because homology assessment can subgen. Fluitantes. be difficult for these repeated nucleotides (Kelchner 2000) The primary aim of this study was to test the monophyly and they might be technical artifacts of the PCR amplifi- of Isolepis fluitans sensu Muasya and Simpson (2002). To cation (Clarke et al. 2001). Ambiguously aligned regions, do so, we performed simultaneous molecular phylogenetic found in trnL, were excluded from the analyses. analyses based on plastid DNA (ptDNA) and nuclear DNA Phylogenetic inference was determined using maximum (nDNA) data sets. For ptDNA markers, two regions (rbcL, parsimony (MP) in PAUP* (ver. 4.0b10; Swofford 2002), trnL) of Muasya et al (2001) and one region (rps16) of maximum likelihood (ML) in the RAxML web-server Muasya and de Lange’s (2010) were chosen and combined program (Stamatakis et al. 2008), and Bayesian inference altogether. The nuclear ITS data set that Muasya and de (BI) in MrBayes (ver. 3.2.2; Ronquist et al. 2012). The Lange (2010) used was expanded and then analyzed incongruence length difference test (Farris et al. 1994) was separately. conducted to test congruence between the four DNA 123 Phylogeny of Isolepis Table 1 Distribution and some morphological characters of Isolepis subgen. Fluitantes : non-monophyly of Distribution Involucral Spikeleta Length of involucral bract over Glumesa,c Glumesd Anthersa,c Anthersd bracta (mm) spikeleta,b Isolepis beccarii Indonesia 4–18 9 0.3–0.7 3.1–7.3 9 1.5–2.7 mm Longer 1.9–3 9 0.9-1.2 mm N/A 0.4–0.7 mm N/A long I. crassiuscula Australasia, 3–10 9 0.6–1.6 3.7–11.2 9 1.9–3.6 mm Equala 2.7–4.5 9 0.8-1.6 mma 3–4 mm 0.7–1.5 mm 0.8–1.5 mm Asia long longa long I. fluitans I. fluitans var. Africa, 2–12 9 0.3–1.3 2.4–9.4 9 0.7–2.7 mm Shortera 1.5–3.4 9 0.5-1.3 mmb N/A 0.4–1.5 mm N/A fluitans Europe longb I. fluitans var. Australasia 2–12 9 0.3–1.3 2.4–9.4 9 0.7–2.7 mm Longera 1.5–3.4 9 0.5-1.3 mmb 1.7–2.8 mm 0.4–1.5 mm 0.5–1.0 mm and resurrection of lenticularis long longb long I. fluitans var. Africa 2–12 9 0.3–1.3 2.4–9.4 9 0.7–2.7 mm Shortera 1.5–3.4 9 0.5-1.3 mmb N/A 0.4–1.5 mm N/A nervosa longb I.