Choosing the right plants to test: The host-specificity of Longitarsus sp. (Coleoptera: Chrysomelidae) a potential biological control agent of Heliotropium amplexicaule

Available online at www.sciencedirect.com

Biological Control 44 (2008) 271–285 www.elsevier.com/locate/ybcon

Choosing the right plants to test: The host-specificity of Longitarsus sp. (Coleoptera: Chrysomelidae) a potential biological control agent of Heliotropium amplexicaule D.T. Briese *, A. Walker CSIRO Entomology, GPO Box 1700, Canberra ACT 2601, Australia Received 14 March 2007; accepted 2 May 2007 Available online 13 May 2007

Abstract Over the past 30 years, protocols for the selection of test plants used to determine the host range of candidate biological control agents have remained largely unchanged. Using the case of the root-feeding flea beetle, Longitarsus sp., a candidate agent for biological control of Heliotropium amplexicaule in Australia, this paper describes a ‘‘modernized’’ protocol, based more strongly on phylogeny, and refined by ecological and biogeographic similarities. Taxonomic nomenclature is de-emphasized in favour of strict phylogenetic relationships and the use of so-called ‘‘safeguard species’’ is abandoned. This is the first time that a biological control agent has been tested for host-specificity and application made for release, based solely on the new protocol, and the changes were acceptable to the regulatory organisations in Australia. The testing showed that adult feeding extended to plant species with up to five degrees of phylogenetic separation from H. amplexicaule, indicating that there would be a moderate risk that more distantly related plants suffer some feeding damage by adult Longitarsus sp. when they co-occur with infestations of the target weed that have large flea-beetle populations. Such damage would not have severe consequences for survival and reproduction of the plants. However, Longitarsus sp. was able to complete its lifecycle on plants related to the target weed by two degrees of phylogenetic separation or less, leaving indigenous Heliotropium and Tournefortia species at some risk of colonisation. While these species had different life-histories and/or only slightly overlapped with the actual and potential range of the target weed, a minority of reviewers were concerned that insufficient information was available on the dispersal abilities of Longitarsus sp. to dismiss this risk. Release was therefore not approved. While disappointing for the biological control project, the outcome was not unexpected, as the assessment was based on factors that modified the effects of host range alone. The new protocols highlighted some problems in the review process concerning an overreliance on taxonomic nomenclature as opposed to actual genetic relationships and an inadequate understanding of the nature of risk. However, they also directed attention to knowledge gaps in biogeography and agent biology that might refine the assessed risk. As such, this process can be considered an improvement in methodology. Moreover, rejection of the application to release Longitarsus sp. demonstrated that fears expressed by some researchers/regulators that the removal of ‘‘safeguard’’ species would somehow weaken the data and allow unsafe agents to be released were unwarranted. Crown copyright  2008 Published by Elsevier Inc. All rights reserved. Keywords: Biological control; Host-specificity testing; Test plant selection; Risk assessment; Heliotropium amplexicaule; Longitarsus sp.

1. Introduction Over the past 30 years, applications for the release of weed biological control agents have tended to follow a fairly rigidly formula; selection, primarily on taxonomic (but often including economic/environmental) criteria, of a list of non*

Corresponding author. E-mail address: [email protected] (D.T. Briese).

target plants for testing the host-specificity of the agent, presentation and interpretation of the results of those tests in terms of agent host-range followed by a recommendation that it is ‘‘safe’’ for release. ‘‘Unsafe’’ agents never reach the application process. Recently though, there has been considerable questioning of the way this testing is done in the light of perceived non-target effects following the release of certain agents (e.g. Gassmann and Louda (2000), McFadyen et al. (2003), Haines et al. (2004)).

1049-9644/$ - see front matter Crown copyright  2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.05.001

272

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

In a recent review of the tensions developing within the discipline of biological control, Briese (2005) noted that there are four key components to addressing the issue of host-specificity and risk to non-target organisms; (1) knowing what plants to test, (2) using the most appropriate methodology for testing, (3) interpreting these data on in the light of a broader knowledge of the evolution and ecology of host-choice behaviour and (4) communicating the results and interpretations clearly to regulators and a concerned public. It was clear that better ways were needed of structuring and presenting test data to regulatory agencies. Since 1974, biological control practitioners have followed Wapshere’s (1974) centrifugal phylogenetic method in selecting test plants for testing candidate control agents. However, as pointed out by Briese (2003), this procedure is anchored in a now outdated state of knowledge and, in practice, focuses more on taxonomic circumscriptions than phylogeny. The practical result is a tendency to include test plants from as many named related taxa as possible rather than being concerned about their precise relationship to the target species. While impressive in size, and giving some degree of comfort because of this, such test lists are probably no more informative than much reduced ones that focus on closely related species. There is now consensus amongst Australian biological control researchers (see Sheppard et al., 2003) that the centrifugal phylogenetic method should be modernised along the lines proposed by Briese (2003) to take advantage of (1) the explosion in published data on plant phylogenetic relationships over recent years, and (2) a better understanding of the evolutionary and ecological drivers of host-usage by specialist herbivores. This method would select plants primarily on the basis of their phylogenetic relationship to the target weed, but with ecological and biogeographic filters applied to ensure that plants to be tested included those species with the highest risk profiles. The key difference with current presentation of plant test lists is that the critical feature for determining relatedness to the target weed is the degree of phylogenetic separation rather than applying taxonomic circumscriptions; i.e. relationships are emphasised rather than categories. It is argued that this is biologically more realistic and would focus attention more on choosing plants likely to be acceptable to an agent. This would place more attention on defining the agent’s host range rather than determining whether or not individual plants were ‘‘safe’’. Moreover the modernised protocol would not include so-called safeguard species (economically or ecologically important plants that are phylogenetically distant from the target weed), which have remained as part of most test plant lists solely to placate regulators for reasons of public acceptability; an example of vertical tensions within the biological control framework. These distract from the real purpose of host-specificity testing, as they do not add information on host-range (see Briese and Walker, 2002). Originally, most host-specificity testing was carried out in the open field, but for regulatory, logistic and environ-

mental reasons, this is no longer feasible (see Clement and Cristofaro, 1995). Host-testing in recent years has been strongly dependent on results obtained in closed quarantine environments. However, the fact remains that no test carried out in the confines of a quarantine cubicle can replicate real world conditions and there can never be certainty about any agent host-range determined under artificial conditions (see papers in Withers et al., 1999). To reduce this problem, Briese (2005) proposed the use of comparative laboratory-based vs open-field host tests against a few key non-target species to proactively calibrate subsequent quarantine data and aid interpretation of results obtained under artificial conditions. Lonsdale et al. (2000) have pointed out that good risk communication is as important as risk assessment and the primary responsibility for this falls on researchers. Their role is not only to provide information to regulators, but to interpret it in a manner that is understandable, i.e. in the language of risk assessment. It is not necessary to quantify risk in absolute terms, and may even be misleading, but it is no longer acceptable to simply declare an agent ‘‘safe’’ for release. At the least, clarification needs to be made whether potential damage might be collateral, such as occasional adult feeding which would be localised with short-term consequences, or whether there is a likelihood of colonisation of the plant by breeding populations with much longer-term ecological and evolutionary ramifications. Few applications for release explicitly break down risk into ecological or evolutionary terms. Risk assessors are used to working in relativity-based systems, where terms such as more and less or limited rating categories are commonplace. A well-presented qualitative assessment of risk based on clearly presented data makes the host-specificity testing process more transparent and leads regulators towards knowledge-based, rather than fear-based decisions. This paper describes the host-specificity testing of Longitarsus sp. (Coleoptera: Chrysomelidae), a candidate biological control agent for the perennial South American herb, Heliotropium amplexicaule Vahl (Heliotropiaceae), an invasive weed in south-eastern Australia. It demonstrates the increased transparency of phylogeny-based test plant selection, the value of calibrating quarantine hostchoice data from open-field data and the importance of explicitly addressing different risk factors. Finally, the value of the approach is discussed in the light of the decision made by Australian regulatory authorities not to approve the release of the agent. 2. Methods 2.1. Target weed Heliotropium amplexicaule Vahl, commonly known as blue heliotrope, is a semi-prostrate perennial herb that reshoots each spring from a long-lived rootstock. It is native

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

to South America, but since its introduction into Australia as an ornamental plant in the 19th century, it has become a widespread weed of cultivation and pasture. Plants contain high levels of pyrrolizidine alkaloids that are toxic to livestock, causing chronic poisoning with associated damage to the liver and loss of condition (Ketterer et al., 1987). Cases of stock death are regularly reported (Glover and Ketterer, 1987). In agricultural systems, production losses occur due to competition by blue heliotrope with more desirable cropping and pasture species and a decline in animal performance as a result of its toxicity. It now occurs widely throughout New South Wales, southern Queensland, South Australia and Victoria (Parsons and Cuthbertson, 1992), infesting over 110,000 ha in New South Wales alone (Da Silva, 1991). H. amplexicaule is continuing to expand both its range and the size of local infestations and, in 1999, was approved as a target weed for biological control in Australia. 2.2. Agent The biological control strategy for blue heliotrope envisages a two-pronged attack against the above ground biomass of the plant and the long-lived perennial root stock (Briese and Zapater, 2002). A leaf beetle, Deuterocampta quadrijuga Sta˚l (Coleoptera: Chrysomelidae) was released in 2002 to target the above-ground biomass. Longitarsus sp., a 2 mm long, dark-brown halticine flea-beetle, was the only agent that attacked the root system of the target weed, H. amplexicaule (Briese et al., 2000; Briese and Zapater, 2001). Aspects of the life cycle of Longitarsus sp. are described by Zapater et al. (2004). It is active from spring to autumn. Female Longitarsus sp. enter gaps in the soil to lay eggs directly onto the roots and emerging larvae then feed on the fine feeder roots that extend from the larger storage roots, impeding the uptake of nutrients and water. After emerging as adults, they feed on the leaf tissue, leaving shot-hole feeding wounds which become infected by saprophytic bacteria. Leaves eventually die and fall from the plant. This can cause complete defoliation of the target weed by the end of the activity period in autumn and, coupled with feeding on the root tissue by larvae, severely weakens the plants. Studies in the home range (Zapater et al., 2004) found that many attacked plants die before the onset of winter. Preliminary host-specificity testing in Argentina, based on a two-phase open field experiment (Briese et al., 2002), suggested that Longitarsus sp. does not attack plants outside the genus Heliotropium, and is most likely limited to a subset of species within that genus. 2.3. Test plant selection The host-specificity test list was based on the approach developed by Briese and Walker (2002) for testing the blue heliotrope leaf-beetle, D. quadrijuga, and expanded more generally in Briese (2003). Selection of test plants was based

273

on three elements of potential risk; phylogenetic relatedness (see Wapshere, 1974), biogeographic overlap and ecological similarity (i.e. life-history, phenology, growth form). As not all test plants with strong taxonomic affinities to the target weed can be tested, the latter two elements were used to select a representative sample of plants with known degrees of phylogenetic relatedness that best measured overall risk from the introduction of the proposed agent (see Briese, 2003). The phylogeny of H. amplexicaule has been become much clearer due to recent studies based on molecular markers. The more distant phylogenetic relationships shown here are based on recent morphological (Takhtajan, 1987) and molecular (Olmstead et al., 1993) studies, which recognise a monophyletic subclass, Lamiidae, within the Asteridae. This comprises four related orders, Boraginales, Gentianales, Solanales and Lamiales s.l. The Solanales are considered a sister clade of the Boraginales by Olmstead et al. (1993) and later studies by Grayer et al. (1999) support this relationship, which is closer than previously thought. Recent molecular studies have shown that that the order Boraginales includes the families Boraginaceae, Heliotropiaceae, Cordiaceae, Ehretiaceae, Hydrophyllaceae and Lennoaceae (not present in Australia) (see Bo¨hle and Hilger, 1997; Gottschling et al., 2001). Diane (2003) and Diane et al. (2002) have further used molecular markers to clarify the relationships of genera within the Heliotropiaceae and of species within the genus Heliotropium. Based on morphology, Craven (1996) had listed 81 species in Australia, including H. amplexicaule, as belonging to the genus Heliotropium. Of these, 75 are regarded as endemic, one cosmopolitan and five of recent introduction. The majority of endemic Australian species (70) belong to the section Orthostachys. However, the molecular studies by Diane et al. (2002) and Diane (2003) showed that this section forms a different phylogenetic clade to other species of Heliotropium, which they have described as a new genus, Euploca. Two other genera of Heliotropiaceae, Tournefortia and Argusia, were considered to be native to Australia. However, recent molecular studies (Gottschling et al., 2001; Diane et al., 2002) show that Tournefortia is in fact paraphyletic within the genus Heliotropium, though its name is currently maintained. Moreover, the sole native representative of Argusia, A. argentea, has recently been redescribed as Heliotropium foertheri Diane & Hilger on the basis of molecular studies (Diane, 2003). These studies have shown that the genus Tournefortia is paraphyletic within the genus Heliotropium, while the vast majority of endemic Australian species from the old section Orthostachys have been excised from the genus Heliotropium and placed in a sister genus, Euploca (Diane, 2003). The current understanding of the phylogenetic relationships of H. amplexicaule clearly demonstrates the importance of choosing test plants on the basis of phylogeny rather than taxonomic circumscription. It should be reemphasised here that the test list is based on relationships

274

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

rather than nomenclature. This application uses the taxon names of Diane (2003), which splits the old genus Heliotropium, in describing taxa within the Heliotropiaceae. While there is not a consensus as to whether parts of the genus should be renamed or not, this is a nomenclatural debate, and the underlying phylogenetic relationships remain valid (L. Craven, personal communication). Following from this understanding of the phylogeny of H. amplexicaule and related plants in Australia, test plants were selected on the basis of ‘‘degrees of phylogenetic separation’’, using the approach outlined by Briese (2003). Separation occurs where a phylogenetic lineage branches into two distinct clades (see Table 1). The following criteria were therefore used for selection of the test list: • At least one representative occurring in Australia from each of the four increasingly distant clades within the genus Heliotropium, • At least one representative of the sister genus, Euploca, within the family Heliotropiaceae, • Selected representatives of native genera belonging to the families Boraginaceae, Hydrophyllaceae, Cordiaceae and Ehretiaceae within the order Boraginales, and

• At least one representative from a genus within the sister order Solanales and the Lamiales/Gentianales clade. This provided testing to the seventh degree of phylogenetic separation, with the majority of test plants within the closest clades. Briese and Walker (2002) found a strong correlation between host-choice behaviour and the degree of phylogenetic separation, while studies by Pemberton (2000) in North America and Willis et al. (2003) in Australia indicate that where non-target use has occurred subsequent to release, this has always been on closely related plants (i.e. those with very few degrees of phylogenetic separation from the target). Hence, choosing a test list on this basis enabled a more realistic assessment of risk. To ensure that the plants selected would maximise the measurement of risk posed by Longitarsus sp., where possible, the representative species were those with similar life-histories and overlapping or allopatric ranges. Biogeographic overlap was based on distribution maps produced from herbarium records of Heliotropium species (Craven, 1996) and ecological similarity was based on descriptions of the life-history and plant form of Heliotropium species in Australia (Craven, 1996).

Table 1 List of test plants proposed for host-specificity testing of Longitarsus sp. for the biological control of blue heliotrope, Heliotropium amplexicaule

The phylogenetic relationships and family circumscriptions of the test plants are also shown. a There are no representatives of the same phylogenetic group (0) as the target weed in Australia. H. nicotianaefolium was tested in Argentina to ensure that the assessment of host range is complete. b H. arborescens, an exotic species, is the sole representative of this group in Australia. c This group is slightly larger than might normally be expected as a representative has been chosen from each Boraginales family, because Boraginaceae, Cordiaceae, Ehretiaceae and Heliotropiaceae were at one time considered subfamilies of the family Boraginaceae s.l.

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

Detailed host specificity of Longitarsus sp. was conducted in the high-security Black Mountain Quarantine Facility at CSIRO Entomology, Canberra, under high light conditions to promote plant growth and health and at a temperature of 20–27 C. The confinement of insects, such as must occur when testing under quarantine conditions, has the potential to modify the normal host-selection behaviour and must be considered in the design of any testing procedure to reduce the risk of errors. No-choice tests occasionally produce false positive results (when the test indicates that the insect uses a plant as a host when it actually doesn’t) (see Hill, 1999), but this means that negative results are very robust, as results err on the conservative side. Hence, the results of no-choice testing give confidence to regulators and the public (Hill, 1999). No-choice tests were therefore used to test both larval feeding and survival, and adult feeding and oviposition for Longitarsus sp. However, as a corollary, positive results stemming from nochoice tests need to be treated with some caution, as they may not reflect field behaviour where the agent has a choice to leave a particular plant (Harris and McEvoy, 1995). Hence, where no-choice tests produced ambiguous results or indicated that there may be some risk, these results were interpreted in the light of data from choice tests, in which Longitarsus sp. was presented with a choice between target and non-target plants. Some choice tests were carried out in the quarantine facility while others had been carried out in the field in Argentina prior to the introduction of Longitarsus sp. (see Briese et al., 2002). As little was known about the biology of Longitarsus sp., initial tests followed the method of Huber (1979) in which plant seedlings were grown in a glass beaker to enable the plant roots and insect activity below the soil level to be observed directly. One seedling of each of four test plant species was transplanted with their root system against the glass wall of a beaker. A sheet of blotting paper was used to press the roots system against the glass and a 50:50 mix of perlite and vermiculite poured into the centre

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

5

0

Eggs laid per week

2.4. Quarantine testing protocol

to hold the roots in place against the glass and to hold water for the plants. An aluminium foil sleeve wrapped around the outside of the beaker kept the roots dark and could easily be removed for periodic observation. Following the sequential control method successfully used for the host-specificity testing of the blue heliotrope leaf-beetle, D. quadrijuga (see Briese and Walker, 2002), ten adult Longitarsus sp. beetles were initially caged on a beaker containing H. amplexicaule seedlings for 5 days as a pre-test control to verify oviposition. They were then transferred to a beaker containing the test plant species for 8 days, followed by transfer back to H. amplexicaule plants for a further 5 days as a post-test control to verify that they were still capable of oviposition. At the end of each period of exposure, all the beakers were examined. The plants were assessed for feeding damage, the number of eggs laid was counted and the position and depth of each egg recorded. As adults feed by chewing small shot-holes into the leaves, feeding could be simply assessed by counting the number of shot-holes. Six replicates were carried out for each test plant species. Once the biology of the beetle was better understood, a second series of no-choice tests was carried out to determine the survival of Longitarsus sp. from eggs laid on the roots of test plants to adulthood. Rearing experiments had shown that the life-span of an adult Longitarsus sp. was ca. 8 weeks with maximum oviposition occurring at 2–3 weeks (Fig. 1), while development from egg to adult also took at least 8 days under quarantine conditions (Fig. 2). Test plants were grown in 25 cm diameter plastic pots and sleeve cages placed over each potted plant. Tenweek old Longitarsus sp. adults were then added to the sleeve cage and were exposed to the plant for 10–14 days, the period of maximum oviposition rate, before being removed. Where possible, six replicates were carried out for each test species. Due to difficulties in obtaining some test plants and ensuring that they were in good condition for testing, testing of the full list of plants was completed in 12 series of tests from September 2002 to April 2004.

Surviving adults

However, the majority of native species (70+) within the family Heliotropiaceae have non-overlapping ranges, being restricted to the dry tropics of northern and north-western Australia or deserts of central Australia. In addition, 38 of these species are ephemeral annuals that would not, in the long-term, sustain a fauna specialised on a perennial species such as the target weed, H. amplexicaule. Only three native species (Heliotropium asperrimum, Euploca ovalifolia and Euploca brachygyne) have a similar life-history and occur within the range of or close to H. amplexicaule. The proposed test list was approved by Biosecurity Australia with the addition of two additional species, Myosotis australis (Boraginaceae) and Euploca pauciflora (Heliotropiaceae), following recommendations by reviewers. The approved test list, showing the phylogenetic separation of test plants, is shown in Table 1.

275

0 0

2

4

6

8

10

No. of weeks

Fig. 1. Survival (open squares) and oviposition rate (closed circles) of adult Longitarsus sp. under quarantine conditions (14L:10D, 20–27 C).

276

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285 450

No. of adults emerging

400 350 300 250 200 150 100 50 0 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

No. of days from oviposition to emergence

Fig. 2. Time between oviposition and the emergence of F1 generation of Longitarsus sp. under quarantine conditions (14L:10D, 20–27 C).

Each series of tests was run in parallel with a control series of H. amplexicaule plants. Following exposure to and removal of adults, the numbers of feeding holes in the leaves of the test and control plants were counted and the cages were then checked twice a week for the emergence of progeny Longitarsus sp. adults from the soil. These observations continued for a period of 20 weeks, by which time all adults had emerged. As the beetles spend much of their time in the soil, it was difficult to remove them all at the end of the exposure period without risking damage to eggs or progeny. Some beetles were collected as early as five weeks after the exposure period, but these could not have been progeny as there was an insufficient timeframe for development from egg to adult. Therefore, emerging beetles were considered to be progeny only if they were collected after 8 weeks, the time by which most parental beetles would have died and the earliest time for egg and larval development to have occurred. Finally, in order to clarify some of the observations, choice experiments were set up as described above, except that Longitarsus sp. adults were confined in a large 1.2 · 0.6 · 0.6 m mesh cage containing H. amplexicaule and several more closely related test plants in 25 cm pots. This enabled the beetles to choose both feeding and oviposition sites. After 14 days exposure, the adults were removed and feeding holes were counted on each plant. The plants were then caged separately to monitor emergence of progeny as described above. 2.5. Risk assessment As mentioned in the introduction, risk assessors work with relativity-based systems. However, qualitative terminology itself has the inherent risk of causing confusion or losing interpretive value if it is used loosely or if different sets of terms are used. Hence, the risk to non-target organisms posed by the introduction of Longitarsus sp. is described using a strictly defined set of terms developed by the New Zealand Environmental Risk Management Authority (ERMA New Zealand, 2004). These comprise (1) a qualitative scale for describing the likelihood of an event occurring using seven levels with precise meanings:

• Highly improbable = almost certainly not occurring but cannot be totally ruled out • Improbable (remote) = only occurring in very exceptional circumstances • Improbable = considered only to occur in very unusual circumstances • unlikely (occasional) = could occur, but not expected to occur under normal conditions • Likely = a good chance that it may occur under normal operating conditions • Very likely = expected to occur if all conditions met • Extremely likely = almost certain and (2) a qualitative scale for describing the magnitude of an effect using five levels with precise meanings: • Minimal = highly localised contained environmental impact, no discernible ecosystem impact • Minor = localised and contained reversible environmental impact, some local plant or animal communities temporarily damaged, no discernible ecosystem impact or species damage • Moderate = measurable long term damage to local plant and animal communities, no obvious spread beyond defined boundaries, medium term individual ecosystem damage, no species damage • Major = long term/irreversible damage to localised ecosystem but no species loss • Massive = extensive irreversible ecosystem damage, including species loss

3. Results and discussion The results of the no-choice beaker tests with sequential controls are summarised in Table 2. These results indicated that, although there appeared to be a strong preference for the host plant and the congeneric species, Heliotropium indicum, both feeding and oviposition did occur on more distantly related plants under these conditions. The beaker method enabled detailed observations to be made of oviposition for the first time. Adult Longitarsus sp. followed roots down below soil-level to lay eggs directly on the roots of H. amplexicaule, up to 15 cm deep in the beaker, though this would probably be dependent on soil texture in the field. Larvae hatched and were observed feeding externally on finer hair roots. Unfortunately, it was difficult to control humidity in the beakers and larvae did not survive to maturity, even on the host plant, H. amplexicaule. It was therefore decided to abandon this technique for specificity testing, and use plants growing in soil. While oviposition could no longer be quantified, the results gave a combined measure for any oviposition and subsequent survival and development to adulthood. This is the critical parameter for oviposition/larval specificity. The results of the no-choice tests for plants grown in soil are summarised in Table 3. These data show that adult

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

277

Table 2 Sequential no-choice tests for Longitarsus sp. on test plant seedlings grown hydroponically in a beaker to enable observation of the roots Species

Verbena citriodora Myosotis discolor Echium plantagineum Heliotropium indicum

Adult feeding (No. of feeding holes)

Reproduction (No. of eggs laid)

Pre-test control

Test plant

Post-test control

Pre-test control

Test plant

Post-test control

402 642 1150 936

4 132 346 708

174 192 336 228

26 5 90 66

8 9 15 74

6 12 6 11

Both pre- and post-test controls were on Heliotropium amplexicaule. Six replicates of 10 adult beetles were exposed to each pre-control, test and postcontrol plant for 8 days.

feeding is less specific than is larval development. In the case of four of the seven congeneric species of Heliotropium, and Tournefortia muelleri, which is paraphyletic within the same clade as Heliotropium (Diane et al., 2002), adult feeding was not significantly different to that observed on the control H. amplexicaule plants (Table 3). However, of these five species, only in the case of Heliotropium curassavicum and T. muelleri did oviposition/larval survival also not differ significantly from the control (Table 3). Both of

these species have only one degree of phylogenetic separation from the target weed (see Table 1). A second group of test plants, including more distantly related members of the family Heliotropiaceae and species from the Boraginaceae, recorded significantly less adult feeding and no or virtually no oviposition/larval survival (Table 3). It should be noted that, even though development of adults was possible in some cases, it was below replacement rate and hence too low to support a sustainable

Table 3 Results of no-choice host-specificity tests for Longitarsus sp. in chronological order Species

Replicates

Adults per replicate

Adults added on

Adults removed on

Mean No. of feeding holes/replicate

Mean No. of adults emerged per replicateb

Verbena citriodora Myosotis discolor Heliotropium indicum Heliotropium arborescens Heliotropium amplexicaule Heliotropium foertheri Heliotropium amplexicaule Heliotropium curassavicum Heliotropium amplexicaule Lycopersicon esculentum Mentha spica Heliotropium amplexicaule Ehretia saligna Heliotropium amplexicaule Phacelia tanecetifolia Heliotropium amplexicaule Heliotropium supinum Heliotropium europaeum Heliotropium amplexicaule Convolvulus sabatius Heliotropium amplexicaule Euploca ovalifolium Cordia dichotoma Heliotropium amplexicaule Heliotropium asperrimum Heliotropium amplexicaule Tournefortia muelleri Heliotropium asperrimum Heliotropium amplexicaule Euploca brachygyne Myosotis australis Heliotropium amplexicaule

6 6 6 6 6 6 2 6 2 3 3 2 6 2 6 2 6 6 6 5 5 6 2 6 3 3 6 2 6 6 6 6

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

10/09/02 10/09/02 10/09/02 10/09/02 10/09/02 20/09/02 20/09/02 2/10/02 2/10/02 31/10/02 31/10/02 31/10/02 6/11/02 6/11/02 13/01/03 13/01/03 6/02/03 6/02/03 7/02/03 12/06/03 12/06/03 29/09/03 29/09/03 29/09/03 14/10/03 14/10/03 4/11/03 4/11/03 4/11/03 25/03/04 25/03/04 25/03/04

1/10/02 1/10/02 1/10/02 1/10/02 1/10/02 14/10/02 14/10/02 22/10/02 22/10/02 10/11/02 10/11/02 10/11/02 18/11/02 18/11/02 23/01/03 23/01/03 20/02/03 20/02/03 21/02/03 23/06/03 23/06/03 13/10/03 13/10/03 13/10/03 27/10/03 27/10/03 17/11/03 17/11/03 17/11/03 8/04/04 8/04/04 8/04/04

0a 23 ± 6a 174 ± 9 125 ± 16 132 ± 15 2 ± 1a 105 ± 16 64 ± 7a 203 ± 7 0a 0a 120 ± 32 1 ± 1a 175 ± 44 0a 170 ± 30 125 ± 10 150 ± 11 153 ± 27 0a 129 ± 28 54 ± 5a 38 ± 7a 227 ± 28 110 ± 17a 209 ± 13 181 ± 26 110 ± 30 160 ± 47 57 ± 13a 77 ± 47a 183 ± 31

0a 0.2 ± 0.2a 0.3 ± 0.2a 0a 33.3 ± 6.7 0.2 ± 0.2a 9.0 ± 2.0 19.0 ± 11.0 9.5 ± 7.0 0a 0a 7.3 ± 2.4 0a 76.0 ± 7.8 0a 21.0 ± 6.0 0.2 ± 0.2a 0.8 ± 0.8a 3.2 ± 1.2 0.2 ± 0.2a 10.2 ± 2.2 0a 0a 29.3 ± 5.3 1.0 ± 0.6a 59.0 ± 30.6 9.3 ± 3.5 2.5 ± 2.5a 12.5 ± 2.9 0a 0.2 ± 0.2a 8.0 ± 0.6

Results followed by a are significantly different from the Heliotropium amplexicaule control. b values for adult emergence of 0.2 indicate that only one adult emerged per set of six replicate plants. These values are included although there it is probable that these could be contaminant adults that managed to get on plants being sampled due to their high mobility and very small size.

278

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

and choice testing, the highest result (i.e. risk was maximised) was used to obtain relative data. These relative results are set out in Figs. 3 and 4. Fig. 3 shows that adults are capable of feeding on plants with up to three degrees of phylogenetic separation to the same extent as on the target weed. Low levels of feeding also occurred on plants phylogenetically separated by up to four and five degrees. This suggests that short-term feeding damage could be sustained by a number of related species in the field. Fig. 4 shows the great specificity of immature stages (the combined measure of oviposition/ development from egg to adult). Here, similar performance to that on the target weed is restricted to those test plants with one degree of separation, with low performance by those with two degrees of separation. The data indicate that plants with three or more degrees of phylogenetic separation could not sustain a viable population of Longitarsus sp. in the field. The ranks of each plant species based on adult feeding data and adult emergence after eight weeks were added together and then re-ranked. The Spearman rank correlation between this combined ranking for adult feeding and larval development and the degree phylogenetic separation was highly significant (rs = 0.88, t21 = 8.17, P < 0.001). The value of using phylogenetic separation as a determinant of host plant utilization is further shown by Fig. 5, which shows the mean rankings for groups of plants of increasing distant phylogenetic relatedness to the target weed.

population on the test plants. There were too few progeny to test the viability or reproductive ability of the F1 generation. Two somewhat anomalous results were recorded. Although H. foertheri was very closely related to the target weed (phylogenetic separation of 1), it sustained virtually no feeding and supported virtually no development. Although closely related, this plant has quite a different form to the other Heliotropiaceae tested, as it is a woody plant with larger and harder leaves, growing to a tree, compared to the softer leaved, herbaceous growth habit of the other Heliotropiaceae test species. Longitarsus sp. may well have been physically unable to attack this plant regardless of any biological acceptability. This result emphasises the importance of testing plants of similar form to ensure more meaningful results. Secondly, H. curassavicum, also having only one degree of phylogenetic separation, showed reduced feeding by Longitarsus sp. adults, but the survival of larvae on its roots appeared to be similar to that on the target weed. Again, this species has a somewhat different leaf form to H. amplexicaule, with smooth succulent leaves compared to the flat, more hairy leaves of the target weed. It seems likely, therefore that different specificity criteria apply to the different insect stages. This result was confirmed in the choice test, where H. curassavicum, was subject to very little feeding, but produced the same number of F1 progeny adults as H. amplexicaule (Table 4). These choice tests also confirmed the no-choice results, by showing that the other congeneric species were still fed on and could support larval development in the presence of the target weed (Table 4). To better compare the test results, the performance of the beetles on test plants in each series was scored relative to that on their concurrent control H. amplexicaule plants, by dividing the test result by that occurring on the target weed. H. amplexicaule therefore had a score of 1 and, if the result for a test plant was greater than for the control, it was set at 1 as well. Heliotropium nicotinaefolium is included on the basis of it being a host plant in the native South American range of Longitarsus sp. that was tested under open-field conditions in Argentina (see Briese et al., 2002). Where differences occurred between no-choice

4. Risk assessment As indicated in the introduction there are two levels of risk associated with the release of an exotic biological control agent into the Australian environment; that of colonisation of non-target plants, with possible widespread, long-term population consequences and irreversible evolutionary consequences for the attacked plant, and that of collateral damage to non-target plants if the agent feeds on them at a particular stage without completing its life-cycle, which might engender localised, short-term consequences.

Table 4 Results of a choice host-specificity test for Longitarsus sp., in which adult weevils were exposed to Heliotropium amplexicaule and several test plant species in the same cage Species

No. of plants

Heliotropium indicum Heliotropium curassavicum Heliotropium europaeum Heliotropium amplexicaule

Adults per replicate

Adults added on

Adults removed on

Mean no. of feeding holes/replicate

Mean no. of adults emerged per replicate

4

100

18/12/03

5/1/04 315 ± 47

1.3 ± 1.2

4

10 ± 1a

4.5 ± 1.8

4

473 ± 96

0.3 ± 0.3a

4

206 ± 35

4.5 ± 2.5

Values followed bya are significantly different from the others at the 5% level.

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

279

Adult feeding Verbena citriodora

Degree of phylogenetic separation

Mentha spica

4-7 3

Lycipersicon esculentum Convolvulus sabatius

2 1 0

Phacelia tanecetifolia Ehretia saligna Heliotropium foertheri Cordia dichotoma Myosotis discolor Euploca ovalifolium Euploca brachygyne Heliotropium curassavicum Myosotis australis Heliotropium asperrimum Heliotropium supinum Heliotropium arborescens Heliotropium europaeum Tournefortia muelleri Heliotropium indicum Heliotropium nicotianaefolium Heliotropium amplexicaule

0.0

0.2

0.4 0.6 Relative performance

0.8

1.0

Fig. 3. Level of adult feeding on leaves of test plant species relative to that occurring on Heliotropium amplexicaule (species ranked according to increasing performance). Different shading of bars shows the degree of phylogenetic separation of the test plant from H. amplexicaule.

Survival (emergence after 8 weeks) Verbena citriodora

Degree of phylogenetic separation

Mentha spica Lycipersicon esculentum

4-7 3

Phacelia tanecetifolia

2 1 0

Ehretia saligna Cordia dichotoma Euploca ovalifolium Euploca brachygyne Heliotropium arborescens Convolvulus sabatius Myosotis discolor Myosotis australis Heliotropium supinum Heliotropium foertheri Heliotropium asperrimum Heliotropium europaeum Heliotropium indicum Tournefortia muelleri Heliotropium curassavicum Heliotropium nicotianaefolium Heliotropium amplexicaule

0.0

0.2

0.4 0.6 Relative performance

0.8

1.0

Fig. 4. Development from egg to adulthood in roots of test plant species relative to that occurring on Heliotropium amplexicaule (species ranked according to increasing performance). Different shading of bars shows the degree of phylogenetic separation of the test plant from H. amplexicaule.

280

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

Phylogenetic relatedness

7 6 5 4 3 2 1 0 0

2

4

6

8

10

12

14

16

18

20

Mean rank Fig. 5. Mean ranking of test plant species performances, grouped according their degree of phylogenetic relatedness to Heliotropium amplexicaule (lines indicate range of rankings).

4.1. Risk of colonisation of non-target plants The test data show that Longitarsus sp. is able to complete its life-cycle on plants related to the target weed, H. amplexicaule, by two degrees of phylogenetic separation or less. Of the test plants that fall within this risk category, H. indicum, Heliotropium supinum and Heliotropium europaeum are invasive exotic species considered to be weeds in a number of regions of Australia. Damage to these species would be considered to be beneficial. H. curassavicum is of South American origin, but now occurs widely on saline soils in Australia. While not considered weedy, it is not a native species and mainly occurs in more arid areas of Australia, well outside the suitable range of H. amplexicaule. The two native species tested that are at risk of colonisation are H. asperrimum and T. muelleri. H. asperrimum belongs to the section Pterotropium of the genus Heliotropium, which also includes H. ammophilum, H. crispatum, H. murinum and H. pleiopterum. These species have the same degree of separation from the target weed and should also be considered at risk of colonisation. While Tournefortia is a genus of South American origin, there are two indigenous Australian species, and both Tournefortia sarmentosa and T. muelleri should be considered at risk of colonisation. The level of risk and the degree to which it is realised for these species is dependent on (a) whether Longitarsus sp. behaviour in the open field reduces the likelihood that it will colonise these species, and (b) to what extent the actual and potential distributions of the species involved modify such risks. With regard to Longitarsus sp. behaviour in the field, surveys in South America (Briese and Zapater, 2002) included seven species of Heliotropium, and the flea-beetle was only ever collected from those species in the same section, i.e. with zero degrees of phylogenetic separation. It seems clear that natural hosts are limited to this closely related group of plants, none of which occur in Australia.

However, open-field experiments, in which a more distantly related species was deliberately planted alongside the natural host plant, did show that Longitarsus sp. would attack these under certain circumstances (Briese and Zapater, 2002). It is impossible to undertake the same tests on Australian native species outside of quarantine, so, this report adopts a precautionary approach and assumes that breeding could occur on plants of this small group of non-target plants, if they occurred in the vicinity of an infestation of H. amplexicaule that had been colonised by the flea-beetle. With regard to distributions of the species relative to the target weed, it should be noted that H. amplexicaule is a temperate/sub-tropical species that requires substantial summer rainfall. The actual and predicted distributions of H. amplexicaule in Australia are shown in Fig. 6. It is not highly competitive in its native South American range and occurs as an early successional plant following disturbance (Zapater et al., 2004). This is also true in its introduced range in Australia, where it has become a weed in open areas that are subject to continual disturbance by grazing and/or cultivation and is largely restricted to these habitats within the distribution range. There is little evidence that it can invade native vegetation and its spread has been restricted to farming and grazing habitats in temperate and sub-tropical eastern Australia. Longitarsus sp. was also only collected in temperate and sub-tropical areas of South America (Briese et al., 2000) and, given that the target weed is its natural host plant, it should also be restricted to similar habitats in Australia. Of the most closely related indigenous Heliotropium species (i.e. those in the section Pterotropium), four occur well outside the actual and potential distribution of the target weed (Fig. 7). Thus it seemed highly improbable that the flea-beetle would colonise these species. H. asperrimum

Fig. 6. Predicted range of blue heliotrope in Australia (from Briese and Zapater, 2001), based on temperature, rainfall quantity and rainfall seasonality, using the Bioclim climate-modelling module of the Biolink informatics software package (the darker the shading the more suitable for blue heliotrope). White squares indicate current distribution records.

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

281

Fig. 7. Distribution of indigenous Heliotropium species and Heliotropium amplexicaule (from Australia’s Virtual Herbarium, http://www.cpbr.gov.au/ avh/).

has some overlap at the extreme limits of its distribution and, while it is likely that Longitarsus sp. would colonise it in localised areas, the risk to H. asperrimum was considered to be locally minor and minimal overall. The two native species of Tournefortia are tropical species, occurring in northern Australia, from Cape York to midway down the Queensland coast (Fig. 8). Due to ecoclimatic limitations, it was considered highly improbable that the flea-beetle would colonise these species in the more northerly tropical part of their range. There is potential for broader overlap in area with the target weed in the southern part of this range, though the habitats of the species are quite different, since Tournefortia species occur mainly in undisturbed areas of native vegetation. It was therefore considered improbable that Longitarsus sp. would colonise these two species, while any impact in the southern part of their range would be localised and minor and unlikely to lead to a broader colonisation of Tournefortia species due to habitat restrictions.

The quarantine testing, however, showed that such adult feeding could also extend to less closely related plant species (with up to five degrees of phylogenetic separation). The open-field tests in Argentina (Briese et al., 2002) indicated that, under natural conditions, this was not likely to be widespread, as only plants with three or less degrees of phylogenetic separation were fed on by adults, even when the natural hosts were removed. Nonetheless, Zapater et al. (2004) did observe a few feeding holes on the more distantly related Convolvulus sp., though only when it grew in the presence of heavily attacked H. amplexicaule. Taking a conservative approach again, there would be a moderate risk that more distantly related plants suffer some feeding damage by adult Longitarsus sp. when they co-occur with infestations of the target weed that have large flea-beetle populations. However, such damage should be minor and temporary and it is highly improbable that it would have any consequences for survival and reproduction of the plants.

4.2. Risk of short-term collateral damage

4.3. Application outcome

In all these cases, it was considered likely that there would be short-term collateral damage due to feeding by adult Longitarsus sp. on the leaves of non-target plants growing in the presence of the target weed. This would primarily occur when heavy feeding damage to the target weed made it less favourable as a host plant.

The data presented here suggested that Longitarsus sp. had a highly restricted host-range. There was a likelihood of short-term damage to a range of non-target plants growing with H. amplexicaule, but the risk of colonisation and serious damage to these plants was considered minimal for all bar three species of importance, H. asperrimum, T.

282

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

Fig. 8. Distribution of Tournefortia species and Heliotropium amplexicaule (from Australia’s Virtual Herbarium, http://www.cpbr.gov.au/avh/).

muelleri and T. sarmentosa. In these cases, risk would be minimal over most of their range and highly localised over part of their range. The overall risk to these three non-target species was therefore considered to be low and, given the potential benefits of Longitarsus sp. for the biological control of H. amplexicaule, its release was therefore proposed in an Application to Biosecurity Australia in July 2004. Although most of the 21 reviewers approved release without comment, five opposed release on the basis of unacceptable risk to Australian native species within the genera Heliotropium and Tournefortia. Release was therefore not approved. While disappointing for the biological control project, the outcome was not unexpected. Clearly, evaluation of risk by the regulatory authorities was not going to be simple, as the assessment was based on factors that modified the effects of host-range alone. The responses of the opposing reviewers are themselves informative. Firstly, some became confused in trying to relate phylogenetic separation to taxonomic nomenclature. This misses one of the major reasons for the rigid emphasis on phylogeny in the new approach, namely to get away from the confusion and dubious reliance on the names of taxa. This project was a particular minefield for anyone relying too much on taxon names, as these have been the subject of revisions at both higher and lower taxonomic levels, largely based on the underlying phylogenetic relationships (see references in Briese et al., 2005). Such relationships are the only constants and it might be less

confusing if higher taxon names were omitted altogether from test lists, though our addiction to nomenclature probably renders this idea too radical for acceptance. Secondly, it was suggested that ‘‘the test list should be expanded to include more species on the basis of attack on those species tested’ as this would ‘‘give a better assessment of risk’’. This was a very worrying point of view, as the original list had already selected those species most likely to be used by the agent (i.e. most similar phylogenetically, ecologically and biogeographically) so that the maximum risk could be identified and a decision based on it. Moreover, the application had assumed that, if a test plant supported the agent’s life-cycle, then all plants with the same degree of phylogenetic separation would do the same. Risk is additive not averaged, so testing additional species would not diminish the existing risk. If sufficient doubt existed about those species found to be most at risk, rejection of the application was a valid response. Further testing of other less exposed species could not alter this fact. A major concern here is that the some of the reviewers responsible for assessing biological control introductions in Australia may not understand the fundamental nature of risk. Perceived risk can, however, be modified by further experiments that better define host usage on those non-target plants in question (e.g. continuation studies over several generations in quarantine as was suggested by one reviewer or open-field tests as was suggested by another). Finally, concerns were expressed about the accuracy and/or stability of biogeographic barriers to the risk of

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

colonization, i.e. what were the chances of Longitarsus sp. encountering biologically susceptible non-target plants that were presently allopatric to the distribution of the target weed or were considered outside the agent’s presumed habitat range. The reviewers correctly identified gaps in our knowledge of the dispersal ability of Longitarsus sp. and the limits of its native range in South America, both good indicators of its distribution limits in Australia. Such information could refine those elements of risk dependent on biogeography. From a project perspective, the need to collect such new data is probably beyond its logistic capabilities, but from the discipline perspective, it shows us the directions needed to improve the science of biological control and the types of supplementary information that we may need to obtain in a society increasingly averse to risk-taking. In one way, rejection of the application to release Longitarsus sp. was a positive result, as it should dispel fears expressed by some researchers/regulators that the removal of ‘‘safeguard’’ species would somehow weaken the data and allow unsafe agents to be released.

5. Conclusions The examples discussed in this paper show that it is possible to modernize the centrifugal phylogenetic method and that regulatory authorities, in Australia at least, do accept the new approach as outlined in Table 5. Release applications based on this approach have led to both acceptance (see Briese and Walker, 2002) and rejection (this paper) Table 5 Features of a modernized centrifugal phylogenetic method for selecting non-target plants to determine the host-choice of candidate biological control agents (after Briese, 2003, 2005) Risk framework used to refine test plant selection to what is logistically possible; • plant phylogeny is the primary criterion (based on knowledge of evolutionary, ecological and behavioral studies of host-usage by specialist insect herbivores) • ecology (similarity of life-history and form) and biogeography (similarity of range and habitat) refine this to ensure that plants most likely to be at risk are tested Strict phylogenetic approach to choosing test plant species • based on phylogenetic (evolutionary) relationships, not on taxonomic circumscription • degrees of phylogenetic separation clearly indicated in test lists and used in interpretation No ‘‘safeguard’’ species, which can be ineffective and distracting • pragmatism would dictate that where economically or environmentally important species form part of the high-risk non-target group selected by the above process, they would be preferentially included in the test list Results and interpretation presented in terms of broader risk, using precisely defined terminology • risk of colonization (long-term, widespread, irreversible, evolutionary and ecological consequences) • risk of collateral damage (short-term, local, reversible)

283

of candidate agents for the biological control of H. amplexicaule. However, by focusing on framing risk in terms of phylogeny, biogeography and ecology, the new approach has shifted the perspective from whether a particular plant may be ‘‘safe’’ or not to a broader concept of the factors influencing the expression of host range. It also has identified some possible flaws to the risk assessment process, namely a misunderstanding of the nature of risk and a deep-rooted reliance on taxonomic nomenclature, which persists even as a more phylogenetic approach is endorsed. In Australia, there are even signs of a ‘‘backlash’’ to a perceived too rapid or too extreme change to existing protocols. At a workshop held in early 2006, there was support for maintaining ‘‘plants known to be attacked by close relatives (=congeneric) of the candidate agent’’ as a safeguard category and recently led to the testing of grass species for a candidate control agent of a woody weed. This is curious, considering Wapshere (1974) himself played down the importance of this criterion. It demonstrates once again the pre-eminence of taxonomic circumscription over phylogeny (in this case insect phylogeny and how it relates to the evolution of host-choice) in current biological control thinking. Examples of large insect genera containing individual species that specialize on relatively distantly related plant species ignore the fact that these genera may comprise several phylogenetically distinct clades, e.g. Chrysolina (Garin et al., 1999) and one of the two insect genera discussed in this paper, Longitarsus (Dobler, 2001). In the application involving Longitarsus sp., no consideration was given to plants fed on by congeneric species and there was no objection to this by the regulatory authority or reviewers. However, if this criterion were maintained, researchers would either have to make arbitrary decisions about which distantly related plants to include, or include all of them, which would negate the whole idea of science-based plant lists for host-specificity testing. More importantly, phylogenetically-based host-specificity testing demonstrates high-risk patterns of host-usage within a few degrees of separation, without resorting to distantly-related ‘‘safeguards’’. Given the current sensitivity to the introduction of exotic organisms, this would be sufficient to reject such an agent. It is true that cases have been described, where populations of an insect herbivore species feed on very different hosts, but these species are disjunct oligophages whose host-plant usage depends on habitat and plant availability (see Thompson, 1993; Gomez-Zurita et al., 2000). Such species would be detected by phylogenetically-based host-specificity testing. This does reinforce, however, the need to better know the candidate agent and, more and more, this will mean its genetic identity and phylogenetic relationships. The ultimate test of any proposed changes to an existing methodology is whether or not it improves the outcomes. This is a difficult test for the host-specificity of biological control as there few instances of errors in host range assessment having occurred. However, the consequences of even a single mistake can be devastating and thus, any process

284

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285

which minimises the chances of this occurring should be adopted. In Australia, there is only one case where non-target damage could be directly attributed to a failure of the test plant selection protocol. The membracid, Aconophora compressa, released for the biological control of Lantana camara (Verbenaceae) in 1995 after host-specificity testing implied a narrow host-range, was recently reported causing severe damage to exotic ornamentals of the closely related Verbenaceae genera, Citharexylon and Duranta (McFadyen et al., 2003). These had not been tested prior to release, but testing subsequent to the incidence of non-target damage found them to be equally suitable hosts as the target weed (Manners et al., 2006). The original protocols failed because the non-target species attacked ‘‘slipped under the radar’’ (i.e. they were not indigenous and were not considered to be of economic importance). With the focus of these protocols on taxonomic nomenclature, rather than phylogenetic relationships, and concerns about testing ‘‘safeguard’’ species, they were easily left off. Had there been a stronger focus on phylogenetic relationship, supported by ecological and biogeographical criteria, as used here, it could be strongly argued that these plants would not have been omitted and the insect’s broader host-range would have been detected. In conclusion, plant selection is only one part of the whole testing procedure; methodology, interpretation of the data and communication are also important. Modernizing the centrifugal phylogenetic method needs to be done in conjunction with a re-examination of the whole process. As Briese (2005) pointed out, biological control is subject to the tensions that exist between the philosophical, regulatory/political and empirical components of the discipline. Modernizing host-specificity testing procedures is philosophically appealing and empirically feasible, but whether and to what extent it will be adopted depends to a large degree on the politics surrounding the reality and perception of risk and accountability. Acknowledgments This work was funded by the Rural Industry Research and Development Corporation. Thanks are due to the following people for the provision of seed or plant materials used in host specificity testing: Bob Bates, Adelaide, SA; Frank Burden, Tintinara, South Australia; Katherine Batchelor, CSIRO Entomology, Perth; Tanya McAndrew and Michael Day, Queensland Department of Natural Resources and Mining, Sherwood, QLD; Tony Grice, CSIRO Sustainable Ecosystems, Townsville, QLD; and the Yuruga Nursery, Atherton, QLD. We thank Shon Schooler for reviewing an earlier draft of this paper. References Bo¨hle, U-R., Hilger, H.H., 1997. Chloroplast DNA systematics of ‘‘Boraginaceae’’ and related families—a goodbye to the old and familiar concept of five subfamilies. Scripta Botanica Belgica 15, 30.

Briese, D.T., 2003. The centrifugal phylogenetic method used to select plants for host-specificity testing of weed biological control agents: can and should it be modernised? Improving the selection, testing and evaluation of weed biological control agents. CRC Technical Series No. 7, pp. 23–33. Briese, D.T., 2005. Translating host-specificity test results into the real world: the need to harmonise the yin and yang of current testing procedures. Biological Control 35, 208–214. Briese, D.T., Walker, A., 2002. A new perspective on the selection of test plants for evaluating the host-specificity of weed biological control agents: the case of Deuterocampta quadrijuga, a potential insect control agent of Heliotropium amplexicaule. Biological Control 25, 273–287. Briese, D.T., Zapater, M., 2001. Biological control of blue heliotrope. RIRDC Publ. No. 01/119, p. 18. Briese, D.T., Zapater, M., 2002. A strategy for the biological control of blue heliotrope (Heliotropium amplexicaule). In: Spafford-Jacob, H., Dodd, J., Moore, J.H. (Eds.), Proceedings of the 13th Australian Weeds Conference, 8–13 September, 2002, Perth, Australia. Plant Prot. Soc., WA, pp. 394–397. Briese, D.T., Walker, A., Zapater, M., 2005. Implementation of the blue heliotrope biological control strategy: Host-specificity testing of Longitarsus sp. RIRDC Publ. No. 05/003, 25pp. Briese, D.T., Zapater, M., Andorno, A., Perez-Camargo, G., 2002. A twophase open-field test to evaluate the host-specificity of candidate biological control agents for Heliotropium amplexicaule. Biological Control 25, 259–272. Briese, D.T., McLaren, D.A., Pettit, W., Zapater, M., Anderson, F., Delhey, R., Distel, R., 2000. New biological control projects against weeds of South American origin in Australia: blue heliotrope and serrated tussock. In: Spencer, N.R. (Ed.), Proceedings of the X Symposium on Biological Control of Weeds, 5–9 July, 1999, Bozeman, Montana. Montana State University, pp. 215–223. Clement, S.L., Cristofaro, M., 1995. Open-field tests in host-specificity determination of insects for biological control of weeds. Biocontrol Science and Technology 5, 395–406. Craven, L., 1996. A taxonomic revision of Heliotropium (Boraginaceae) in Australia. Australian Systematic Botany 9, 521–657. Da Silva, E., 1991. The ecology and control of blue heliotrope (Heliotropium amplexicaule Vahl). Unpublished Final Report to the Wool Research Corporation, p. 34. Diane, N., 2003. Systematic analysis of the Heliotropiaceae based on molecular and morphological-anatomical data. PhD thesis, Free University Berlin.. Diane, N., Fo¨rther, H., Hilger, H.H., 2002. A systematic analysis of Heliotropium, Tournefortia and allied taxa of the Heliotropiaceae (Boraginales) based on ITS1 sequences and morphological data. American Journal of Botany 89, 287–295. Dobler, S., 2001. Evolutionary aspects of defense by recycled plant compounds in herbivorous insects. Basic and Applied Ecology 2, 15–26. ERMA New Zealand, 2004. User guide to making an application for approval for the release of a qualifying organism by rapid assessment under the HSNO Act 1996. ER-UG-NO1Q-1, 03/04, p. 37. http:// www.ermanz.govt.nz/resources/publications/pdfs/ER-UG-NO1Q1.pdf. Gassmann, A., Louda, S.M., 2000. Rhinocyllus conicus: initial evaluation and subsequent ecological impacts in North America. In: Wajnberg, E., Scott, J.K., Quimby, P.C. (Eds.), Evaluating Indirect Ecological Effects of Biological Control. CABI Publishing, Wallingford, pp. 147–183. Garin, C.F., Juan, C., Petitpierre, E., 1999. Mitochondrial DNA phylogeny and the evolution of host-pant use in palearctic Chrysolina (Coleoptera: Chrysomelidae) leaf beetles. Journal of Molecular Evolution 48, 435–444. Glover, P.E., Ketterer, P.J., 1987. Blue heliotrope kills cattle. Queensland Agricultural Journal 113, 109–110.

D.T. Briese, A. Walker / Biological Control 44 (2008) 271–285 Gomez-Zurita, J., Petitpierre, E., Jusn, C., 2000. Nested cladistic analysis, phylogeography and speciation in the Timarcha goettingensis complex (Coleoptera: Chrysomelidae). Molecular Ecology 9, 557–570. Gottschling, M., Hilger, H.H., Wolf, M., Diane, N., 2001. Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny of Boraginales. Plant Biology 3, 629–636. Grayer, R.J., Chase, M.W., Simmonds, M.S.J., 1999. A comparison between chemical and molecular characters for the determination of phylogenetic relationships among plant families: an appreciation of Hegnauer’s ‘‘Chemotaxonomie der Pflanzen’’. Biochemical Systematics and Ecology 27, 369–393. Haines, M.L., Syrett, P., Emberson, R.M., Withers, T.M., Fowler, S.V., Worner, S.P., 2004. Ruling out a host-range expansion as the cause of the unpredicted non-target attack on tagasaste (Chamaecytisus proliferus) by Bruchidius villosus.. In: Cullen, J.M., Briese, D.T., Kriticos, D., Lonsdale, W.M., Morin, L., Scott, J.K. (Eds.), Proceedings of the XI International Symposium on Biological Control of Weeds. CSIRO, Canberra, pp. 271–276. Harris, P., McEvoy, P., 1995. The predictability of insect host plant utilisation from feeding tests and suggested improvements for screening weed biological control agents. In: Delfosse, E.S., Scott, R.R. (Eds.), Proceedings of the 8th Symposium on the Biological Control of Weeds, Lincoln, February 1992. CSIRO Publishing, Melbourne, pp. 125–131. Hill, R.L., 1999. Minimising uncertainty: in support of no-choice tests. In: Withers, T.M., Barton Browne, L., Stanley, J. (Eds.), Host Specificity Testing in Australasia: Towards Improved Assays for Biological Control. Scientific Publishing, Indooroopilly, pp. 1–10. Huber, J.T., 1979. A method for observing and rearing the root-feeding larvae of Longitarsus albineus (Foudras) (Col. Chrysomelidae). Mitteilungen der Schweizerischen Entomologischen Gesellschaft 52, 431– 433. Ketterer, P.J., Glover, P.E., Smith, L.W., 1987. Blue heliotrope (Heliotropium amplexicaule Vahl) poisoning in cattle. Australian Veterinary Journal 64, 115–117. Lonsdale, W.M., Briese, D.T., Cullen, J.M., 2000. Risk analysis and weed biological control. In: Wajnberg, E., Scott, J.K., Quimby, P.C. (Eds.), Evaluating Indirect Ecological Effects of Biological Control. CABI Publishing, Wallingford, pp. 185–210. Manners, A.G., Walter, G.H., Palmer, W.A., Dhileepan, K., Hastwell, G.T., 2006. Retrospective testing of Aconophora compressa; reproduc-

285

tion and survival on the target weed, Lantana camara, and non-target species. In: Preston, C., Watts, J.H., Crossman, N.D. (Eds.), Proceedings of the 15th Australian Weeds Conference. Weed. Management Society of South Australia, Adelaide, pp. 595–599. McFadyen, R., Day, M., Palmer, W., 2003. Putting a price on exotic ornamentals. Biocontrol News and Information 24, 48N–49N. Olmstead, R.G., Bremer, B., Scott, K.M., Palmer, J.D., 1993. A parsimonious analysis of the Asteridae sensu lato based on rbcL sequences. Annals of the Missouri Botanical Gardens 80, 700–722. Parsons, W.T., Cuthbertson, E.G., 1992. Noxious Weeds of Australia. Inkata Press, Melbourne, Australia. Pemberton, R.W., 2000. Predictable risks to native plants in weed biological control. Oecologia 25, 489–494. Sheppard, A.W., Heard, T.A., Briese, D.T., 2003. Workshop recommendations: The selection, testing and evaluation of weed biological control agents. In: Improving the selection, testing and evaluation of weed biological control agents. CRC Technical Series No. 7, pp. 89– 100. Takhtajan, A.L., 1987. Diversity and Classification of Flowering Plants. Columbia University Press, New York. Thompson, J.N., 1993. Preference hierarchies and the origin of geographic specialization in host use in swallowtail butterflies. Evolution 47, 1585– 1594. Wapshere, A.J., 1974. A strategy for evaluating the safety of organisms for biological weed control. Annals of Applied Biology 77, 20–211. Willis, A.J., Kilby, M.J., McMaster, K., Cullen, J.M., Groves, R.H., 2003. Predictability and acceptability: potential for damage to non-target native plant species by biological control agents for weeds. Improving the selection, testing and evaluation of weed biological control agents. CRC Technical Series No. 7, pp. 36–49. Withers, T.M., Barton Browne, L., Stanley, J., 1999. Host Specificity Testing in Australasia: Towards Improved Assays for Biological Control. Scientific Publishing, Indooroopilly. Zapater, M., Bartoloni, N., Perez-Camargo, G., Briese, D.T., 2004. Natural impact of the flea-beetle, Longitarsus sp., on Heliotropium amplexicaule in Argentina and its potential for use as a biological control agent in Australia. In: Cullen, J.M., Briese, D.T., Kriticos, D., Lonsdale, W.M., Morin, L., Scott, J.K. (Eds.), Proceedings of the XI International Symposium on Biological Control of Weeds. CSIRO, Canberra, pp. 208–214.