Accepted Manuscript

Scrutinizing biological control survey data from the native range – the phylog- eny and Lygodium fern host associations of

Graham A. McCulloch, Jeffrey R. Makinson, Ryan Zonneveld, Matthew F. Purcell, Dean R. Brookes, Komal Gurdasani, Ellen C. Lake, S. Raghu, Gimme H. Walter

PII: S1049-9644(19)30115-X DOI: https://doi.org/10.1016/j.biocontrol.2019.04.004 Reference: YBCON 3966

To appear in: Biological Control

Received Date: 12 February 2019 Revised Date: 11 April 2019 Accepted Date: 12 April 2019

Please cite this article as: McCulloch, G.A., Makinson, J.R., Zonneveld, R., Purcell, M.F., Brookes, D.R., Gurdasani, K., Lake, E.C., Raghu, S., Walter, G.H., Scrutinizing biological control survey data from the native range – the phylogeny and Lygodium fern host associations of Musotiminae moths, Biological Control (2019), doi: https:// doi.org/10.1016/j.biocontrol.2019.04.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Scrutinizing biological control survey data from the native range – the phylogeny and

Lygodium fern host associations of Musotiminae moths

Graham A. McCulloch1,2*, Jeffrey R. Makinson3, Ryan Zonneveld3, Matthew F. Purcell3, Dean

R. Brookes1, Komal Gurdasani1, Ellen C. Lake4, S. Raghu3, Gimme H. Walter1

1 School of Biological Sciences, The University of Queensland, St. Lucia, Queensland,

Australia, 4072

2 Department of Zoology, University of Otago, PO Box 56, Dunedin, 9054, New Zealand

3 United States Department of Agriculture, Agricultural Research Service, Australian

Biological Control Laboratory, c/o CSIRO, GPO Box 2583, Brisbane, Queensland, Australia,

4001

4 United States Department of Agriculture, Agricultural Research Service, Invasive Plant

Research Laboratory, 3225 College Avenue, Fort Lauderdale, FL, 33314, USA

*To whom correspondence should be addressed. Email: [email protected]

Running title: Lygodium systematics

Abstract

Lygodium microphyllum is considered one of the most damaging environmental weeds in

Florida. Despite substantial efforts to control this fern, it continues to spread rapidly through the Greater Everglades ecosystem and other regions of Florida. Biological control is considered a critical component of the management strategy to control this weed, and foreign exploration for natural enemies is ongoing. A number of crambid moths from the subfamily Musotiminae are considered the most promising of the potential biological control agents found to date, because they are relatively abundant on Lygodium and apparently host-specific. We amplified three genes (COI, 18S rRNA and 28S rRNA) to assess the phylogenetic relationships among these moths in relation to geography. Limited genetic structuring was typical within each moth species, and no obvious signals of unrecognised host-specific cryptic species were detected, though further investigation is required, particularly for Eugauria albidentata. Our results emphasize the value of complementing initial field surveys with molecular screening, as such an approach provides valuable information on the biogeographic distribution, genetic structuring, and field host range of potential biological control agents.

Keywords

Florida, Lygodium microphyllum, Molecular screening, Native range surveys, Old World climbing fern, , Systematics

1. Introduction

2

Foreign exploration for natural enemies of an invasive weed is usually an essential initial phase of any successful biological control program (Purcell et al., 2004; Zwölfer et al., 1976).

These surveys are typically difficult, both technically and logistically, and can be very time- consuming and expensive (Goolsby et al., 2006). The primary aim of such surveys is to provide a list of herbivores associated with the target weed (Purcell et al., 2004), and thus provide the background for making decisions about the potential of different organisms as biological control agents. However, the surveys from foreign exploration are seldom comprehensive because political, practical and/or financial considerations often intrude

(Goolsby et al., 2006). Nevertheless, vital information can be derived from these surveys, especially in terms of providing a framework for testing crucial aspects of the host associations, species status and evolutionary history of the species concerned.

Biological control practitioners increasingly incorporate modern molecular techniques to assess the relationships within and among potential biological control agents (see Gaskin et al., 2011). Such genetic screening, however, is seldom employed in the initial stages of foreign exploration for biological control agents. Here we assess the value of incorporating molecular screening at the foreign exploration stage of a biological control project, focusing on one subfamily of the lepidopteran natural enemies of the invasive fern Lygodium microphyllum (Pteridophyta: Lygodiaceae).

Lygodium microphyllum, also known as Old World climbing fern, is a damaging invasive environmental weed that was first reported as naturalized in Florida, USA, in 1965 (Beckner,

1968). It has since spread across a broad area of southern and central Florida. Range expansion is ongoing, with establishment as far north as Jacksonville (EDDMapS, 2018;

3

Pemberton, 1998; Rodgers et al., 2014). This fern grows rapidly and indeterminately into a vine with individual fronds capable of reaching 30m in length (Hutchinson et al., 2006;

Pemberton and Ferriter, 1998). The twining fronds of L. microphyllum climb over trees and shrubs, forming thick rachis mats that smother or shade understorey vegetation (Nauman and Austin, 1978; Pemberton and Ferriter, 1998). Dense rachis mats on the ground and thick skirts of rachises climbing trees can carry fire into habitats that do not normally burn , and if the canopy burns the tree may die (Hutchinson et al., 2006; Pemberton and Ferriter, 1998).

These climbing ferns are particularly damaging in the Greater Everglades ecosystem, where their presence results in the destruction of entire tree islands. Lygodium microphyllum is therefore considered one of the most serious invasive weeds impacting the state (FLEPPC,

2017; Volin et al., 2004).

Lygodium microphyllum continues to spread rapidly through Florida (EDDMapS, 2018;

Humphreys et al., 2017; Rodgers et al., 2014; Volin et al., 2004), despite substantial control efforts against it (Hutchinson et al., 2006; Hutchinson, 2010; Stocker et al., 2008).

Traditional management tools such as herbicide applications, prescribed fire, and mechanical controls can be expensive, damage native plant communities, and may be difficult to apply to remote infestations of L. microphyllum (Hutchinson et al., 2006).

Biological control can complement existing management tools, and the integration of weed management techniques may provide more effective control (Hutchinson et al., 2006; Lake and Minteer, 2018; Pemberton and Ferriter, 1998).

The native range of L. microphyllum includes tropical and subtropical areas of Asia,

Australia, the Pacific Islands and Africa (Pemberton, 1998). The search for natural enemies

4 has focused on the Asian and Australian regions of this range, partially due to early molecular work suggesting that the L. microphyllum present in Florida was more closely related to plants in this region than those in Africa (Goolsby et al., 2003). Also, the diversity of Lygodium species is high in Asia and Australia, increasing the likelihood of the presence of specialist herbivores (Goolsby et al., 2003). Foreign exploration across Australia and Asia has identified a number of herbivores of Lygodium, including more than 20 species (Goolsby et al., 2003). The most prominent herbivores associated with L. microphyllum are crambid moths from the subfamily Musotiminae (Goolsby et al., 2003), being present in well over 1000 collections (made across 21 years) from across the native distribution of the fern. This large crambid subfamily includes 193 described species whose larvae feed entirely on ferns and bryophytes (Nuss et al., 2003; Regier et al., 2012). Nine

Musotiminae species (two yet to be described) have been found feeding on L. microphyllum

(Table 1). Two additional Musotiminae species, fuscolinealis and Siamusotima aranea, have been found associated exclusively with the congeneric L. japonicum and L. flexuosum respectively (Table 1; Bennett and Pemberton, 2008; Solis et al., 2005).

Larvae of these Musotiminae moths are either defoliators of the pinnae (leaflets) or are

‘stem borers’, feeding on the protostele within the rachises and into the rhizome (Solis et al., 2017; Solis et al., 2005; Yen et al., 2004). Two defoliators, N. conspurcatalis and

Austromusotima camptozonale, were approved for release by regulatory authorities in

Florida following host-range testing, but only N. conspurcatalis established (Boughton and

Pemberton, 2008; Boughton and Pemberton, 2009; McCulloch et al., 2018; Smith et al.,

2014). Neomusotima conspurcatalis is currently being mass-reared and released in Florida as part of the Comprehensive Everglades Restoration Plan (CERP). An additional

5

Musotiminae defoliator, Lygomusotima stria, is undergoing evaluation in the USDA ARS

Invasive Plant Research Laboratory quarantine in Florida. The stem borers are difficult to rear, and as a result, sustainable laboratory cultures have never been established, but because of the potential damage they can cause they are of particular interest to biological control.

Understanding genetic structuring within potential biological control agents is important for successful biological control (McCulloch et al., 2018; Rauth et al., 2011), particularly for the discovery and resolution of cryptic species complexes (Gaskin et al., 2011; Paterson et al.,

2016; Smith et al., 2018). Because cryptic species are likely to have different host- associations from one another (Fernando and Walter, 1997; Paterson, 1991; Rafter et al.,

2013), the unknowing release of a mixture of cryptic species may lead to unanticipated non- target impacts (Paynter et al., 2008; Withers et al., 2008). Several Musotiminae associated with Lygodium have broad geographic ranges (Fig. 1), and the species status of the individual populations has not yet been assessed. It thus possible that these species may in fact consist of several cryptic species, potentially with smaller geographic ranges. Likewise, several of these moth species have been collected feeding on more than one Lygodium species (Table 1). Although it is not uncommon for moths to feed on more than one fern species (Weintraub et al., 1995), the possibility of unrecognised host-specific cryptic species must be considered and tested (see Paterson, 1991; Rafter et al., 2013). Molecular screening is often the first step in identifying cryptic species, though subsequent population genetic screening (Hereward et al., 2017; Rafter et al., 2013; Toon et al., 2016) or behavioural tests (e.g. Fernando and Walter, 1997) are invariably required.

6

In this manuscript we examine the phylogenetic relationships among the Musotiminae

Lygodium fern moths collected during foreign exploration. We determine the extent of genetic structuring within each species, and assess evidence for unrecognised host-specific cryptic species. In addition, we expand on the value of incorporating molecular approaches at an early stage of foreign exploration.

2. Methods

2.1. Sample collection

Extensive surveys of L. microphyllum, L. japonicum, L. flexuosum, and L. reticulatum have been conducted by USDA ARS Australian Biological Control Laboratory (ABCL) throughout

Indonesia, Singapore, Malaysia, Thailand, Hong Kong, China, New Caledonia and through subtropical and tropical regions of Northern Australia since 1996. Less extensive surveys have also been conducted in India and Papua New Guinea. Collections involved a combination of searches above and below ground, sweep nets, beating trays, and light traps

(as described by Goolsby et al., 2003). Specimens were identified morphologically, and either dried on pins or stored in 95% ethanol prior to DNA extraction.

2.2. DNA extraction, amplification and sequencing

DNA was extracted from between one and nine specimens from each of the Musotiminae moth species (Table 1; Table S1), following the DIY spin column protocol of Ridley et al.

(2016). Three DNA regions were amplified: a 732-bp portion of the mitochondrial

7 cytochrome oxidase (COI) region, a 1158-bp region of 28S rRNA, and a 698-bp region of 18S rRNA. DNA amplification of COI and 28S rRNA followed McCulloch et al. (2018), with the 18S rRNA region amplified using the primers 18Sa2.0 and 18S9R (Whiting et al., 1997), following the same protocol. PCR products were cleaned using Exonuclease I and Antarctic

Phosphotase (New England Biolabs, Massachusetts, USA), with sequencing performed by

Macrogen (Korea).

2.3. Sequence analysis

All new sequences were submitted to GenBank (GenBank accession numbers MK776974 -

MK776994). Sequences were aligned in Geneious 11.1.5 (https://www.geneious.com,

Kearse et al., 2012) using the MUSCLE plugin (Edgar, 2004). Substitution saturation in COI sequences was assessed using Dambe v6.4.31 (Xia, 2017). Indels in the 28S rRNA and 18 rRNA were treated as missing data. The most appropriate model of sequence evolution for each region was assessed using jModelTest 2 (Darriba et al., 2012) using the AIC criterion.

Compatibility of the plastid data sets was analysed using a partition homology test (200 replicates) in PAUP v4.0610 (Swofford, 2002). As all data were congruent, the three genes were concatenated into a single dataset. The analysis of multiple data partitions in a concatenated form may mislead phylogenetic inference (Kubatko and Degnan, 2007), but this is most likely to occur during studies at shallow phylogenetic levels, where incomplete lineage sorting can produce discordant gene trees among individual data partitions.

Phylogenetic relationships were estimated using MrBayes v3.12 (Huelsenbeck and Ronquist,

2001). Four Markov chains were run for 3,000,000 generations, with the first 2,000 trees discarded as burn-in. Three crambid moths were selected as outgroup taxa: Ostrinia

8 nubilalis, Loxostege sticticalis, and Conogethes punctiferalis. TRACER 1.7 (Rambaut et al.,

2018) was used to confirm that effective sample size was greater than 200 for all parameters, and to select the appropriate number of burn-in cycles. Four runs were completed for each dataset, and then concatenated using LogCombiner to estimate posterior distributions.

3. Results

COI sequences lacked any ambiguous sites or stop codons, consistent with true mitochondrial origin (Zhang and Hewitt, 1996). No sequence saturation was detected in the

COI dataset. The concatenated dataset comprised 2562 bp from 53 individuals (50 ingroup,

3 outgroup). Sequence statistics and substitution models are provided in Table 2.

Bayesian inference of the combined dataset (Fig. 2) indicated that each species was monophyletic (PP = 1.00). Removing the outgoups had no effect on the basic topology or branch support. The four stem boring taxa formed a well-supported monophyletic clade (PP

= 0.96), although the relationships among these species were poorly resolved (Fig. 2).

Likewise, four of the defoliating taxa formed a well-supported clade (PP = 0.98; Fig. 2). The two Neomusotima species were monophyletic (PP = 1.00), however the

Lygomusotima was paraphyletic (Fig. 2).

Only one species (N. conspurcatalis) showed clear evidence of phylogeographic structuring, though it was the only species in the study with a very broad geographic range (Fig. 1, 2).

The undescribed Lygomusotima moths from Thailand and Malaysia (Fig. 1) differed by only

9

0.7% at COI. By contrast the undescribed Siamusotima species from Bamaga differed from the described Siamusotima species by about 9.5% (Fig. 2). No genetic differences were found across the moths collected from L. microphyllum and those obtained from the other

Lygodium species (Fig. 2). That is, no obvious evidence of host-specific cryptic species was apparent in the taxa Siamusotima disrupta, Lygomusotima n sp., or A. camptozonale.

4. Discussion

Pteridophagy (feeding on ferns) is considered rare amongst the lepidopterans, with no lepidopteran taxon known to feed on only a single fern lineage (Weintraub et al., 1995).

These Lygodium Musotiminae moths are, therefore, apparently exceptional. Although the host-range of many of these species has not yet been fully quantified in the field, laboratory host-range testing has demonstrated that the larvae of many of these moth species are able to develop fully only on plants from the genus Lygodium (Boughton et al., 2009; Boughton et al., 2011; Goolsby et al., 2003).

4.1. Field host range

Limited genetic structuring was detected within the moth species studied (Fig. 2), providing no evidence for previously undetected cryptic species. This lack of obvious evidence of host- specific cryptic species within these Lygodium moths may be overly suggestive; too few specimens were sequenced from more than one host to be entirely certain. The stem boring species Lygomusotima n. sp. and Siamusotima disrupta have been collected on L. japonicum and L. flexuosum, but they are found feeding on these plants only in regions where they grow sympatrically with L. microphyllum. This suggests that L. microphyllum is their primary

10 host plant, and further quantitative sampling on all these ferns across areas of sympatry and allopatry is required.

Eugauria albidentata has previously been collected in Taiwan and Malaysia feeding on

Nephrolepsis ferns (Yen et al., 2004), suggesting that this species may have a broader host- range than the other Lygodium moths. Perhaps consistent with this is that E. albidentata is phylogenetically somewhat distinct from the remaining Lygodium moths (which all appear to be host specialists on Lygodium; Fig. 2). However, the geographic ranges of the Lygodium feeding (Sumatra) and Nephrolepsis feeding (Taiwan, Malaysia) E. albidentata moths do not appear to overlap. This may indicate there are distinct (cryptic) E. albidentata species feeding on Lygodium and Nephrolepsis. This hypothesis should be tested with additional sampling of Nephrolepsis (particularly in Sumatra) and L. microphyllum in Taiwan and

Malaysia. Initial genetic screening of E. albidentata from Nephrolepsis may give an indication that there are cryptic species, but subsequent genetic screening (with nuclear genes, microsatellites, or GBS) of sympatric populations would be required for confirmation

(see Hereward et al., 2017). If additional surveys confirm that the Nephrolepsis feeding E. albidentata moths occur only in allopatry their species status may be best determined by investigating their pheromones comparatively, or with appropriately designed cross-mating tests (Walter, 2003).

Most species in this study appear to be specialists on L. microphyllum (Table 1), with species feeding on other Lygodium species only in regions where L. microphyllum is not naturally abundant. For instance N. fuscolinealis, which feeds solely on L. japonicum, is primarily found in northern regions of Asia where L. microphyllum is rare (Japan, Taiwan, Hong Kong,

11 and South East China; Fig. 1; Yen et al., 2004; Yoshiyasu, 1985). Neomusotima fuscolinealis is sister to the widespread N. conspurcatalis (Fig. 2), which feeds primarily on L. microphyllum, although host-testing reveals that N. conspurcatalis can complete development on L. japonicum (Boughton et al., 2009). The mean genetic divergence across N. conspurcatalis and N. fuscolinealis was 15% at the COI gene; based on the average insect COI mutation rate

(3.54% per million years; Papadopoulou et al., 2010) this value suggests that these two species have been separated for about 4 million years.

Likewise, the stem boring S. aranea, which feeds solely on L. flexuosum, is found only in the

North of Thailand where L. microphyllum does not occur (Fig. 1). This species was placed sister to Siamusotima n. sp. (from Cape York, Australia), though this relationship is not well- supported (pp = 0.55; Fig. 2). Indeed, the relationships among the four stem boring species are poorly resolved (Fig. 2), perhaps indicating multiple speciation events have occurred in this group over a relatively short evolutionary time-frame.

4.2. Evolution of distinct larval feeding modes

The phylogenetic placement of the Lygodium moths within the subfamily Musotiminae could not be established in this study, as too few Musotiminae sequences are available for comparison. Musotiminae species have a range of feeding modes, including leaf mining, leaf feeding, and stem boring (Nuss et al., 2003), but the problems with the Musotiminae phylogeny mean it is uncertain which of these feeding modes is ancestral. If the Lygodium moths do form a monophyletic assemblage within Musotiminae it would indicate that there has been a single switch in feeding modes within this clade of Lygodium feeding specialists.

Conversely, if the Lygodium moths are not monophyletic (i.e. the stem boring and leaf

12 feeding clades are not sister clades on the Musotiminae phylogenetic tree) then distinct stem boring and leaf feeding Musotiminae lineages have evolved independently as

Lygodium specialists.

The stem boring species appear to have smaller geographic ranges than their leaf feeding counterparts (Fig. 1). Some of this apparent difference in geographic range may be an artefact of sampling, as the characteristic damage of leaf feeding moths is easier to locate and identify during field surveys than the damage caused by stem borers. In addition, much of the broad geographic surveying throughout Asia was conducted before field researchers were familiar with the characteristic damage associated with the stem borers. However, the larger geographic ranges of leaf feeding moths may indeed be real, and reflect differing ecological requirements across leaf feeders and stem borers. These difference could impact their relative efficacy as biological control agents, so further research on the ecological requirement of the species in these two groups is required.

4.3. Taxonomic considerations

Our results suggest that the undescribed Lygomusotima moths from Thailand and Malaysia are likely the same species. By contrast, the undescribed Siamusotima species from Bamaga likely represent a new species (Fig. 2), and thus its potential as a biological control agent must be assessed independently of the other Siamusotima species.

Austromusotima metastictalis has been found mainly in Papua New Guinea (Fig. 1), though it has been suggested that this species may exist in sympatry with the congeneric A. camptozonale in the Australian Wet Tropics (Yen et al., 2004). Despite extensive surveys

13 across Northern Australia this species has not been identified again in Australia (see

McCulloch et al., 2018), suggesting it is confined to Papua New Guinea. The two

Lygomusotima species sequenced in this study have been found feeding on the same stands of L. microphyllum in Singapore. Our results, however, suggest that the genus

Lygomusotima is not monophyletic, with the undescribed Lygomusotima from Singapore and Malaysia instead part of the well-supported stem boring clade (which has no other

Lygomusotima species), while Lygomuostima stria is included in the defoliator clade (Fig. 2).

This implies that preliminary taxonomic evaluation placing the undescribed stem borer from

Singapore in the genus Lygomusotima may need a more thorough evaluation.

4.4. Conclusions

Our results suggest there is little genetic structuring within each of the moth species, and so provide no obvious evidence that host-specific cryptic species are present in these taxa.

Likewise, we identify several areas where additional taxonomic evaluation is necessary (e.g. in the case of Lygomuostima n. sp. and A. metastictalis). The identification of these considerations thus emphasise the value of incorporating molecular screening at an early stage of foreign exploration projects, even when the initial survey dataset appears fragmented.

Acknowledgements

We thank Kylie Galway, Tony Wright, and Bradley Brown (USDA ARS Australian Biological

Control Laboratory) for their collections of the insect and plant specimens. We also thank

James Hereward (UQ) for assisting with the initial molecular screening of these moths. We wish to acknowledge the support of the Florida Fish and Wildlife Conservation Commission,

14 the South Florida Water Management District, USDA ARS National Programs, and the staff of the USDA ARS Invasive Plant Research Laboratory. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity employer and provider. This research was also supported by a University of

Queensland Strategic Grant awarded to Gimme Walter and Graham McCulloch.

15

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Figure Captions

Figure 1. Distribution of Musotiminae moths whose larvae feed on Lygodium ferns across

Australia and Asia. Data points derived primarily from collections made during the current survey for natural enemies of Lygodium ferns.

Figure 2. Bayesian maximum clade consensus phylogeny of Lygodium fern moths

(Musotiminae) based on three genes (COI, 18S rRNA, 28S rRNA). Outgroups (Ostrinia nubilalis, Loxostege sticticalis, and Conogethes punctiferalis) have been removed for diagrammatic clarity. Posterior probabilities are noted above each node. Colours represent the host plant from which the insects were collected (green = L. microphyllum, blue = L. japonicum, red = L. flexuosum).

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Tables

Table 1. Geographic distribution, feeding mode, and host-range of Lygodium fern moths (Musotiminae). The undescribed Lygomusotima species was identified as such by Alma Solis (pers. comm.), while the undescribed Siamusotima species was named on the basis of its morphological similarity to Siamusotima disrupta. *See section 4.3, where reasons are given to explain why this species probably does not occur in Australia.

Feeding Species Collection sites Host-range (field collections) Defoliating Lygomusotima stria Solis & Yen Singapore, Thailand, Malaysia L. microphyllum Austromusotima camptozonale Hampson Australia L. microphyllum

Austromusotima metastictalis Hampson PNG, northern Australia* L. microphyllum

Neomusotima conspurcatalis Warren Australia, PNG, South East Asia L. flexuosum, L. microphyllum

Neomusotima fuscolinealis Yoshiyasu Japan, Hong Kong, China L. japonicum

Eugauria albidentata Hampson Indonesia L. microphyllum

Stem boring Siamusotima aranea Solis & Yen Thailand L. flexuosum Lygomusotima n. sp. Singapore, Malaysia L. microphyllum, L. flexuosum, L. circinnatum

Siamusotima disrupta Solis Hong Kong, China L. microphyllum, L. japonicum, L. flexuosum

Siamusotima n. sp. Australia (Cape York) L. microphyllum

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Table 2. Sequence statistics and model parameters for each gene region

COI 28S rRNA 18S rRNA Number of sites 675 1156 677 Variable sites (incl. outgroups) 226 237 21 Parsimony informative sites (incl. outgroups) 207 206 17 Variable sites (excl. outgroups) 211 224 23 Parsimony informative sites (excl. outgroups) 196 199 17 Optimal substitution model GTG +γ+I GTR+γ HKY

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GM, JM, MP, and GW conceptualised the manuscript. JM, RZ, and MP conducted the field-work. GM, DB, and KG conducted the molecular laboratory work. GM analysed the molecular data. GM drafted the initial manuscript, with significant input from GW. All authors contributed to the submitted version of the manuscript.

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Highlights

- Ten crambid species have been collected from Lygodium during foreign exploration

- We assessed phylogenetic relationships among these moths

- Stem boring and leaf feeding species formed well-supported monophyletic clades

- There was no evidence of host-specific cryptic species

- Our results emphasize the value of incorporating molecular screening at an early

stage of biological control projects

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