See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257053219 Identity and origins of introduced and native Azolla species in Florida Article in Aquatic Botany · July 2013 DOI: 10.1016/j.aquabot.2013.07.009 CITATION READS 1 287 6 authors, including: Paul T. Madeira Julie Angela Coetzee United States Department of Agriculture Rhodes University 26 PUBLICATIONS 289 CITATIONS 59 PUBLICATIONS 485 CITATIONS SEE PROFILE SEE PROFILE Matthew F Purcell Martin P. Hill The Commonwealth Scientific and Industrial … Rhodes University 61 PUBLICATIONS 746 CITATIONS 197 PUBLICATIONS 1,639 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Nutritional ecology of Erebid moths View project Biological Control of Weeds View project All content following this page was uploaded by Paul T. Madeira on 24 March 2017. The user has requested enhancement of the downloaded file. 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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Aquatic Botany 111 (2013) 9–15 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot Identity and origins of introduced and native Azolla species in Florida Paul T. Madeira a,∗, Ted D. Center a, Julie A. Coetzee b, Robert W. Pemberton a, Matthew F. Purcell c, Martin P. Hill b a United States Department of Agriculture, Agriculture Research Service, Invasive Plant Research Laboratory, 3225 College Avenue, Ft. Lauderdale, FL 33314, United States b Department of Zoology and Entomology, Rhodes University, Grahamstown, South Africa c Matthew Purcell. CSIRO Ecosystem Sciences, US Department of Agriculture, Agriculture Research Service, Australian Biological Control Laboratory, GPO Box 2583, Brisbane, Qld 4001, Australia article info abstract Article history: Azolla pinnata, an introduced aquatic fern, is spreading rapidly causing concern that it may displace native Received 6 August 2012 Azolla. It is now present in the Arthur R. Marshall Loxahatchee National Wildlife Refuge, the northern- Received in revised form 26 July 2013 most portion of the Florida Everglades. Because A. pinnata subspecies are native to Africa, Southeast Accepted 29 July 2013 Asia, and Australia, determining the actual geographic origin of the Florida exotic is important to the Available online 7 August 2013 discovery of efficacious biological control agents. Both the exotic and native Azollas were examined using morphological and molecular criteria. Both criteria distinguished three A. pinnata subspecies with the Keywords: Florida exotic matching the Australian A. pinnata subsp. pinnata. Molecular divergence between the A. Azolla Biocontrol pinnata subspecies indicates the three types should be considered separate species. The Florida native Exotic was characterized by both molecular and morphological methods as Azolla caroliniana. The discovery of Invasive a previously uncharacterized Ecuadorian Azolla, which appears to be a paternal ancestor of A. caroliniana, Listed Federal Noxious Weed indicates that A. caroliniana is a hybrid species. Molecular taxonomy Published by Elsevier B.V. Morphology 1. Introduction Florida’s native Azolla has historically been considered to be Azolla caroliniana Willd. (Godfrey et al., 1961; Wunderlin and The floating aquatic fern Azolla pinnata R. Br. (Azollaceae), a Hansen, 2003). However there is a paucity of information as to how listed Federal Noxious Weed (U.S.A.), was discovered in Palm Beach this identification was determined. Evrard and Van Hove (2004) County, Florida during 2007 (Bodle, 2008). The South Florida Water have noted that the taxonomy of New World Azolla species has Management District immediately but unsuccessfully sought erad- been “the subject of much debate and remains unsatisfactory”. The ication by treatment with diquat dibromide. The District’s concern status of A. caroliniana is perhaps the most debatable within the stemmed from the invasive potential demonstrated by various genus because Willdenow (1810) described the holotype A. carolin- species of Azolla worldwide. Quick regeneration, rapid growth, iana (“habitat in aquis Carolinae”) from a sterile specimen. This has broad distribution, and dense surface mats of Azolla can obstruct led to various interpretations including that the original specimen weirs, locks, and water intakes impeding irrigation, boating, fishing, was actually Azolla filiculoides Lamarck (Dunham and Fowler, 1987; and recreational activities (McConnachie et al., 2003; Hashemloian Evrard and Van Hove, 2004). Regardless of what the holotype rep- and Azimi, 2009; Baars and Caffrey, 2010). A dense surface cover resents, an “A. caroliniana” species with a unique megaspore perine may also reduce aquatic oxygen levels (Janes et al., 1996) and sub- structure exists (Perkins et al., 1985; Pereira et al., 2001). mersed animal populations (Gratwicke and Marshall, 2001). There The broad geographic range of A. pinnata encompasses Africa, is also concern that if A. pinnata becomes invasive, it will compete Madagascar, India, China, southeast Asia, Japan, Malaysia and for the same niche and suppress native populations of Azolla in Australia. Saunders and Fowler (1992), in a morphological taxo- Florida. This concern seems warranted inasmuch as introduced A. nomic revision of Azolla Lam. section Rhizosperma, identified three pinnata has largely replaced the native Azolla rubra R. Br. in northern geographically distinct subspecies of A. pinnata: (1) A. pinnata New Zealand (Owen, 1996). R.Br. subsp. pinnata R.Br. from Australia and New Caledonia; (2) A. pinnata R.Br. subsp. asiatica Saund. & Fowl. from India, south- ern China, Southeast Asia, and southern Japan; and (3) A. pinnata ∗ Corresponding author. Tel.: +1 9544756553; fax: +1 9544769169. subsp. africana (Desv.) Saund. & Fowl., from tropical Africa and E-mail address: [email protected] (P.T. Madeira). Madagascar. However, Pereira et al. (2011), using morphological 0304-3770/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquabot.2013.07.009 Author's personal copy 10 P.T. Madeira et al. / Aquatic Botany 111 (2013) 9–15 and molecular (RAPD) criteria, suggested that A. pinnata is actu- Fresh material of the Florida native sample was also examined ally best described as two varieties, A. pinnata R.Br. var. imbricata using the vegetative criteria in Pereira et al. (2011). Additionally (Roxb.) Bonap. from Asia and A. pinnata var. pinnata R.Br. from Africa a VHX-600 3-dimensional digital microscope (Keyence Corpora- and Australia. tion, USA) was used to examine several microsporocarps and a Classical biological control is often used to address the com- single megasporocarp. Massulae were isolated from microspores plex and multi-faceted problems of exotic alien plant invasions and their glochidia (barbed hairs) examined. A single megaspore by introducing specialized natural enemies derived from native was teased from its megasporocarp wall and examined. regions. This process usually begins with determinations of the identity of the plant and its native range followed by surveys for potential agents, an expensive and often difficult process. Because 2.3. Molecular methods exotic invasive species frequently have large native ranges (Lodge, 1993) the actual geographic origin may not be apparent. Biologi- DNA was extracted using the DNeasy Plant Mini kit (Qiagen Inc., cal control agents that have coevolved with the invasive host plant Valencia, CA, USA). Two plastid genes, trnL-trnF (includes the trnL are most likely to be successful in suppressing it (Hufbauer and intron and the trnL-F intergenic spacer) and trnG-trnR (includes Roderick, 2005; Ward, 2006; Mills and Kean, 2010). When regional trnG and the trnG-R intergenic spacer), as well as the nuclear ITS1 intra-specific genetic variation is ignored, both within the invasive sequence were amplified and sequenced. Primers used for ampli- plant and co-evolved natural enemies, it has the potential to con- fication and sequencing were TrnLC, TrnLD, TrnLE and TrnLF for found the efficacy of the insects chosen (Goolsby et al., 2006; Le trnL-trnF (Taberlet et al., 1991) and TrnG1F, TrnG43F1, TrnG63R, and Roux and Wieczorek, 2009) resulting in failure to control the target TrnR22R for trnG-trnR (Nagalingum et al., 2007). The nuclear ITS1 weed (Palmer and Witt, 2006). Weed biological control programs primers were the ITS-A, ITS-B, ITS-C and ITS-D primers of Blattner are increasingly using molecular techniques to determine the ori- (1999). PCR annealing temperatures were 56 ◦C for trnL-trnF,52◦C gins of the targeted plants and to geographically match biotypes of for trnG-trnR and 58 ◦C for ITS1. Plastid reaction mixtures con- prospective agents with the target weed (Von Senger et al., 2000; tained 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Bond et al., 2002; Goolsby et al., 2006; Paterson et al., 2009). Triton X-100, 0.5 mM Betaine, 0.001% BSA, 0.2 mM dNTPs, 0.5 ␮M Identification of the subspecies (or variety, implied hereafter)of each primer, and 0.06 U/␮l EconoTaq polymerase (Lucigen Corp., A. pinnata recently found in Florida (Pemberton and Bodle, 2009) Middleton, WI, USA). The ITS1 reaction contained 10 mM Tris–HCl would enable a search for biocontrol agents in the appropriate (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 10% DMSO, geographic area.