Molecular Ecology (2006) 15, 287–297 doi: 10.1111/j.1365-294X.2005.02788.x MatchingBlackwell Publishing Ltd the origin of an invasive weed for selection of a herbivore haplotype for a biological control programme JOHN A. GOOLSBY,*† PAUL J. DE BARRO,‡ JEFFREY R. MAKINSON,†,‡ ROBERT W. PEMBERTON,§ DIANA M. HARTLEY¶ and DONALD R. FROHLICH** *United States Department of Agriculture, Agricultural Research Service, Beneficial Insects Research Unit, 2413 E. Hwy. 83, Weslaco, TX 78596, USA, †United States Department of Agriculture, Agricultural Research Service, Australian Biological Control Laboratory, CSIRO Long Pocket Laboratories, Indooroopilly, Queensland 4068, Australia, ‡CSIRO Entomology, Long Pocket Laboratories, 120 Meiers Rd, Indooroopilly, Queensland 4068, Australia, §United States Department of Agriculture, Agricultural Research Service, Invasive Plant Research Laboratory, 3205 College Ave. Ft. Lauderdale, FL 33314, USA, ¶CSIRO Entomology, Black Mountain Laboratories, Clunies Ross St., ACT 2601, Australia, **Department of Biology, University of St Thomas, 3800 Montrose Blvd, Houston, TX 77006, USA Abstract The Florida Everglades have been invaded by an exotic weed fern, Lygodium microphyllum. Across its native distribution in the Old World tropics from Africa to Australasia it was found to have multiple location-specific haplotypes. Within this distribution, the climbing fern is attacked by a phytophagous mite, Floracarus perrepae, also with multiple haplotypes. The genetic relationship between mite and fern haplotypes was matched by an overarching geographical relationship between the two. Further, mites that occur in the same location as a particular fern haplotype were better able to utilize the fern than mites from more distant locations. From a biological control context, we are able to show that the weed fern in the Everglades most likely originated in northern Queensland, Australia/Papua New Guinea and that the mite from northern Queensland offers the greatest prospect for control. Keywords: agent selection, Australia, biological invasions, co-evolution, Floracarus, Florida Everglades, Lygodium, phylogeography Received 5 April 2005; revision received 19 July 2005; accepted 4 October 2005 However, local adaptation is not always apparent, and the Introduction reverse, local maladaptation, where locally derived species The interaction between phytophagous insects and their perform worse than their nonlocal counterparts, occurs hosts, and the resultant evolution of means by which to (Kaltz et al. 1999). The idea here is that a herbivore that has thwart or enhance the capacity of herbivores to feed on no direct experience with a particular plant species may plants, raises the question of the importance of co- bring with it the capacity to nullify the target’s defences evolution in enabling insects to feed on particular plant as it in turn has had no experience with the herbivore species. One school of thought considers that locally and therefore no opportunity to develop the necessary occurring herbivores with shorter generation times than defences (Hokkanen & Pimentel 1984, 1989; Pimentel their host have the opportunity to adapt more quickly to 1991). Roy (1998) identified two examples, one involving a that host than those not in regular exposure (Kniskern & species of Puccinia, a rust pathogen on Arabis holboellii, Rausher 2001 for a review). In such cases, these herbivores while the other involved the larvae of a species of Pieris are described as exhibiting local host adaptation (Ebert that also feeds on A. holboellii. The two examples cited here 1994; Gandon & Van Zandt 1998). Karban (1989) are successful examples of biological control — the act of demonstrated local adaptation between the host, Erigeron deliberately introducing an exotic species to control an glaucus, and the thrips herbivore, Apterothrips secticornus. exotic species of pest, in these cases both weeds. In short, the aim of weed biological control is to introduce a species Correspondence: John A. Goolsby, Fax: +1-956-969-4888; E-mail: that has the capacity to avoid or overcome the defences of [email protected] the host, and so dramatically reduce the numbers of the © 2006 Blackwell Publishing Ltd 288 J. A. GOOLSBY ET AL. target weed to a point where it is no longer a problem. were removed from the galls and placed in 70% alcohol for Practitioners of biological control thus face a problem in analysis. In total there were 25 collection sites. For 15 sites selecting control organisms; where to go to find effective two or more ferns were collected, however, at nine sites natural enemies? Is it best to search the geographical origin only a single fern was found and the fern from Simar- of the weed for herbivores, or do they take the local jarunjung, Sumatra, Indonesia, produced no amplification maladaptation approach and search areas away from the product. origin for ‘new associations’? To date attempts to shed light on which is the most prof- DNA extraction itable approach have been to a large extent undertaken after releases have taken place. A strategy that enables this One–square-centimetre portions of the fern fronds were decision to be made prior to release may overcome one of processed using Bio101® Systems FastDNA® Kit for the principal weaknesses of the practice of biological con- plants in conjunction with FastPrep® Instrument. Pooled trol — its lack of predictability in terms of establishment samples of 5–15 mites were processed using QIAGEN and success (Greathead 1986; Ehler 1990; Harris 1998). DNeasy® Tissue Kit. While predictive tools based on pre-release evaluation of prospective agents — by default the assessment of the her- Amplification and sequencing bivore’s ability to avoid the host’s defences (Ehler 1990; Roush 1990; Waage 1990), climate matching between the For the fern, fast-evolving genes such as mitochon- pest and prospective control agents (DeBach 1964; Roush drial cytochrome oxidase 1 (CO1), ribosomal RNA ITS1 1990; Sutherst et al. 1991; Cameron et al. 1993) and assess- and amplified fragment length polymorphisms (AFLPs) ment of intraspecific variation within the agent (Roush showed no differences between the non-African accessions. 1990; Goldson et al. 1997) — have been promoted, rarely has Sequence data from two chloroplast introns were therefore their value been tested as part of the pre-release phase of used to compare the Lygodium microphyllum samples. classical biological control programmes. Our study seeks These regions were chosen as being the fastest-evolving to do this by investigating the interaction between a range regions that could be targeted for sequencing that showed of geographically distinct haplotypes of the Old World some variation within the non-African accessions. One of climbing fern, Lygodium microphyllum (Pteridophyta: Lygo- these introns lies between trnL and trnF genes, splitting the diaceae), and a suite of genetically similar, but geograph- trnL gene (Taberlet et al. 1991). The other lies between the ically distinct lines of phytophagous mite, Floracarus chloroplast small ribosomal protein (rps4) and trnS genes. perrepae Knihinicki & Boczek (Eriophyidae). The fern is an For the mite, sequence data from the domain 2 gene invasive weed in southern Florida, USA (Pemberton & region (D2) of the 28s rRNA gene (Campbell et al. 1993; Ferriter 1998) and is indigenous to the wet tropical and sub- Goolsby et al. 2001) and a portion of the CO1 were used to tropical regions of the Old World including Australia, compare samples of Floracarus (Navajas et al. 1996) (see Africa, Asia and Oceania (Pemberton 1998). The origin of Table S1, Supplementary material, for primer sequences). the Florida fern is unknown. The mite is known to occur Again, these regions were selected for the speed with across Asia and Australasia, but appears to be absent from which they evolve. Africa (Goolsby et al. 2003). Floracarus perrepae feeds on the Polymerase chain reaction (PCR) was used to amplify leaflets of L. microphyllum and in doing so induces leaf roll the specific regions for each specimen. All reaction vol- galls which in turn act to stunt fern growth and eventually umes were 50 µL containing 20 pm of each primer, 200 µm µ kill the leaves (Goolsby et al. 2004; Ozman & Goolsby each dGTP, dATP, dCTP, dTTP, 2.5 mm MgCl2, 4 L DNA, 2005). Without this gall the mite is unable to reproduce 1X supplied buffer and 1 U Taq (QIAGEN Taq PCR Core normally. The capacity to induce galls is therefore a major kit). PCR was carried out using a Hybaid thermocycler determinant of mite fitness. We can therefore use this relation- with the following parameters. Denaturation step for ship to investigate the role of co-evolution in the development 5 min at 94 °C, followed by the addition of Taq. Then 35 of host–herbivore relations. cycles of 1 min at 94 °C, 1 min at 52 °C, and 1.5 min at 72 °C followed by a final post-extension at 72 °C, except the CO1 where the annealing temperature was 47 °C. Materials and methods PCR amplicons were visualized and excised for sequen- Samples of the fern were collected from Africa, Asia, cing by electrophoresis in 0.8% TAE agarose gels contain- Australasia and North America (Table 1). Mite samples ing 10 µg/mL ethidium bromide. Excised PCR products were collected from Asia and Australasia (Table 1). Samples were purified for sequencing using Eppendorf Perfect- of the fern were collected live and held in silica tubes for Prep® Gel Cleanup kit. Up to 4 µL of purified product and analysis. Marginal leaf roll galls containing the mites were the appropriate PCR primers were used for sequencing collected from the field and held in 70% alcohol. Mites according to the Beckman Coulter Dye Terminator © 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 287–297 © 2006Blackwell PublishingLtd, Table 1 Populations of Floracarus perrepae collected from Lygodium microphyllum and associated fern haplotypes based on the sequence results of the combined trnL – trnF and rps4 – trnS chloroplast intron sequences (1215 base pairs in total). Mite samples from Hainan and fern samples from Indonesia failed to amplify as the DNA had degraded.