Lycium Ferocissimum) – Characterising the Target Weed for Biological Control T ⁎ G.A

Biological Control 143 (2020) 104206

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Biological Control

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Genetic diversity and morphological variation in African boxthorn (Lycium ferocissimum) – Characterising the target weed for biological control T ⁎ G.A. McCullocha,b, , E.V. Maudac, L.D. Charic, G.D. Martinc, K. Gurdasanib, L. Morind, G.H. Walterb, S. Raghue a Department of Zoology, University of Otago, PO Box 56, Dunedin 9054, New Zealand b School of Biological Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia c Centre for Biological Control, Department of Zoology and Entomology, Rhodes University, PO Box 94, Makhanda 6140, South Africa d CSIRO Health and Biosecurity, Black Mountain Science & Innovation Park, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia e CSIRO Health and Biosecurity, Ecosciences Precinct, GPO Box 2583, Brisbane, Queensland 4001, Australia

ARTICLE INFO ABSTRACT

Keywords: Lycium ferocissimum (African boxthorn) is a Weed of National Significance in Australia. Biological control may Australia have potential to manage this weed, but taxonomic uncertainty needs to be addressed first to facilitate searches Chloroplast and nuclear markers for potential agents. We sampled putative L. ferocissimum (i.e. tentatively identified morphologically in the field) Lycieae across its native range in South Africa and introduced range in Australia. Morphometric and genetic analyses Native range survey were conducted to confirm the species identity of these samples, and to assess morphological and genetic variation across both ranges. All samples collected in Australia were confirmed as L. ferocissimum, with no evidence of hybridisation with any other Lycium species. Nuclear and chloroplast genetic diversity within L. ferocissimum across both South Africa and Australia was low, with no evidence of genetic structure. One of the two common chloroplast haplotypes found across Australia was found at only two sites in South Africa, both near Cape Town, suggesting that the Australian lineage may have originated from this region. Ten samples from South Africa putatively identified in the field as L. ferocissimum were genetically characterised as different (unidentified) Lycium species. Our morphometric analyses across different Lycium species in South Africa did not identify any leaf or floral characteristics unique to L. ferocissimum, and thus morphological identification of the latter species in its native range may remain problematic. To ensure the correct Lycium species is surveyed for candidate biological control agents we suggest that individuals should be permanently tagged and putative morphological determinations supplemented with genetic analyses to confirm species identity.

1. Introduction Control of L. ferocissimum in Australia is challenging. It has a broad distribution across a range of environments and it is resilient to both Lycium ferocissimum Miers (African boxthorn; Solanaceae) is a large chemical and mechanical management techniques (Noble and Rose, spine-covered shrub native to South Africa (Arnold and De Wet, 1993; 2013). Biological control is considered a potential option to comple- Venter, 2000). It was widely planted in Australia as a hedge plant ment conventional control methods for L. ferocissimum in Australia during the mid-1800s, but has subsequently developed into an ag- (Adair, 2013; Ireland et al., 2019a). A prerequisite during the initiation gressive invader of pastures, roadsides and reserves across large areas of of a biological control program is the clear taxonomic identification of the country (Parsons and Cuthbertson, 1992). Lycium ferocissimum can the weed in question, in both its native and introduced ranges (Gaskin form dense thickets that are impenetrable barriers to grazing animals. et al., 2011). Interspecific hybrids in plants are said to be particularly These thickets reduce the value of grazing land significantly and can successful invaders (Ellstrand and Schierenbeck, 2000; Lee, 2002; harbour pests such as rabbits and foxes (Noble and Rose, 2013). The Schierenbeck and Ellstrand, 2009), so it is paramount to determine if extensive environmental and economic damage caused by L. fer- the invasive Australian L. ferocissimum lineage is of hybrid descent, ocissimum is one of the reasons this species was designated a Weed of especially considering that hybridisation among Lycium species is con- National Significance in 2012 (Australian Weeds Committee, 2013). sidered common in South Africa (Minne et al., 1994; Venter et al.,

⁎ Corresponding author at: Department of Zoology, University of Otago, PO Box 56, Dunedin 9054, New Zealand. E-mail address: [email protected] (G.A. McCulloch). https://doi.org/10.1016/j.biocontrol.2020.104206 Received 15 October 2019; Received in revised form 15 January 2020; Accepted 21 January 2020 Available online 25 January 2020 1049-9644/ © 2020 Elsevier Inc. All rights reserved. G.A. McCulloch, et al. Biological Control 143 (2020) 104206

2003a,b). In addition, hybridisation between invasive and native spe- measurements were taken from plants from an additional 21 hapha- cies is not uncommon (Paul et al., 2010), and can significantly affect zardly selected sites distributed along regional roads and on farms in potential biological control strategies (Gaskin et al., 2011). Lycium Queensland and New South Wales in February 2019. Flowering in- australe F. Muell., the single Lycium species native to Australia, has an dividuals were found at only six of these sites during this survey, so data overlapping distribution with L. ferocissimum across parts of Western on floral traits were recorded only from these sites in Australia. Australia, South Australia, Victoria and New South Wales (Adair, 2013). The introduced “Goji berries” (either L. barbarum L. or L. chi- 2.2. DNA extraction, amplification, sequencing, and genetic analyses nense Mill.; Wetters et al., 2018), of Eurasian origin, are also grown in southern and eastern regions of the country where L. ferocissimum oc- We sequenced samples identified morphologically as L. ferocissimum curs, with L. barbarum and L. chinense known to have escaped cultiva- from 41 sites across its native range (South Africa), and 43 sites across tion and become environmental weeds (Randall, 2007). Likewise, Ly- the invaded range (Australia; Fig. 1; Table S1). One to four samples cium afrum L., of African origin, has been introduced and is naturalised were sequenced per site. DNA was initially extracted from leaf tissue in Australia (Randall, 2007). using the CTAB extraction protocol (Doyle and Doyle, 1987). This was Molecular tools are commonly used to complement morphological followed by a do-it-yourself spin column extraction with EconoSpin characters for the taxonomic identification of weeds (Gaskin et al., spin columns from Epoch Life Sciences (Missouri City, Texas), using the 2011). In addition, these tools can be used to assess evidence of hy- protocol of Ridley et al. (2016). Four genomic regions were amplified: bridisation, and to determine the provenance of invasive weeds (Gaskin three chloroplast markers (trnH-psbA intergenic spacer, maturase K, and et al., 2011; Sutton et al., 2017; Williams et al., 2005). However, ac- the trnT-trnL intergenic spacer), and one nuclear marker (granule bound curate taxonomic identification of the target weeds using morpholo- starch synthase; GBSSI). The trnH-psbA intergenic spacer region was gical characters in the field remains vital, particularly when conducting amplified using primers trnHf and psbA3_f (Sang et al., 1997; Tate and comprehensive native range surveys for candidate biological control Simpson, 2003), for maturase K we used matK-1RKIM-f and matK- agents across broad geographic regions (Goolsby et al., 2006; Witt, 3FKIM (Kuzmina et al., 2012), for the trnT-trnL intergenic spacer we 2004). Species within the genus Lycium are highly plastic, so delimiting used trnL-a and trnL-b (Taberlet et al., 1991), and for GBSSI we used and identifying species within the genus with morphological characters waxyF and waxy2R (Levin et al., 2006a; Miller et al., 1999). PCRs can be difficult (Levin et al., 2006b; Venter, 2000). Morphological (12 µL) contained 7.6 µL water, 2.0 µL 5x buffer (Bioline, London, UK), variation within L. ferocissimum is substantial (Venter, 2000), perhaps 0.24 µL each of 10 mM forward and reverse primer, 0.08 µL Taq influenced by the broad range of climatic and environmental conditions polymerase (Bioline, London, UK) and 2 µL of DNA template. Cycling across its distribution in South Africa (Fig. 1). Identification of this conditions for the chloroplast markers involved an initial denaturation species in the field is therefore challenging. step (95 °C for 3 min) followed by 40 cycles of 95 °C for 20 s, 50 °C for In this study, we collected samples from putative L. ferocissimum 30 s, and 72 °C for 45 s, with a final annealing step of 72 °C for 10 min. plants (i.e. tentatively identi fied morphologically in the field) from A touchdown procedure was used to amplify GBSSI, following the across its native range in South Africa and invaded range in Australia. protocols of Levin and Miller (2005). PCR amplification was confirmed Genetic analyses (three chloroplast markers, one nuclear marker) were by gel electrophoresis, with amplified DNA cleaned with 1 µL of Exo- conducted in order to: (a) confirm plant identity, (b) assess genetic nuclease I (New England Biolabs Inc, Ipswich, United States) and 1 µL structuring across the native and invaded ranges (to explore the pro- of Antarctic Phosphatase (New England Biolabs Inc, Ipswich, United venance of the invasive lineage), and (c) assess evidence for hy- States) per sample. Samples were sequenced in both directions by bridisation between L. ferocissimum and other Lycium species that occur Macrogen Inc. (Korea). in Australia. In addition, we undertook morphometric measurements of Sequence chromatograms were edited in Geneious v11.1.5 (https:// a number of plants from South Africa and Australia. This allowed us to www.geneious.com; Kearse et al., 2012), and aligned with additional quantify the extent of morphological variation within L. ferocissimum, sequences of South African and Asian Lycium species, and two out- determine whether reliable morphological traits can aid in-field iden- groups (Capsicum lycianthoides Bitter and Solanum tuberosum L.) from tifications of plants, and assess whether morphological differences exist GenBank (Table S2), using the MUSCLE plugin (Edgar, 2004). We used across plants from South Africa and Australia. JModeltest v2.1.2 (Darriba et al., 2012) to select the best model of sequence evolution for each marker. The three chloroplast regions were 2. Material and methods concatenated into a single dataset, as each exhibited low levels of sequence divergence, and there should be no incongruence among re- 2.1. Sampling gions of the plastid genome as they cannot assort independently or recombine. The native distribution of L. ferocissimum in South Africa was col- Phylogenetic relationships were reconstructed in MrBayes 3.2.6 lated from South African herbarium records and the South African (Huelsenbeck and Ronquist, 2001). The concatenated chloroplast da- National Biodiversity Institute online database (https://www.sanbi. taset was partitioned by DNA region, allowing independent estimation org/)(Fig. 1). Plants were subsequently located during roadside sur- of parameters for each partition. Indels were treated as missing data. veys, and putatively identified as L. ferocissimum based on fruit char- Four Markov chains were run for five million generations, with chains acteristics and flower shape (sensu Venter, 2000). At least one plant sampled every 200 generations. The first 2,000 trees were discarded as was tagged at all sites (Fig. 1). At several sites significant morphological burn-in. We used Tracer v1.7.0 (Rambaut et al., 2018) to confirm that variation was evident, so two or more plants were tagged. Global Po- all parameters had converged, that burn-in was sufficient, and that the sitioning System (GPS) co-ordinates were recorded for each tagged effective sample size was greater than 200 for each prior. plant, and morphometric measurements taken (see Section 2.3). Leaf samples for genetic analyses were taken from tagged plants, and from a 2.3. Morphometric analyses maximum of ten surrounding plants growing within 100 m of the tagged individual. Leaf samples were stored dry in envelopes with silica The following morphometric measurements were recorded for each gel to preserve the DNA. of 48 plants from South Africa, including more than one plant per site in The distribution of L. ferocissimum across Australia was obtained some instances, and 21 plants from different sites in Australia (Tables from the Atlas of Living Australia (ALA, 2016). Leaf samples were taken S3, S4): for genetic analysis from up to 12 individual plants per site from across Plant. Plant height (measured as the highest leaves on the plant in the species’ distribution in Australia (Fig. 1). Morphometric relation to the ground), plant width (measured at its widest part from

2 G.A. McCulloch, et al. Biological Control 143 (2020) 104206

Fig. 1. (a) Lycium ferocissimum distribution records from the GBIF database (GBIF.org, 2019; open circles) and the location of sites where morphometric assessments were performed and samples for the genetic analysis were taken from individual plants across South Africa (cyan circles = Eastern Cape Province, magenta circles = Western Cape Province), and (b) location of sites where L. ferocissimum samples were collected for the genetic analysis in Australia (yellow circles). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) branch tip to branch tip), and stem diameter (recorded at ground level). Leaves. Ten of the largest leaves from ten haphazardly selected branches were excised, and we measured leaf length (measured from lamina tip to the petiole along the lamina midrib), and leaf width (measured at the widest part of the leaf). In addition, we removed a branch from each plant (which was deposited in the herbarium; as detailed below) and counted the number of leaves per fascicle. Flowers. A mature flower, where present, was haphazardly excised from each plant and dissected to show a transverse section of the flower. A photograph of the section with scale bars was then taken, and this was used to determine flower shape (tubular or bell), and sexuality, and to measure flower dimensions (Fig. 2). These floral characters have previously been used to differentiate between distinct Ly- cium species (Venter, 2000). Herbarium specimens were taken from all plants in South Africa for fl which morphometric characters were recorded in the field. Specimens Fig. 2. A transverse section of a Lycium ferocissimum ower showing the measurements used to characterise floral morphology. The letters A, B, C and D were examined in the Selmar Schonland Herbarium, Grahamstown, represent different measuring points on a transverse section of the flower South Africa to verify in-field identifications using a dichotomous key (AB = length from the top of the stalk to the highest point of corolla, to African Lycium species (Venter, 2000), and voucher specimens BD = width of the corolla, BC = length between the corolla petals, and fi lodged (Tables S3, S4). The de ning characters for L. ferocissimum were AC = length from the top of the stalk to the corolla petals). shiny bright green obovate to elliptic leaves, 12–35 mm long, 4–10 mm wide and a flower calyx 2/3 the length of the corolla tube (Venter 2000). Representative voucher specimens of Australian L. ferocissimum

3 G.A. McCulloch, et al. Biological Control 143 (2020) 104206 plants were deposited in the Australian National Herbarium, Canberra included in the well-supported clade with L. ferocissimum (clade A; (accession codes: CANB912039 and CANB912040; Tables S3, S4). Fig. 3). Samples from two of these sites (WC14, WC16) shared a hap- Based on DNA sequencing results, morphometric measurements lotype that was also common in Australian samples (haplotype AU made in South Africa were allocated to two groups: L. ferocissimum and common 2; Fig. 3; see Table S5 for a list of sites where this haplotype “Lycium sp. unknown”. A third group consisted of measurements made was found). Lycium ferocissimum samples in clade A were not mono- on L. ferocissimum in Australia. Statistical differences in plant mor- phyletic, with Clade A also including three other Lycium species (Fig. 3). phology among groups were assessed using STATISTICA 10 (Hill and For the purposes of the morphometric analyses (Section 3.2) all of the Lewicki, 2007). Analysis of Variance (ANOVA) were conducted for each samples from Clade A were considered to be L. ferocissimum. of the plant characters measured. Where significant differences were Plants from eight sites in South Africa, putatively identified mor- detected, Fisher’s Least Significant Difference (LSD) tests were used to phologically as L. ferocissimum, were not included in clade A with the make post hoc comparisons across groups. Differences in flower shape remaining L. ferocissimum samples. These putative L. ferocissimum se- and sexuality were assessed using Chi-Square statistics. quences did not match any sequences in GenBank, and their phylogenetic position suggests that they are not L. ferocissimum. These samples were part of an unresolved polytomy containing 12 other Lycium spe- 3. Results cies (Fig. 3). These plants were classified as “Lycium sp. unknown” and placed in a separate group for the morphometric analyses (see Section 3.1. Genetic analyses 3.2). A sample from site WC1 had a unique chloroplast haplotype, but the phylogenetic placement of this sample was not well resolved We sequenced three chloroplast markers from 217 plants across 84 (Fig. 3). Only a single Lycium species was recorded at most of our col- sites – 89 plants from 41 sites in South Africa and 128 plants from 43 lection sites across South Africa (Table S1). However, at three sites sites in Australia (GenBank accession numbers: (EC2, EC3, WC1) both L. ferocissimum and “Lycium sp. unknown” were MN910317–MN910965). Following concatenation of these distinct recorded (Table S1). markers, the edited DNA alignment was 2,152 bp in length. Limited We sequenced the GBSSI gene from 199 plants across 84 sites – 79 genetic diversity was detected in the concatenated chloroplast dataset, plants from 41 sites in South Africa and 120 plants from 43 sites in with only eight haplotypes identified across the material from South Australia (GenBank accession numbers: MN910966–MN911163). Africa and Australia. All plants at most sites were characterised by only Eighteen discrete GBSSI haplotypes were identified (Fig. 4). Only one of a single haplotype, though at some sites two haplotypes were recovered these haplotypes was common across Australia and South Africa (la- (see below). belled SA common and AU common; see Table S5 for a list of sites Plants tentatively identified with morphological characters in the where this haplotype was found). The remaining haplotypes were ty- field as L. ferocissimum did not form a monophyletic clade in the pically restricted to only a single site (Fig. 4). A single GBSSI haplotype Bayesian phylogeny (Fig. 3). Most of the South African samples, and was found at most sites, though two haplotypes were recorded at some many of the Australian samples, were identical across the three chlor- sites (e.g. EC3; Fig. 4). The relationships among the GBSSI haplotypes oplast markers, and matched sequences of L. ferocissimum accessions were not well resolved (Fig. 4), and the GBSSI and chloroplast phylo- from GenBank (haplotypes SA common and AU common 1; Fig. 3; see genies were not completely congruent. However, all of the specimens Table S5 for a list of sites where this haplotype was found). Samples from clade A in the chloroplast phylogeny (considered to be L. fer- from several locations in the Eastern (EC15, EC19) and Western Cape ocissimum) were included in a single clade in the GBSSI phylogeny (WC10, WC11, WC13, WC14, WC16; see Fig. 1) Provinces of South (which also includes the L. ferocissimum accession from GenBank). This Africa did not match any Lycium accessions on GenBank but were

Fig. 3. Bayesian phylogeny of samples collected in this study and Lycium spp. from GenBank based on three concatenated chloroplast regions (trnH-psbA intergenic spacer, maturase K, trnT-trnL intergenic spacer; see Table S2 for GenBank accession numbers). Posterior probabilities are noted above each node. Species in bold are found in Australia. Sites from which samples were collected are labelled with the region code (EC = Eastern Cape, South Africa, WC = Western Cape, South Africa and AU = Aus- tralia) followed with a reference number (Table S1). The number of samples with the same haplotype found at a site, if more than one, is noted in parentheses. Coloured bars summarise the geographic region where the haplotype was found (cyan = Eastern Cape, magenta = Western Cape, yellow = Australia; see Fig. 1). The most common South African haplotype (SA common) matched the L. ferocissimum accession on GenBank (see Table S5 for a list of sites where this haplotype was found). The two common haplotypes found in Australia (one that matched the L. ferocissimum GenBank accession) are labelled AU common 1 and AU common 2 (see Table S5 for a list of sites where these haplotypes were found.). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

4 G.A. McCulloch, et al. Biological Control 143 (2020) 104206

Fig. 4. Bayesian phylogeny of samples collected in this study and Lycium spp. from GenBank based on the nuclear GBSSI gene (see Table S2 for GenBank accession numbers). Posterior probabilities are noted above each node. Species in bold are found in Australia. Sites from which samples were collected are labelled with the region code (EC = Eastern Cape, South Africa, WC = Western Cape, South Africa and AU = Australia) followed with a reference number (Table S1). The number of samples with the same haplotype found at a site, if more than one, is noted in parentheses. Coloured bars summarise the geographic region where the haplotype was found (cyan = Eastern Cape Province, magenta = Western Cape Province, yellow = Australia; see Fig. 1). Chloroplast clade A refers to the clade that includes L. ferocissimum in Fig. 3 (Clade A). SA common and AU common were the most commonly found haplotypes in South Africa and Australia respectively, and matched the L. ferocissimum accession in GenBank (see Table S5 for a list of sites where these haplotypes were found). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) clade, however, was only weakly supported (PP = 0.70). The clade also (PP = 0.60). The sample from site EC9, however, was most closely includes L. afrum and L. schizocalyx (as it did in the chloroplast phy- related to L. oxycarpum (Fig. 4). logeny), but not L. tenue (Fig. 4). All samples classified as “Lycium sp. No evidence of a genetic disjunction within L. ferocissimum across unknown” in the chloroplast phylogeny were again genetically distinct the Western and Eastern Cape Provinces of South Africa was apparent, from those confirmed as L. ferocissimum (Fig. 4). Most of these samples with a single common chloroplast and GBSSI haplotype shared across were included in a clade in the GBSSI phylogeny with L. tenue, L. gar- the two regions. There was no obvious conflict between the placement iepense, and L. australe, though this clade was poorly supported of our L. ferocissimum samples in the GBSSI and chloroplast phylogenies

5 G.A. McCulloch, et al. Biological Control 143 (2020) 104206

Table 1 Comparisons of morphological characteristics across plants of Lycium ferocissimum from South Africa (n = 37) and Australia (n = 21), and “Lycium sp. unknown” (n = 10) examined in South Africa (Tables S3, S4). The identification of L. ferocissimum and “Lycium sp. unknown” in South Africa is based on results from the genetic analysis. Results of ANOVAs and χ2 tests across the three groups are also displayed, with superscript letters denoting a statistically significant difference across groups at p < 0.05.

Lycium ferocissimum “Lycium sp. unknown” ANOVAs and χ2 tests

South Africa Australia1

Floral characteristics Sexuality (no. of sites) Bisexual (34), asexual (3)a Bisexual (6)a Bisexual (4), asexual (6)b χ2 = 16.42, d.f. = 2, p < 0.001 Flower shape (no. of sites) Bell (29), tubular (8)a Bell (6)a Bell (2), tubular (8)b χ2 = 15.6, d.f. = 2, p < 0.001 2 a b a Length AC ( ± 1SE) [mm] 17.0 ( ± 3.9) 11.8 ( ± 2.3) 17.8 ( ± 5.2) F2,65 = 46.0, p < 0.001 2 a b a Width BD ( ± 1SE) [mm] 9.4 ( ± 2.0) 10.8 ( ± 2.6) 8.4 ( ± 1.7) F2,65 = 58.6, p < 0.001 2 a b a Length AB ( ± 1SE) [mm] 12.7( ± 3.2) 8.8 ( ± 1.0) 10.8 ( ± 2.3) F2,65 = 49.8, p < 0.001 2 a b a Length BC ( ± 1SE) [mm] 8.0 ( ± 2.4) 7.4 ( ± 2.0) 8.2 ( ± 2.8) F2,65 = 54.7, p < 0.001 Stem characteristics a b a Diameter ( ± 1SE) [mm] 63.9 ( ± 46.0) 28.5 ( ± 16.0) 68.3 ( ± 32.5) F2,65 = 11.1, p < 0.001 Plant characteristics

Height ( ± 1SE) [m] 1.80 ( ± 0.80) 2.0 ( ± 0.8) 2.0 ( ± 0.6) F2,65 = 0.5, p = 0.642

Width ( ± 1SE) [m] 2.5 ( ± 1.1) 2.4 ( ± 1.0) 3.1 ( ± 0.90) F2,65 = 1.6, p = 0.214 Leaf characteristics

Leaves per fascicle 7.5 ( ± 2.1) 7.1 ( ± 1.0) 8.4 ( ± 2.2) F2,65 = 1.7, p = 0.192 a b a Length ( ± 1SE) [mm] 28.1 ( ± 8.6) 21.0 ( ± 5.5) 24.8 ( ± 4.6) F2,65 = 4.0, p = 0.022

Width (mm) 8.1 ( ± 3.8) 8.6 ( ± 2.2) 5.0 ( ± 2.3) F2,65 = 1.3, p = 0.290

1 Floral characteristics were assessed for only six individuals plants, because of lack of ﬂowering at the time to survey was conducted. 2 See Fig. 2.

(Figs. 3, 4), providing no evidence for hybridisation in the native range. resulting from incomplete lineage sorting. However, these samples Likewise, no evidence of hybridisation was apparent in Australian group together and are genetically distinct from samples confirmed as plants, with all plants from Australia having only L. ferocissimum L. ferocissimum in both the nuclear and chloroplast phylogenies, even at chloroplast and nuclear DNA. sites where the discrete chloroplast lineages occur in sympatry (EC2, EC3, WC1). This suggests an absence of gene-flow across lineages, in- dicating that these samples are most likely from different Lycium spe- 3.2. Morphometric analyses cies. Identifying these non-L. ferocissimum plants to species level was not possible based on sequence data currently available in GenBank. Both asexual and bisexual, and bell-shaped and tubular flowers Nonetheless, the genetic analysis suggests that more than a single were recorded from South African plants molecularly identified as L. unidentified Lycium species was included in our sampling. Several of ferocissimum, though bisexual and bell-shaped flowers were the most these plants may represent interspecific hybrids between L. ferocissimum common (Table 1; Table S3). In contrast, most of the “Lycium sp. un- and one or more other Lycium species, though further well-designed known” plants had asexual and tubular flowers, with Chi-squared tests tests of this conjecture are required. Indeed, these unidentified species revealing significant differences in these characteristics across L. fer- could potentially represent undescribed Lycium species. South Africa is ocissimum and “Lycium sp. unknown” (Table 1). There were, however, considered one of the centers of diversity for the genus Lycium (Venter, no significant differences across these two groups in any other mor- 2000), but few studies have been conducted on the genus across the phological trait measured (Table 1). country. A more thorough investigation of the genus across South Africa Only bisexual bell-shaped L. ferocissimum flowers were recorded in – using a combination of alpha taxonomy and DNA sequencing – is Australia (Table 1). Differences in a number of floral characteristics clearly required. were detected across L. ferocissimum plants from South Africa and Confirmation that several of the plants putatively identified as L. Australia (Table 1). Most notably, plants from Australia had a sig- ferocissimum in the field using morphological characters were from nificantly wider corolla’s, but significantly shorter AC (length from the different (unidentified) Lycium species is cause for concern in the con- top of the stalk to the corolla petals) and AB (length from the top of the text of surveying for candidate biological control agents. Whereas floral stalk to the highest point of corolla) measurements (see Fig. 2). In ad- traits, such as shape and sexuality, may assist to distinguish L. fer- dition, South African L. ferocissimum plants had significantly larger ocissimum from other Lycium species, they may still not do so with diameter stems and longer leaves than those of Australian plants complete accuracy. Furthermore, flowers are not always present, and (Table 1; Table S4). our analyses highlighted that identification based on other morphological characters is not reliable. To overcome this problem, we have been 4. Discussion surveying for potential biological control agents on permanently tagged individual L. ferocissimum plants for which the identity has been con- 4.1. Genetic and morphological variation across Lycium samples collected fi rmed by DNA sequencing. Further, we have deemed it prudent to keep in South Africa specimens and colonies of candidate biological control agents collected from different plants separate, until their identity as L. ferocissimum is Our study highlights the challenges that must be dealt with when confirmed. searching for candidate biological control agents for L. ferocissimum across its native range. While most specimens collected across South Africa that were morphologically identified as L. ferocissimum were 4.2. Genetic and morphological variation across L. ferocissimum from South confirmed as that species, genetic analyses revealed that several spe- Africa and Australia cimens (particularly from the Eastern Cape Province) were not included in the clade that included L. ferocissimum. It is possible that these plants The identification of L. ferocissimum in the field in Australia was not are indeed L. ferocissimum, with the discrete chloroplast lineages as problematic as in South Africa. This is because there are only four

6 G.A. McCulloch, et al. Biological Control 143 (2020) 104206 other Lycium species that are present, outside of cultivation, but with review & editing. G.H. Walter: Conceptualization, Writing - review & restricted distributions: the native L. australe and the naturalised L. editing. S. Raghu: Conceptualization, Investigation, Funding acquisi- barbarum, L. chinense, and L. afrum (Randall, 2007). Our genetic ana- tion, Project administration, Writing - review & editing. lyses confirmed that all samples morphologically identified as L. ferocissimum were indeed that species. No evidence of hybridisation be- Declaration of Competing Interest tween any of the Lycium species in Australia was detected in the analyses. This is encouraging because invasive weeds that are hybrids The authors declare that they have no known competing financial are typically considered to be more challenging to target with biological interests or personal relationships that could have appeared to influ- control (Williams et al., 2014; Zalucki et al., 2007). ence the work reported in this paper. Few morphological differences were detected in L. ferocissimum leaf or stem characteristics across South Africa and Australia. However, Acknowledgements significant differences in flower structure were evident among plants from South Africa and Australia. These differences could: (a) indicate We thank Andrea Bowden, Nicola Brookhouse, Stephen Butler, that only plants with genetic material for shorter, bell-shaped flowers Gareth Evans, Patrick Gleeson Jr., Patrick Gleeson Sr., John Heap, Peter were introduced into Australia, (b) be the result of phenotypic plasticity Hennig, Scott Herring, Craig Hunter, Mike Jones, Raelene Kwong, related to environmental conditions (see Gratani, 2014), or (c) suggest Matthew Lowry, Peter Michelmore, Kumaran Nagalingam, Jason that the reduced selection pressure in Australia (e.g. due to fewer Neville, Michael Noble, Ian Quinn, Michelle Rafter, Henry Rutherford, herbivores; see Adair, 2013) resulted in plants investing in reproduction John Scott, Tim Vance and Jason Walter for collecting L. ferocissimum and growth rather than herbivore defenses (see Blossey and Notzold, samples from across Australia for the genetic study. In addition, we 1995), leading to larger flowers. These morphological differences in thank Tony Dold (Selmar Schonland Herbarium in the Albany Museum, flower shape and size are, however, unlikely to affect future biological Grahamstown, South Africa) for assisting in plant identification. We are control agents, unless flower-feeding agents are released. grateful to Kylie Ireland and Gavin Hunter for constructive feedback Genetic diversity across L. ferocissimum in South Africa was low that greatly improved the manuscript. based on the markers used in this study, and even less genetic diversity was found in L. ferocissimum in Australia, a phenomenon commonly Appendix A. Supplementary data observed in invasive plants (Ward et al., 2008). The lack of any detected genetic diversity and structure across L. ferocissimum in South Africa Supplementary data to this article can be found online at https:// makes inferring the introduction history of the invasive lineage chal- doi.org/10.1016/j.biocontrol.2020.104206. lenging. 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