Matching the Origin of an Invasive Weed for Selection of a Herbivore

Molecular Ecology (2006) 15, 287–297 doi: 10.1111/j.1365-294X.2005.02788.x

BlackwellMatching 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

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

MATCHING THE ORIGIN OF AN INVASIVE WEED FOR BIOLOGICAL CONTROL 289

Haplotype Lygodium microphyllum

19 Lygodium microphyllum sequenced 13 8 lify as the DNA had degraded. No mites were found to Haplotype designation CO1 was found in the region between southeast Queensland and wet

Genotype designation D2 L. microphyllum

Floracarus perrepae sequenced 11 2111 11 41 3 41 54 8 and associated fern haplotypes based on the sequence results of combined trnL – trnF rps4 trnS

Lygodium microphyllum collected from

Ban Klong Manao, Trat, Thailand 2 1 9 2 7 Big headed mite, Iron Range National Park,Queensland, northern Australia Logan Carbrook Creek, Queensland, southern Australia Indooroopilly, Queensland, southern Australia 1, outgroup Daintree, Queensland, northern AustraliaMartyville, Queensland, northern AustraliaKatherine, Northern Territory, northern Australia 1Mabel Downs, Western Australia, northern Australia Litchfield National Park, 1 1Northern Territory, northern Australia 1Iron Range National Park, Queensland, northern Australia Jupiter, Florida, USA 1Kiunga, Western, Papua New GuineaBulolo, Markham, Papua New Guinea 1Palau Ubin, Singapore 1 1 1 3 1 1 4 18 17 1 2 1 1 1 1 2 2 4 1 5 1 6 3 1 1 1 3 7 3 3 8 1 8 7 Simarjarunjung, Sumatra, Indonesia 2 1 10 — — Ban Tul, Cha Uat, Thailand 2 1 8 1 7 la Coulée, Sud, New Caledonia 2 2 11 2 1 Ponérihouen, Nord, New Caledonia 1 2 11 2 1 Quilon, Kerala, India 2 3 15 1 5 Nagercoil, Tamil Nadu, India 2 3 16 2 6 Boralegamura, Sri LankaShing Mun, Hong Kong, ChinaLamma Island, Hong Kong, ChinaQuanghai, Hainan, China 1 1 2 1 6 6 4 5 13 12 14 — 2 2 1 1 11 11 10 4 Axim, Western, Ghana Floracarus perrepae E E E E E ′ ′ ′ ′ ′ E E E E Tyagarah, New South Wales, southern AustraliaE E E 1E E E 1E E 1 5 2 E E W E E E E E W ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 57 47 08 08 31 08 34 16 59 59 04 11 31 08 24 17 34 34 27 ° ° ° ° ° 06 54 58 41 25 54 14 ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° Populations of S, 131 S, 153 S, 153 S, 152 S, 145 S, 146 S, 132 S, 153 S, 131 S, 143 N, 80 S, 141 S, 146 N, 103 N, 102 N, 98 N, 99 S, 166 S, 165 N, 76 N, 77 N, 79 N, 114 N, 114 N, 110 N, 02 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 19 36 41 30 13 36 10 29 19 37 56 07 16 24 57 43 02 14 06 51 19 50 23 12 02 52 ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° ° tropics region in northern Queensland Coordinates Location 02 Table 1 13 28 27 18 17 14 28 12 07 01 11 27 13 26 06 be present on the fern in Africa. Further, distribution of Australia is disjunct as no chloroplast intron sequences (1215 base pairs in total). Mite samples from Hainan and fern Indonesia failed to amp 08 22 21 08 08 06 22 22 19 04

Sequencing with Quick Start Kit. Both strands of each frag- unidentified eriophyid mite at the root. The species T. urticae ment were sequenced on a Beckman Coulter CEQ 8000 and P. mori were therefore omitted from the subsequent capillary sequencer. analyses. The ingroup and outgroup sequences were aligned using clustal w (Thompson 1994) and required no adjust- ment by hand. Analysis of the chloroplast introns Evolutionary trees were estimated using both distance, Chloroplast sequences were analysed with the software parsimony and maximum-likelihood methods. Analyses package paup (Phylogenetic Analysis Using Parsimony) were undertaken using paup. In each instance, CO1 and D2 (Swofford 2002) for Macintosh, using parsimony, dis- sequences were analysed both as individual data sets and tance (NJ — neighbour-joining), and maximum-likelihood as a single concatenated data set. Again, the PHT option in methods. The PHT (partition homogeneity test) option in paup measured no significant difference in phylogenetic paup measured no significant difference in phylogenetic signal between the two sequences (P = 0.964), thus the two signal between the two sequences (P = 1.00), thus the two sequences were combined for a total length of 976 base chloroplast intron sequences were combined for a total pairs. An estimate of a most parsimonious phylogenetic length of 1596 bases. Eleven unique sequences from 24 reconstruction was made by bootstrapping (64 parsimony- accessions were obtained. Fern GenBank accession nos for informative characters, 1000 replicates, 10 random addi- trnL-trnF are AY832860–AY832911, DQ167308–DQ167321 tion sequence replicates, TBR, MULTREES option in and for rps4-trnS AY827158–AY827200, DQ167322– effect). Prior to maximum-likelihood analysis, modeltest DQ167343. Of 1596 bases, 1531 were constant and 52 were 3.06 (Posada & Crandall 1998) was used to select the appro- parsimony informative. Because of the small number of priate evolutionary model parameters. Optimal para- taxa, an exhaustive search was run to generate parsimony meters corresponded to the K81uf + I + G model (–ln L results. Prior to maximum-likelihood analysis, modeltest = 2786.8193) where the proportion of invariable sites 3.06 (Posada & Crandall 1998) was used to select the (I) = 0.4230 and the gamma distribution shape parameter appropriate evolutionary model parameters. Optimal para- (G) = 0.0913. Estimated base frequencies were A = 0.2723, meters corresponded to the F81 model (–ln L = 2538.1487) C = 0.1559, G = 0.2255, T = 0.3463. Bootstrapping was per- with no assumed proportion of invariable sites, and equal formed with the heuristic search option for 500 replicates distribution of among-site rate variation at variable sites. (10 random addition sequence replicates, branch swap- Estimated base frequencies were A = 0.3314, C = 0.1673, ping by TBR, and MULTREES in effect). The big-headed G = 0. 1990, T = 0.3023. Bootstrapping was then performed mite sequence was the outgroup in all cases. with the heuristic search option for 500 replicates [10 random addition sequence replicates, branch swapping Herbivore transfer experiment — the capacity to induce by tree-bisection–reconnection (TBR), and MULTREES leaf roll galls in effect]. The chloroplast sequence from Ghana was the outgroup in all cases. The capacity to induce leaf roll galls is a strong indicator of fitness in F. perrepae as mites can only reproduce on leaves that are sufficiently rolled. To assess the capacity Analysis of ribosomal D2 and mitochondrial CO1 from of different mite haplotypes (i.e. those from different Floracarus perrepae geographical locations and clades) to induce leaf roll galls, A total of six unique D2 sequences were obtained from mites from southern Australia (Logan, Indooroopilly), 24 accessions and 18 unique CO1 sequences from 23 col- New Caledonia (la Coule), China (Lamma Island), Thailand lections. The outgroup (big-headed mite) was an unidentified (Ban Tul, Ban Klong, Manao), India (Nagercoil), and northern sympatric species of eriophyid mite feeding inside the leaf Australia (Iron Range, Far North Queensland) were screened roll galls on Lygodium reticulatum obtained from Far North against two fern haplotypes, the southern Australian Queensland. The unidentified species is morphologically Logan Accession, and the northern Australian Iron Range distinct and based on morphometric measures represents accession, which was genetically identical to the Florida a new genus in the Eriophyidae (D. Khininicki, personal weed fern. Mites were field collected and under local communication). Preliminary analysis using as the out- conditions, transferred to sporelings. Ten mites were group species and two distantly related taxa, Tetranychus added to a single leaf on a sporeling fern. Mites were held urticae and Panonychus mori together with the unidentified for 3–4 weeks until completion of leaf rolling, oviposition species, demonstrated that inclusion or exclusion of T. and development of progeny. The numbers of full leaf rolls urticae and P. mori had no influence on the subsequent at the end of each test was recorded for each mite sample. structure of the relationships between the members of The number of full leaf rolls was used as an indicator of the in group or between the in group and the outgroup; acceptance of the fern haplotype by the mite haplotype. A the Floracarus taxa formed a monophyletic group with the presence-absence analysis of variance was carried out on

Fig. 1 Distribution of Lygodium microphyllum (fern). Sites enclosed by the green lines indicate identical haplotypes. Only 10 of the 11 haplotypes are shown; the African haplotype is not shown. Distribution of Floracarus perrepae (mite) clades. The red lines show the distribution of the Floracarus perrepae (mite) clades supported by bootstraps ≥ 75. The circles (hatched lines) show the relationships as defined using D2 alone. the proportion of replicates with full leaf rolls; means African accession which differed by 23 bases, the remain- separation was by least significant difference (LSD). ing fern accessions differed from the Florida samples by between 1 and 6 base pairs only which represents between 0.0008 and 0.0048 total sequence variation (Table 2). Results Parsimony, NJ and maximum-likelihood analyses all indicate the same relationship and only the NJ tree is provided Analysis of chloroplast introns (Fig. 2). The Asian populations cannot be separated, with Eleven Lygodium microphyllum haplotypes were found the exception of the China/Hong Kong population, but using the two chloroplast introns (Fig. 1). At the 15 locations always form a polytomy with 100% support. Likewise, the where two or more ferns were collected, only a single New South Wales and New Caledonia/New South Wales/ haplotype was found. Of the nine locations where only a Queensland populations are always together with 100% single fern was collected, the three locations in the Northern support. The southern Australian population (haplotype Territory yielded a single haplotype as did the two ferns #1) belongs to this group. As these groups separate out, the from separate locations in Hong Kong. This suggests that China/Hong Kong, Florida and Queensland, Iron Range/ as far as our data allow, ferns from a single location belong Papua New Guinea, which includes the northern Australian to the same haplotype and show no detectable genetic fern (haplotype #8), are left as an unresolvable polytomy variability. The accession from Queensland, Iron Range (Fig. 2). However, the sequences for Florida and Queensland/ (Table 1) was identical to those obtained from collections Iron Range/Papua New Guinea are identical and differ by of the invasive fern in Florida. With the exception of the one base from the China/Hong Kong population (Fig. 2).

© 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 287–297 292 J. A. GOOLSBY ET AL. Queensland, Iron Range/ Papua New Guinea Florida China/ Hong Kong India, Quilon being ‘southern Australia’, while the remaining accession d, Iron Range accession which is more closely related to

trnL–trnF and rps4–trnS. These data, in combination with that Thailand/ Singapore India, Nagercoil L. microphyllum China, Hainan Sri Lanka Western Australia/ Northern Territory New South Wales accessions based on a comparison of sequences from the New Caledonia/ New South Wales/ Queensland Lygodium Ghana Sequence similarity matrix for presented in Table 1, suggests that the accessions across Australia are all closely related with exception of Queenslan presented in Table accessions from SE Asia. We have therefore described the ferns in Australia all regions, except Queensland, Iron Range, as is referred to as being ‘northern Australia’ Haplotype designationGhanaNew Caledonia/New 9South Wales/Queensland New South Wales —Western Australia/ 1Northern Territory Sri Lanka 1.000China, Hainan — — 1.000India, NagercoilThailand/Singapore 0.980 2India, Quilon — — 0.999China/Hong KongQueensland, Iron Range/ — — 0.998 — —Papua New Guinea 3 — 0.979Florida — 1.000 — — — 0.982 — — — 0.996 — 0.997 10 — 0.995 1.000 — 0.982 0.995 — — 4 — 0.983 0.995 — — — 0.994 0.996 — 0.983 0.994 — — — — 0.997 — 5 0.994 — — — 0.984 0.997 0.996 — 0.994 — — — 7 1.000 0.995 — 0.996 0.984 — — 0.999 1.000 — 0.994 0.995 — — 0.985 — 0.998 0.997 — 0.998 6 — 0.994 0.985 — — 1.000 0.994 0.997 0.994 — 0.998 0.999 11 — 0.996 — — 0.997 0.985 1.000 0.998 0.994 0.999 — 8 — — 0.996 0.999 0.997 0.998 — 0.998 0.996 0.998 — 0.997 — 0.997 8 — 0.997 0.998 1.000 — 0.996 0.997 — 0.999 1.000 0.997 0.998 0.998 1.000 0.999 — 0.998 1.000 0.999 — — 1.000 Table 2

Fig. 2 Neighbour-joining phylogram inferred from fern intron data. The Florida and Queensland/Iron Range/Papua New Guinea sequences were identical, whereas the China/ Hong Kong sequence differs by a single base. The longest branch represents 51 base changes.

Asia–northern Australia clade, a New Caledonia sister to Analysis of ribosomal D2 and mitochondrial CO1 from the whole Australasia–SE Asia clade and a polytomy of Floracarus perrepae India–China (China, India, Sri Lanka) clade at the base of Analysis of the D2 gene region indicated a sequence length the tree (Figs 1 and 3). of 627 bp and six distinct F. perrepae genotypes (Table 1). This represents between 0.092 and 0.288 total sequence Herbivore transfer experiment — the capacity to induce variation. Both the NJ tree (not shown) and the maximum- leaf roll galls likelihood tree estimates support the existence of three major groups (i) subcontinent (India and Sri Lanka), (ii) The leaf-rolling ability of representative mite haplotypes Asian (China) and (iii) Asia–Australasia–Pacific (Australia, from across the phylogenetic tree of mites is shown in Indonesia, New Caledonia, Papua New Guinea, Singapore, Fig. 3. Rolling performance against the northern Australia Thailand) each supported by a bootstrap score ≥ 91%. The haplotype (haplotype #8) of the fern from Iron Range relationship between the three groups is unclear as the varied, with the mite from Iron Range having the strongest suggested relationship between the subcontinent and effect. These show that mites from any of the tested Asia–Australasia–Pacific is weak at bs = 47%. samples can roll the leaves of the southern Australian fern For CO1 there were 18 distinct haplotypes each rep- (haplotype #1) indicating the ability is inherited from the resented by a sequence length of 349 bp (Table 1) which time when there was a single, ancestral population of fern represents between 0.244 and 0.267 total sequence variation. and mite. Mites from China and India have lost their ability The structure of the trees derived from all analyses was to roll the leaves of the northern Australian fern. The New equivalent (trees not shown). The CO1 tree identified the Caledonia mite can roll leaves of the northern Australian same major groups revealed by D2, but provided con- fern, but at a reduced level in comparison to the Thai and siderably more resolution enabling the Asia–Australasia– northern Australian mites. This is unsurprising except that Pacific clade to be split into three groups, SE Asia, northern mites from southern Australia cannot induce leaf rolls Australia/Papua New Guinea and southern Australia on the northern Australian fern either. The southern (overlapping within the southern end of the northern Aus- Australian mites are more closely related to mites from tralia/Papua New Guinea distribution). northern Australia than are the New Caledonia mites. The two sets of sequences were then combined into a single Either the ability to roll the northern Australian fern has concatenated data set. Distances among the accessions varied been lost twice, once in mainland Asia and once in from 1 to 23 bp in a combined sequence length of 973 bp southern Australian mites, or else the relationships among (0.001–0.023 total sequence difference). NJ, parsimony, and populations of the fern do not quite match those of the maximum-likelihood analysis produced a consistent mite. Similarly, the southern Australian mite was the most pattern. The tree inferred from parsimony reconstruction efficient at inducing leaf roll galls on the southern is presented in Fig. 3 and contains the bootstrap values Australian fern (Fig. 3). from the maximum-likelihood analysis. The estimated relationship identifies the existence of a SE Asia (Indonesia, Discussion Singapore, Thailand) sister group to the northern Australia– Australasia (Northern Territory, northern Queensland, The herbivore transfer experiments demonstrated that Papua New Guinea) group, a southern Australia (New Floracarus perrepae from Iron Range in northern South Wales, southern Queensland extending to the south- Queensland was the most effective at inducing leaf roll ern end of northern Queensland) sister group to the SE galls on the northern Australian/Florida fern haplotype

Fig. 3 Phylogeny of the 15 Floracarus perrepae mite populations based on mitochondrial CO1 and the D2 region of the 28S rDNA gene. The outgroup is an undescribed eriophyid mite also from Lygodium. Each genotype is identified by the country and, if appropriate, region of origin. The populations marked with an * were those tested for their capacity to induce leaf rolling (25 replicates). The terms ‘roller’ and ‘nonroller’ indicate whether the individuals from that group were able to induce leaf roll galling. The number to the right is the initiation index, or mite numbers needed to induce rolling. Numbers in columns followed by the same letter are not statistically different (LSD, P < 0.05). Numbers above the branches are bootstrap scores from the parsimony analysis, the numbers below the branches are for the maximum-likelihood bootstrap. The northern Australia Lygodium microphyllum from Iron Range accession (haplotype #8, Table 1) is genetically identical to the Florida pest fern. The southern Australia (haplotype #1, Table 1) L. microphyllum is the Logan accession.

from the same location. Similarly, the mite from southern reduces the fitness of the herbivore thereby invoking a Queensland was more effective at inducing galls on the selective pressure on the herbivore which in turn induces fern haplotype from southern Queensland than mites from a fitness deficit. This selects for a resistant phenotype in the elsewhere. In other words, mites that occur in the same herbivore which in turn confers a selective pressure back location as a particular fern haplotype were better able to onto the plant (Ehrlick & Raven 1964; Dawkins & Krebs induce galls on the local fern haplotype. As this capacity 1979; Kniskern & Rausher 2001; Rausher 2001). has a direct impact on their fitness we conclude that the From the practical perspective of determining which strong geographical pattern of mite–fern associations mite haplotype should be considered as a candidate reflects an overall pattern of optimal host exploitation by for biological control against the pest fern in Florida, the this mite and supports the idea of local adaptation (Ehrlick results, subject to the limitation that not all mites have been & Raven 1964; Dawkins & Krebs 1979; Kniskern & Rausher tested against the weed fern, point strongly at the choice of 2001; Rausher 2001). Based on the arguments outlined by the Iron Range mite. Using the phylogenetic tree of the Kniskern & Rausher (2001), our herbivore transfer experi- mites as a guide and mapping the leaf-rolling effectiveness ments support the role of arms-race co-evolution operating of mites onto that tree, the probability is low that the leaf- in this system. Here, assuming that climatic and other rolling ability of another, untested mite will be more effec- abiotic factors are not constraints to successful utilization tive at inducing leaf roll galls. of a plant species, factors underlying local adaptation lie In recent years, phylogenetic analysis of invasive species within interactions between plants and herbivores that and their relatives has at times been effective in identifying involve processes of cyclical advantage/disadvantage in its geographical origin. For example, Milne & Abbot (2004) which one party gains at the expense of the other. The showed the source of the invasive privet, Ligustrum robus- consequence is a cyclical antagonistic interaction involving tum, in the Mascarene Islands was Sri Lanka. Our case is the evolution of a resistance trait in the plant which slightly different in that the weed is naturally widely

© 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 287–297 MATCHING THE ORIGIN OF AN INVASIVE WEED FOR BIOLOGICAL CONTROL 295 occurring and our task was to identify a source. Popula- We chose to examine fast-evolving regions of the tions tend not to be related hierarchically, but often can be genomes of fern and mite, yet the genetic distances among identified by unique markers, for example, by minor vari- geographically disparate samples turned out to be surpris- ations in gene sequences, and this proved to be the case. ingly small. This suggests that mites from India and China Fortunately, one of our fern accessions was an exact match, through to Australia and New Caledonia have not been within the genes examined, to the Florida weed population. separated for very long in evolutionary time. Any attempt The relationship between the different fern samples at age estimates based on a molecular clock is fraught shows that ferns from New Caledonia and southern Aus- with difficulty, but as a ‘ball-park’ figure the maximum tralia are more similar to each other than they are to those observed 2% distance among mite accessions for CO1 and from northern Australia and Asia (Fig. 2). Similarly, Asian D2 suggests a common origin within the past million years, ferns are more similar to the northern Australian fern perhaps within the past half million. Intron sequences in (haplotype #8) than they are to the southern Australian fern the fern appear to be evolving about 10 times slower than (haplotype #1). The size of the difference between the CO1 and D2 sequences in the mite. This could be a real northern and southern ferns suggests a period of separa- relative-rate difference either because the fern in general tion and points towards the spread of an exotic Asian fern evolves more slowly than the mite or the introns evolve into northern Australia. The level of divergence between more slowly than the genes. Or, it may suggest that the this fern and the southern fern was sufficiently great that ferns are less well separated in time than the mites. The the southern Australian mite was unable to utilize this mites may be less vagile and disperse relatively poorly invader. This is supported by the observations that both compared to the ferns, such that the mites are more separ- the northern and southern Australian mites co-occur in the ated one from another than the ferns. One thing, however, Daintree-Martyville region of northern Australia, which is clear. The distribution of both the ferns and the mites marks the northern limit of the southern Australian fern across India, Southeast Asia and Australasia must stem distribution. What happened then is problematic. How- from relatively recent long-range dispersal, not from ever, the phylogenetic relationship between the Southeast ancient vicariance events. Our results further support the Asian mites and the northern Australian mite hints at a fur- argument that long-range dispersal and not vicariance is ther incursion into northern Australia, this time involving the major contributor to plant biodiversity and agree with the mite. So then, why did the northern Australian mite, recent findings such as those of Beier et al. (2004) and which can utilize both southern and northern ferns, not Knapp et al. (2005). A continental drift explanation of the spread back into the southern range? The answer here lies patterns seen, for example between the New Caledonia in the incapacity of this mite to develop and reproduce at and Australia–Southeast Asia clades, would require us to 21 °C (Goolsby, unpublished), an attribute that would postulate an 800- to 1000-fold slowdown in each of these effectively prevent its spread into cooler southern Australia. four molecules compared to the speed at which they are Did the Florida pest fern come from the remote Iron known to evolve in many other organisms (e.g. for CO1, Range of Cape York, Queensland? It seems improbable 2% in 80 million years compared to approximately 2–5% that collections would have been made in this area given its per 1 million years in other organisms). Such a hypothesis remoteness and actual accounts of plant collecting in the is not tenable. Iron Range do not begin until the 1920s. However, consid- The common identity between the Florida fern and the erable botanizing did take place in this region of Cape York fern from Iron Range is explicable if the fern was intro- near Somerset and Lockerbie Scrub during the latter half of duced into Florida via the route we have outlined. Given the 19th century. Kew Gardens has a record of J. G. Veitch the naturally small genetic distances between ferns from collecting Lygodium microphyllum in 1867 at Somerset (R. widely separate localities, we cannot be sure that the Flor- Cowan, personal communication). Veitch is also known to ida fern actually is from Iron Range. It may differ from the have provided Charles Darwin with Lygodium scandens, a Iron Range fern in some part of its genome we have not synonym of L. microphyllum, for his work studying the sequenced. However, there is no evidence at present to movements and habits of climbing plants (Darwin 1875). suggest it is anything but the Iron Range fern, introduced This confirms that live L. microphyllum was imported into into Florida after having spent several decades in English England and cultured by Veitch. Nursery plant catalogues glasshouses in the mid 19th century. from the period list the availability of L. microphyllum in the tropical plant trade in Florida as early as 1888 and Veitch Nursery is known to have exported material to the USA Acknowledgements during this period (Pemberton & Ferriter 1998; Shephard The authors would acknowledge the following people for their 2003). While circumstantial, the available evidence would contributions to the research: John Curran, Matthew Purcell, Tony seem to point at the fern reaching Florida from north Wright and Ryan Zonneveld (CSIRO Entomology); Ted Center Queensland via England. (USDA-ARS, Ft. Lauderdale, FL); Sebahat K. Ozman and E. Walter

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