New Zealand Journal of Botany ISSN: 0028-825X (Print) 1175-8643 (Online) Journal homepage: http://www.tandfonline.com/loi/tnzb20 Molecular evidence for a natural hybrid between Isolepis crassiuscula and Isolepis lenticularis (Cyperaceae) in New Zealand O Yano, N Tanaka & Y Ito To cite this article: O Yano, N Tanaka & Y Ito (2016): Molecular evidence for a natural hybrid between Isolepis crassiuscula and Isolepis lenticularis (Cyperaceae) in New Zealand, New Zealand Journal of Botany, DOI: 10.1080/0028825X.2016.1205106 To link to this article: http://dx.doi.org/10.1080/0028825X.2016.1205106 Published online: 25 Jul 2016. Submit your article to this journal Article views: 57 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tnzb20 Download by: [FU Berlin] Date: 18 November 2016, At: 05:03 NEW ZEALAND JOURNAL OF BOTANY, 2016 http://dx.doi.org/10.1080/0028825X.2016.1205106 RESEARCH ARTICLE Molecular evidence for a natural hybrid between Isolepis crassiuscula and Isolepis lenticularis (Cyperaceae) in New Zealand O Yanoa, N Tanakab and Y Itoc,d aDepartment of Biosphere–Geosphere Science, Faculty of Biosphere–Geosphere Science, Okayama University of Science, Okayama, Japan; bTsukuba Botanical Garden, National Museum of Nature and Science, Tsukuba, Japan; cSchool of Biological Sciences, University of Canterbury, Christchurch, New Zealand; dXishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Kunming, PR China ABSTRACT ARTICLE HISTORY Isolepis R.Br. (Cyperaceae) is a genus of aquatic, wetland, or Received 6 February 2016 ephemeral sedges that comprises 76 species, mostly in the Accepted 10 June 2016 southern hemisphere, especially Africa and Australasia. Isolepis KEYWORDS crassiuscula Hook.f., Isolepis lenticularis R.Br. and Isolepis producta Aquatic plants; Australasia; (C.B. Clarke) K.L.Wilson are among the Australasian members of a fi Fluitantes chloroplast DNA; Cyperaceae; morphologically well-de ned subgenus . Specimens hybridisation; New Zealand; belonging to this subgenus, collected from Mount Tongariro, New Isolepis; internal transcribed Zealand, showed ambiguous yet somewhat intermediate spacer morphology compared with these three species. We sequenced directly, or cloned and sequenced, nuclear DNA (nrITS) and chloroplast DNA (rbcL, rps16 and trnL) regions of these collections and samples of the three aforementioned species of subgenus Fluitantes. Ribotypes and chloroplast haplotypes were compared to resolve if the Tongariro collection was of hybrid origin and, if so, to address which of the three species may have been involved in the hybridisation event. The Tongariro plants had the species- specific ribotypes of I. crassiuscula and I. lenticularis and the chloroplast haplotype of I. lenticularis, strongly suggesting hybrid origin between them. No data support the inclusion of I. producta as a parent. The morphologically uniform population from which the Tongariro plants were collected is likely to be derived from a single hybridisation event, although this hypothesis merits further investigation. Introduction Hybridisation is recognised as a widespread and important mode of evolution in plants (e.g. Rieseberg 1995; Yakimowski & Rieseberg 2014). However, spontaneous hybridisation is not distributed evenly across plant groups, but seems to be more common in taxa that have a perennial life cycle, outcrossing breeding systems, and the possibility of clonal reproduction (Ellstrand et al. 1996). Cases of interspecific hybridisation are reported in the sedge family (Cyperaceae), especially in the genus Carex L. (e.g. Smith & Waterway 2008; Volkova et al. 2008; Gizaw et al. 2016), yet the frequency and extent of hybridisation CONTACT Okihito Yano [email protected] © 2016 The Royal Society of New Zealand 2 OYANOETAL. in the other genera of the family has been only partially explored (Košnar et al. 2010; Yano et al. 2010). Isolepis R.Br. (Cyperaceae) is a genus of aquatic, wetland or ephemeral sedges that com- prises 76 species mostly in the southern hemisphere, especially Africa and Australasia (Australia and New Zealand) (Muasya & Simpson 2002; Govaerts et al. 2011). This per- ennial, clonal genus (with unknown reproductive system) includes putative hybrids in New Zealand: Isolepis aucklandica Hook.f. × Isolepis cernua (Vahl) Roem & Schult., Isole- pis distigmatosa (C.B.Clarke) Edgar × Isolepis prolifera (Rottb.) R.Br. and Isolepis inundata R.Br. × I. prolifera (Edgar 1970). In Africa, Gordon-Gray (1995) hypothesised that ‘Isolepis angelica B.L.Burtt may have arisen by … interspecific hybridisation’ because of its mor- phology that combines the characters of Isolepis fluitans (L.) R.Br. and Isolepis setacea (L.) R.Br., without discussing whether I. angelica is a homoploid hybrid, allotetraploid, or a hybrid species (Gordon-Gray 1995). No data suggesting that allopolyploid have occurred in Isolepis. Subgenus Fluitantes is one of the four subgenera of Isolepis and is a group disjunctively distributed in Africa and Australasia that includes, among other species, Isolepis crassius- cula Hook.f., Isolepis lenticularis R.Br. (formerly I. fluitans var. lenticularis (R.Br.) Muasya) and Isolepis producta (C.B.Clarke) K.L.Wilson (Muasya & Simpson 2002; Ito et al. 2016). These three species are characterised by the morphological characters ‘involucral bract shorter than spikelet’, ‘spikelet always terminal, bract shorter or as long as spikelet’ and ‘style always bifid ’, and are distinguishable from one another by the morphological fea- tures of the spikelets, anthers and fruit (Ito et al. 2016; Table 1). The habitat preferences of these species are similar in Australasia, though no evidence is available that they occur synpatrically, as, for instance, Wilson (1994) mentions that ‘…I. crassiuscula occurs at higher altitudes than either of those species (I. producta and I. lenticularis (I. fluitans in Wilson 1994))’: I. crassiuscula grows in high-altitude bogs, at water margins or submerged, at 700–3700 m altitude; I. lenticularis in shallow pools and seepages at c. 800 m altitude (I. fluitans var. lenticularis in Muasya & Simpson 2002); I. producta in ponds at 800– 1400 m in altitude (Muasya & Simpson 2002). We recently made collections of Isolepis subgenus Fluitantes from a single population of c. 2 m × 30 cm clumped in a small Table 1. A morphological comparison of three Isolepis species and a hybrid among them in New Zealand. Taxon Anthera Glumeb Spikeletb Spikeletb Spikeletb Spikeletc Stamenc Isolepis (0.7–) 1.5– (2.4–) 3.4– Ovate to broad- 12–40 5–9mm (2–)3– 3 crassiusculad 2.5 mm 4.5 mm elliptic in outline; flowered long 5mm only slightly flatted wide Isolepis (0.4–)1– 1.7– More or less elliptic in 5–10 (–15) 3–5mm 1–2mm 2 (rarely lenticularise 1.5 mm 2.8 mm outline; slender; flowered long wide 3) strongly flatted Isolepis 1.5– 2.5– More or less elliptic in 5–10 (–15) 3–5mm 1–2mm 3 producta 2.7 mm 3.3 mm outline; slender; flowered long wide strongly flatted Isolepis hybrid 0.7– 2.5– Elliptic; flatted 8–12 4–5mm 1–2mm 3 1.3 mm 3.4 mm flowered long wide aMuasya & Simpson (2002). bWilson (1994). cEdgar (1970). dAs Scirpus crassiusculus in Edgar (1970). eAs Scirpus fluitans in Edgar (1970). NEW ZEALAND JOURNAL OF BOTANY 3 stream at c. 1100 m in altitude in Mount Tongariro in New Zealand that show ambiguous yet somewhat intermediate morphology among these three species, although I. producta does not occur in New Zealand (Figure 1, Table 1). Because natural hybrids often display intermediate morphology between putative parental taxa (Rieseberg et al. 2000), and this also applies to Cyperaceae hybrids (Košnar et al. 2010; Yano et al. 2010), this Figure 1. Isolepis crassiuscula × Isolepis lenticularis from Mount Tongariro, New Zealand. 4 OYANOETAL. could be a product of inter-specific hybridisation among the morphologically and ecolo- gically closely related species. This hypothesis does not disagree with the fact that our Mt Tongariro Isolepis collections at least do not set mature fruits but only immature and apparently malformed ones. Molecular markers have been proven useful in detecting hybrids and determining the parents (Soltis et al. 1992). For this purpose, the internal transcribed spacer (ITS) region of nuclear ribosomal (nr) DNA (hereinafter called nrITS) is of particular merit because this can provide evidence of reticulate evolution when the hybrid retains multiple sequence copies contributed by each parent (Baldwin et al. 1995; Soltis et al. 2008), and numerous studies have confirmed hybrid origin using nrITS (e.g. Sang et al. 1995; Campbell et al. 1997; Fuertes et al. 1999; Moody & Les 2002; Saito et al. 2006; Pan et al. 2008; Lee et al. 2012). Additionally, assuming maternal inheritance of the chloroplast genome in Cyperaceae, as in the majority of plant groups (Birky 1995), the simultaneous usage of nrITS and chloroplast DNA (here- inafter called cpDNA) markers will clarify the direction of pollen flow in hybridisation. The aim of this study was to test the morphology-based hypothesis that this population of Isolepis from Mt Tongariro in New Zealand is of hybrid origin. To do so, we employed DNA sequence comparison of nuclear DNA (nrITS) and cpDNA (rbcL, rps16 and trnL) and sought evidence of hybridisation, i.e. polymorphism pattern of nrITS. We further determined which of the three aforementioned species from subgenus Fluitantes were involved in the hybridisation event. Materials and methods Taxon sampling