A molecular, morphological and cytological

A molecular, morphological and cytological investigation of the identity of non-native Ludwigia (Onagraceae) populations in Britain ¨ nyves2, J. P. Bailey3, J. C. David1, A. Culham2 J. D. Armitage*1, K. Ko 1

RHS Garden, Wisley, Woking, Surrey, GU23 6QB, UK, 2Centre for Plant Diversity and Systematics, Harborne Building, School of Biological Sciences, University of Reading, Reading, RG6 6AS, UK, 3Department of Biology, University of Leicester, Leicester, LE1 7RH, UK

Published by Maney Publishing (c) Botanical Society of the British Isles

A combined molecular, morphological and cytological analysis was used to study the identity and number of species of Ludwigia section Oligospermum occurring in British waterways. Only one taxon was identified for which the name L. grandiflora subsp. hexapetala (Hook. & Arn.) G.L. Nesom & Kartesz is preferred. A chromosome count of 2n580 was made for all plants tested and DNA evidence demonstrates that at least two clones are present in Britain. Morphological characters to differentiate L.grandiflora subsp. hexapetala and L. peploides subsp. montevidensis (Spreng.) P.H. Raven are provided. Though the production of fruit in Britain by apparently isolated populations is noted, repeated introduction into the wild from gardens is judged to be primarily responsible for the British distribution of the taxon. Legislative implications are considered and an amendment to Schedule 9 of the UK Wildlife and Countryside Act (1981) is strongly advocated. Keywords: chromosome numbers, identification, invasive species

Introduction Alien plants in the UK flora have been documented as curiosities for many years (Dunn, 1905; Salisbury, 1961; Clement & Foster, 1994), but only recently have examples such as Fallopia japonica (Houtt.) Ronse Decr. and Rhododendron 6 superponticum Cullen (Cullen, 2011) prompted a greater recognition of their impact on native habitats and the economic cost of their eradication. The role of gardens as the source of many alien plants was reviewed by Nelson (1994), and is now seen as critical, with some of the most significant non-native plants in the UK being derived from horticulture. Plants grown as aquatics are considered to pose a particular threat and Thomas (2010) has ranked members of the genus Ludwigia section Oligospermum that are retailed in the UK as large-flowered pond ornamentals a critical invasive risk. These plants have been recorded outside cultivation since the late 1990s (Burton, 1999) and, as is often the case with alien species when they first appear as adventives, have had numerous names applied to them. There are various reasons why this occurs: plants may have come from a horticultural source more concerned with aesthetic considerations than correct nomenclature; local botanists may be unfamiliar with the genus and not *Corresponding author: [email protected]

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ß Botanical Society of Britain & Ireland 2013 DOI 10.1179/2042349713Y.0000000023

have access to appropriate Floras; plants may belong to a complex that is difficult even in its native range; or specialist techniques such as chromosome counting may be required. These issues come to bear heavily when addressing taxonomically confused species. Correct identification of new adventives is critical if the distributions of separate, but morphologically similar species are to be accurately assessed and their source controlled. The primary purpose of this paper is to establish the identity and correct nomenclature of invasive, large-flowered Ludwigia in the UK.

Ludwigia section Oligospermum as invasive plants The species of Ludwigia belonging to section Oligospermum are attractive plants of aquatic ecosystems, native to South-East USA and parts of temperate South America and often known by the common name water primrose. In several places where they have been introduced for ornament, notably California and areas of Europe (Zardini et al. 1991b; Okada et al., 2009; Brunel et al., 2010), species in this section have become almost ineradicable weeds of waterways. In France, it has been shown that two taxa are present, identified as L. grandiflora (Michx.) Greuter & Burdet subsp. hexapetala (Hook. & Arn.) G.L. Nesom & Kartesz and L. peploides (Kunth) P.H. Raven subsp. montevidensis (Spreng.) P.H. Raven

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(Dandelot et al., 2005). Though similar, they are distinguishable by their different chromosome numbers and, morphologically, by characters given in Table 1. L. grandiflora subsp. hexapetala became naturalised first after it was introduced to the Lez river, Montpellier, in about 1830. However, in the southern part of France, this taxon has been displaced by L. peploides during the twentieth century (Dandelot et al., 2005). Studies of populations in France and California suggest that the spread of vegetative material provides much the most important means of dispersal (Dandelot et al., 2005; Okada, et al., 2009). However, Ruaux et al. (2009) demonstrated that seeds are resistant to low temperatures and submersion and that 25% of fruits were buoyant after three months, indicating that seed could be a means of long distance dispersal and that the risk of recolonisation by seed is high. Dandelot et al. (2005) reported that, though L. peploides is self-compatible, L. grandiflora subsp. hexapetala is self-incompatible. They hypothesised that the failure of plants in the South East of France to produce seed was due to their being a monoclonal population while the fruitful plants of the South West were of genetically mixed stock. The earliest report that has been traced of a species of water primrose in Britain is 1812 which Don (1832) gives as the date of their introduction to British cultivation. In providing a description and illustration of L. grandiflora, Anderson (1820) also states that the plant had flowered in the open air for the previous 2 years at the Chelsea Physic Garden, London. The first record of a non-native Ludwigia growing outside gardens in the UK is from 1998 when a plant was reported from Barn Elms in Barnes, London (Burton, 1999). This was followed in 1999 by records from two ponds on Barton on Sea golf course, South Hampshire (Clement, 2001). Subsequent reports have been scattered over a wide range from Dorset to Yorkshire and have shown a steady increase in frequency over time.

Systematics The genus Ludwigia is widely distributed through the old and new world and comprises around 82 species, divided into 23 sections (Zardini et al., 1991a).

Molecular data indicate that the genus is the earliest diverging member of the family Onagraceae and it is assigned to the subfamily Ludwigioideae by Wagner et al. (2007). There is no monograph of the genus, though Wagner et al. (2007) provide a complete treatment to sectional level and some sections have received comprehensive assessment elsewhere (Ramamoorthy, 1979; Peng et al., 2005). The genus has been treated narrowly and broadly, and at times Isnardia L., Oocarpon Micheli, Ludwigiantha Small, and Jussiaea L. have been recognised, the latter gaining widest use, being readily distinguished by possessing 8–12 stamens opposed to 4 in Ludwigia. The invasive species here under consideration have been included in Jussiaea and are frequently retailed under that name. They are currently classified within Ludwigia in section Oligospermum, a group noted for its high level of variability and potential for interbreeding (Peng, 1990; Zardini et al., 1991b), which has led to difficulties in defining species. Zardini et al. (1991a) examined Ludwigia uruguayensis (Cambess.) Hara, a widely distributed North and South American taxon, and concluded that it comprised two separate species, the hexaploid L. grandiflora (including the type of L. uruguayensis) and the decaploid L. hexapetala (Hook. & Arn.) Zardini, H.Y. Gu & P.H. Raven. However, they noted that in Brazil hybrids with intermediate chromosome number were known and in a table of morphological characters provided to distinguish their species all measurements given exhibit considerable overlap and two of the three qualitative characters are qualified by ‘usually’. The only unqualified character offered is that L. grandiflora is villous and L. hexapetala glabrous, though the holotype of L. hexapetala, held at K, features several ramets all of which show distinct, long, straight hairs, quite dense on the stems and scattered on the leaves (pers. obs.). Nesom and Kartesz (2000) questioned the complete separation of the taxa as species and stated that ‘The few morphological distinctions between the two…are strictly quantitative and broadly overlapping’. Based on analysis of 62 collections, they also qualified the pubescence character, reporting that ‘Plants with larger leaves and flowers and less dense

Table 1 Morphological characters that distinguish L. grandiflora subsp. hexapetala and montevidensis according to van Valkenburg (2007) and observations from the present study

Emergent leaves Leaf surface Venation Stipules Sepals Pneumatophores

Non-native Ludwigia in Britain

L.

peploides

L. grandiflora subsp. hexapetala

L. peploides subsp. montevidensis

Cuneate to base Dull, pale green Lateral veins conspicuous Small, sharply triangular, flat, usually less than 1 mm long Longer than 10 mm Frequently produced

Petiolar to some degree Glossy Lateral veins inconspicuous Larger, blunt-ovate, fleshy, approximately 1 mm long Shorter than 10 mm Rarely produced


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vestiture are L. hexapetala…those with smaller leaves and more dense vestiture are L. grandiflora’. They added that in their examination of American material glabrous specimens of L. hexapetala were relatively uncommon and concluded by recombining L. hexapetala as a subspecies of L. grandiflora. However, some, including Okada et al. (2009), continue to recognise two species.

Identity of Ludwigia in the UK


Europe has one native species of Ludwigia, L. palustris (L.) Elliott, which belongs to section Isnardia, and in Britain is confined to Hampshire and Dorset (Stace, 2011). A further member of this section, L. 6 kentiana E.J. Clement, bearing minute, deciduous petals, has become naturalised in a pond in South West London and is apparently a hybrid between L. palustris and L. repens J.R. Forst. of horticultural origin (Clement, 2000; Armitage & Bailey, 2011). Large-flowered Ludwigia have had numerous names indiscriminately applied to them by the horticultural trade, including L. grandiflora, L. hexapetala, L. peploides, L. uruguayensis, and their equivalent combinations under Jussiaea, leading to widespread confusion. This varied nomenclature, in combination with the phenotypic plasticity of the plants, has given rise to confusion concerning how many species are present in the UK and the right names to apply to them. Clement (2001) identified the large-flowered escapees as L. grandiflora but in so doing was simply following the name then most commonly used for plants naturalised in continental Europe (E. Clement, pers. comm.). Stace (2010) also considered that only one species was present in Britain for which he too offered the name L. grandiflora, though later amended this to L. hexapetala (Stace, 2011). A number of Ludwigia names appear in UK legislation. Included among the 34 taxa to be added to Schedule 9 of the UK Wildlife & Countryside Act (1981), which came into force in April 2010, were the names Ludwigia grandiflora, L. peploides, and L. uruguayensis. In addition, the first Invasive Species Action Plan produced by the Non Native Species Secretariat is for the eradication of L. grandiflora. It is essential to ensure names are correct before they become embedded in legislation. The use of incorrect names devalues efforts to control invasive non-native plants and undermines trust in the legislative mechanism, making it difficult to enforce.

Chromosome number The existence of chromosome races within species is well known and in many instances these races are not recognised at any taxonomic rank. However, where they coincide with a distinct ecology or geographic range, they are sometimes separated taxonomically,

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though morphological boundaries may not be certain, as in some of the infraspecific taxa of Festuca ovina L. or the segregates of Polypodium vulgare L. In spite of the various morphological and nomenclatural difficulties in assigning taxa in Ludwigia section Oligospermum, cytological differences provide much clearer separation. Chromosome numbers have been provided for the taxa most commonly implicated in European populations by Zardini et al. (1991b) who give counts for L. hexapetala (2n580), L. grandiflora (2n548), and four subspecies of L. peploides (all 2n516).

Barcoding DNA barcoding has become an established technique to help differentiate and identify species, whether morphological or cryptic. The sequencing of a small number of chloroplast DNA regions has been shown to distinguish flowering plant species around 70% of the time (Hollingsworth et al., 2009). However, for challenging plant groups such as Ludwigia additional chloroplast DNA regions beyond those conventionally used for barcoding need to be added. The DNA sequences are compared and examined to establish whether there is sequence identity within a species and, more importantly, consistent differences of DNA sequence between one putative taxon and another (Lahaye, 2008).

Questions and techniques In order to formulate informed management plans to deal with large-flowered Ludwigia occurring outside gardens in the British Isles, it is necessary to know the number and identity of taxa represented in cultivated and wild populations. A combined molecular, cytological, and morphological approach was used to address these questions. In addition, a nomenclatural assessment was made which it is hoped will allow robust and unambiguous legislation to be framed.

Materials and methods Collection of plant material Fresh plant material was collected from naturalised populations across the UK and from nurseries selling Ludwigia as ornamental plants. A further 15 samples of Ludwigia from California were kindly provided by Brenda Grewell of University of California, Davis. Voucher specimens for UK collections are deposited at WSY. Details are listed in Table 2. Samples from Ludwigia section Oligospermum (Ludwigia grandiflora subsp. grandiflora, Ludwigia grandiflora subsp. hexapetala, Ludwigia peploides subsp. peploides, and Ludwigia peploides subsp. montevidensis), the ingroup samples, were analysed against L. palustris and L. glandulosa Walter, both outside this section and therefore treated as the outgroup.

Ludwigia Ludwigia Ludwigia Ludwigia

Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia

Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia Ludwigia

Anglo Breamore Bromfield Brook

Copythorne Gla Gra 1 Gra 2 Gra 3 Hex 1 Hex 2 Hex 3 Hex 4 Knowle Lydney

Mimmack Mon 1 Mon 2 Mon 3 Mon 4 Pal Pep 1 Pep 2 Pep 3 Pep 4 Scarborough Shaftesbury Stapley West Bay Wildwoods Wisley

Sample Name

subsp. subsp. subsp. subsp.

grandiflora grandiflora grandiflora hexapetala hexapetala hexapetala hexapetala hexapetala hexapetala

hexapetala

hexapetala hexapetala hexapetala hexapetala

grandiflora subsp. hexapetala peploides subsp. montevidensis peploides subsp. montevidensis peploides subsp. montevidensis peploides subsp. montevidensis palustris peploides subsp. peploides peploides subsp. peploides peploides subsp. peploides peploides subsp. peploides grandiflora subsp. hexapetala grandiflora subsp. hexapetala grandiflora subsp. hexapetala grandiflora subsp. hexapetala grandiflora subsp. hexapetala grandiflora subsp. hexapetala

grandiflora subsp. glandulosa grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp. grandiflora subsp.

grandiflora grandiflora grandiflora grandiflora

Taxon

SH BG BG BG BG SH BG BG BG BG SH SH SH SH SH SH

SH SH BG BG BG BG BG BG BG SH SH

SH SH SH SH

Collector

Anglo Aquarium Plant Co., Enfield, Middlesex Breamore, New Forest, Hampshire, v.c. 10, SU155183 Paul Bromfield Aquatics, Hitchin, Hertfordshire Brook, Isle of Wight, Hampshire, v.c. 10 SZ3950083864 Copythorne, New Forest, Hampshire, v.c. 11, SU3070514382 Maidenhead Aquatics, Twyford, Berkshire San Diego River, California San Diego River, California San Diego River, California Russian River at Wohler Pool, Sonoma County, California Russian River at Wohler Pool, Sonoma County, California Russian River at Wohler Pool, Sonoma County, California Russian River at Wohler Pool, Sonoma County, California Sweet Knowle Aquatics, Stratford upon Avon, Warwickshire Lydney Harbour, Gloucestershire, v.c. 34 SO6361901868 Mimmack Aquatics, Stock, Essex Lake Hennessey, Napa County, California Lake Hennessey, Napa County, California Lake Hennessey, Napa County, California Lake Hennessey, Napa County, California Maidenhead Aquatics, Twyford, Berkshire Kesterson National Wildlife Refuge, San Joaquin Valley, California Kesterson National Wildlife Refuge, San Joaquin Valley, California Kesterson National Wildlife Refuge, San Joaquin Valley, California Kesterson National Wildlife Refuge, San Joaquin Valley, California Scarborough, Yorkshire, v.c. 62, TA0339090001 Shaftesbury, Dorset, v.c. 9, ST846217 Stapley Water Gardens, Nantwich, Cheshire West Bay, Dorset, v.c. 9, SY466905 Wildwoods Water Garden Centre, Enfield, Middlesex RHS Garden Wisley, Woking, Surrey

Source

80 80 80 80 80 80

80

80 80

80

80 80 80

Chromosome Number


KC996789

KC996788

KC996814

KC996815

KC996777 KC996778 KC996779 KC996780 KC996781 KC996782 KC996783 KC996784 KC996785 KC996786 KC996787


KC996767 KC996768 KC996769 KC996770 KC996771 KC996772 KC996773 KC996774 KC996775 KC996776

KC996765 KC996766

KC996791 KC996792 KC996793 KC996794 KC996795 KC996796 KC996797 KC996798 KC996799 KC996800 KC996801 KC996802

KC996764

rbcL

KC996790

matK

KC996867

KC996866


KC996845 KC996846 KC996847 KC996848 KC996849 KC996850 KC996851 KC996852 KC996853 KC996854

KC996843 KC996844

KC996842

phyC

GenBank Accession No.

Table 2 Details of samples and collectors (BG5Brenda Grewell, SH5Stephen Heaton), chromosome counts of material from the UK and GenBank accession numbers


KC996841

KC996840


KC996819 KC996820 KC996821 KV996822 KC996823 KC996824 KC996825 KC996826 KC996827 KC996828

KC996817 KC996818

KC996816

trnH-psbA

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Cytology Root collection: actively growing roots were collected in the morning and pre-treated overnight at 4uC in either saturated L bromo napthalene or 0.002 M 8 Hydroxyquinoline, fixed and stored in freshly made up 3 : 1 ethanol/glacial acetic acid. Staining; fixative removed and replaced by 5 N HCl for 10 minutes at room temperature, the HCl was then replaced with 70% alcohol. Root tips were then dissected, tapped, and squashed in a small drop of 2% Sigma-certified orcein in 45% acetic acid. Preparations were examined using a Zeiss 663 oil objective and images captured on a GXDC-3 digital microscope camera.

DNA analysis Published by Maney Publishing (c) Botanical Society of the British Isles

Total genomic DNA was extracted from fresh and silica-dried plant material using a modified CTAB protocol (Bakker et al., 1999). PCR amplifications were performed in 50 ml reactions containing 16 Bioline Biomix (final concentrations), 0.35 mM of each primer, 0.2 mg/ml bovine serum albumin, and 2–3 ml of DNA template. 4% v/v dimethyl sulphoxide was added to all reactions amplifying matK and to reactions of trnH–psbA, where the products were weak/unsatisfactory. PCR amplification was performed with primers and PCR programmes listed in Table 3. Direct sequencing of PCR products was performed by Macrogen Inc. (Seoul, Korea). Raw sequences were assembled and edited using Seqman II (DNAStar, Inc., Madison, WI, USA). Sequences were aligned with ClustalW implemented in eBioX 1.6 (Martıńez Barrio, et al., 2009) and manually adjusted using Mesquite v. 2.72 (Maddison & Maddison, 2011).The beginning and end of alignments as well as one character at position 60 of phyC, where base callings were ambiguous, were excluded from the analyses. The alignment of trnH–psbA contained a 68 bp long complex insertion/deletion event which was excluded from the analysis. The phyC sequence of the Wisley sample was missing 42 bp at the 39 end. The internal transcribed spacer (ITS) data quality was too low to be of use in any further analysis. Therefore, the analysis is of the combined cpDNA data from three separate regions only.

Distance analysis Distance analysis using the neighbour-joining (NJ) method (Saitou & Nei, 1987) in PAUP* 4.0b 10

(Swofford, 2003) was used to assess the clustering of sequences separately for each dataset. NJ analyses were performed with the distance set to Kimura-2parameter and with the best-fit model of evolution as identified by Modeltest v. 3.7 (Posada & Crandall, 1998). Node support was assessed by running 1000 bootstrap (Felsenstein, 1985) replicates of NJ with the same distance options as used for the original search.

Bayesian inference The combined dataset was analysed using Bayesian Inference (BI) in MrBayes v. 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). BI was performed according to the best-fit model of evolution for each individual region, identified by MrModeltest2 v. 2.3 (Nylander, 2004). The analysis was conducted with two separate runs (each of four chains) until the average standard deviation of split frequencies reached 0.01, sampling every 1000 generations. Burn-in was identified by plotting the posterior probabilities and tree lengths against the generations. Trees from the first 10 000 generations were discarded.

Results Cytology Samples from the UK were received under the names L. grandiflora, L. hexapetala, L. peploides, and L. uruguayensis but all proved to be decaploid with a chromosome count of 2n580, as shown in Table 2.

DNA analysis The properties of the aligned sequences are listed in Table 4. Of the four sequenced regions trnH–psbA was the most variable. This region performed the best (22%) in distinguishing the ingroup. Maturase K was close second with 20%, but the variable characters within the ingroup were exclusive to the L. peploides samples. RbcL had only 1% of the total number of characters within the ingroup in 1374 bp. Amplifying the first 550– 600 bp of rbcL, rbcL-a (Kress et al., 2007), recommended for DNA barcoding, would have resulted in only nine variable characters in total and only one in the ingroup. Phytochrome C performed worst having variable characters only in the outgroup. The excluded ambiguous character, at position 60, could have given one character to the ingroup if resolved. The consensus tree from the Bayesian analysis of the combined dataset is shown in Fig. 1. The trees

Table 3 List of DNA regions, PCR primers, and programmes used

Region matK1 phyC 2 rbcL3 trnH–psbA4 ITS5

Genome

Primers (forward then reverse)

Chloroplast Chloroplast Chloroplast Chloroplast Nuclear

390F & 1326R PHYC-F & PHYC-R 1F & 1460R psbAF & trnHR 17SE & 26SE

Initial denaturation Denaturation Annealing Extension Final extension No. of temp./time temp./time temp./time temp./time temp./time cycles 94uC/120 94uC/120 94uC/120 94uC/120 94uC/120

1

s s s s s

94uC/60 94uC/40 94uC/60 94uC/30 94uC/60

s s s s s

48uC/30 48uC/30 51uC/30 53uC/60 48uC/30

s s s s s

72uC/60 s 72uC/60 s 72uC/120 s 72uC/90 s 72uC/60 s

72uC/7 72uC/7 72uC/7 72uC/7 72uC/7

min min min min min

26 30 30 30 26

Cueńoud et al. (2002); 2Samuel et al. (2005); 3Fay et al. (1997) & Olmstead et al. (1992); 4Sang et al. (1997); 5Sun et al. (1994).

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(not shown) resulting from the separate trnH–psbA analyses had the same topology as the combined analyses. The trees from the other regions (not shown) were inferior in identifying the UK material due to the lack of variation, i.e. were poorly resolved. The ingroup was distinguishable from the outgroup (L. glandulosa, L. palustris) in all regions, as 87% of all variable sites changed between the ingroup and outgroup. There was a posterior probability of 1.00 in support of monophyly of the ingroup. L. peploides was clearly distinguished from all other samples in matK and trnH–psbA by single nucleotide polymorphisms. None of the regions distinguished the two different subspecies sampled for L. peploides. The posterior probability of the clade including both subspecies of L. peploides was 1.00. Material labelled as L. hexapetala and L. grandiflora had identical sequences in all regions. The UK material was completely identical to L. hexapetala and L. grandiflora from California in matK, rbcL, and phyC. TrnH–psbA made one clade of Bromfield, Brook, Mimmack, and Copythorne, separated from the rest by a single unique character state. Scarborough appeared as sister to the L. peploides clade, having no unique single nucleotide polymorphism by itself, but sharing three of the variable characters of that clade. The rest of the UK samples (Anglo, Knowle, Stapley, and Wisley) formed an unresolved clade with the material labelled as L. hexapetala and L. grandiflora. The uncoded indel of trnH–psbA followed the same pattern as the DNA sequence substitutions. The ITS was not analysed due to allelic variation, which resulted in ambiguous base callings.

Discussion The number and identity of the species of largeflowered Ludwigia at large in British waterways is revealed using chromosome data. Only one taxon in L. section Oligospermum has a recorded chromosome count of 2n580, that is, L. grandiflora subsp. hexapetala. The results presented here indicate that only one species of Ludwigia section Oligospermum is presently available in the British horticultural trade and occurring in British waterways. This matches the plant recognised by Zardini et al. (1991b) and Okada et al. (2009) as L. hexapetala and by Nesom & Kartesz (2000) and Dandelot et al. (2005) as L. grandiflora subsp. hexapetala. A congruence of morphology and

Figure 1 Consensus tree from Bayesian analysis of the combined dataset. Branches coloured according to chromosome numbers: 2n516 (red), 2n548 (blue), 2n580 (green).

chromosome counts differentiate L. grandiflora subsp. hexapetala and L. peploides. Though significant behavioural differences may not always be expressed morphologically, the value of maintaining species that can only be distinguished with confidence by ploidy level is questionable. We

Table 4 Details of the analysed sequences Length matK phyC rbcL trnH–psbA

817 582–624* 1374 492–567

Number of variable sites Percentage (%) Number of variable sites within ingroup Percentage (%) 15 33 20 46

1.8 5.3 1.5 7.8

3 0 2 10

20 0 1 22

*Length variation due to shorter Wisley sequence.


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recommend the use of the name Ludwigia grandiflora subsp. hexapetala for the plant naturalised in the British Isles to reflect the clear distinction from L. grandiflora subsp. grandiflora in chromosome number though clear morphological separation appears lacking (Nesom & Kartesz, 2000). These findings have legislative relevance. The amended Schedule 9 includes three species names in Ludwigia — L. grandiflora, L. peploides, and L. uruguayensis — all of which, along with their earlier combinations in Jussiaea, are widely used in the British horticultural trade. However, it is clear that at present only one taxon has escaped into the wider environment in the UK, for which we here establish the correct name, although other species, such as L. peploides, have the potential to become invasive if not monitored. It is hoped that these results will feed into the literature produced by those concerned with monitoring and control of invasive non-native species and ultimately inform any future amendments to Schedule 9. As it stands, if L. hexapetala is recognised as a species it is not included in Schedule 9, though our view that it is a subspecies of L. grandiflora does provide coverage under this legislation. The DNA sequence data indicate that at least two genotypes of Ludwigia grandiflora subsp. hexapetala are represented in the British Isles. Plants grown for use in the present study set copious fruit but, as the DNA analysis identified more than one clone among these plants, this is consistent with the assertion of Dandelot et al. (2005) that Ludwigia grandiflora subsp. hexapetala is self-incompatible. However, isolated clones do appear to have set fruit in Britain as Clement (2001) includes a detailed illustration of a fruiting sample collected from the golf course at Barton on Sea. Further, a specimen in WSY, taken in 1999 from the population growing at RHS Garden Wisley, which has developed from a single plant, shows mature fruit. It is known that self-incompatibility systems are often imperfect and sometimes allow limited seed set (Lovett-Doust & Lovett-Doust, 1988). In common with a number of invasive plants, nonnative Ludwigia was present in the UK for a long time before being found in natural or unmanaged habitats. There has been some discussion of this phenomenon, widely referred to as a ‘lag phase’ (Child & Wade, 2000; Sakai et al., 2001). In the case of Ludwigia, it is not clear whether the recent spate of occurrences represents the take-off phase following a long period of persistence at low levels, or whether a slight change in climate has enabled it to be more successful in Britain. Alternatively, it might reflect a greater tendency for people clearing out domestic ponds to deposit excess material in unmanaged waterways or simply a rise in the popularity of Ludwigia as a garden plant.

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The distribution of Ludwigia grandiflora subsp. hexapetala in the wild in the British Isles suggests that there have been multiple introduction events, probably from careless disposal of excess garden material, though animal dispersal over relatively short distances might explain the proximity of some populations. The majority of records are from coastal or urban areas, perhaps indicating that establishment is favoured by warm winter temperatures. Cultivated plants of Ludwigia grandiflora subsp. hexapetala recovered quickly and fully from the cold winters of 2009–2010 and 2010–2011 and naturalised populations must be considered a severe threat to aquatic ecosystems in Britain.

Conclusions This study has demonstrated that only one taxon of non-native Ludwigia, L. grandiflora subsp. hexapetala, is currently occurring in the UK. We recommend that guidance on identifying non-native Ludwigia be revised in the light of this finding. This study emphasises the critical importance of careful observation and research to establish the correct identity of non-native plants before decisions are taken concerning their inclusion in regulatory measures. If the taxonomy of Stace (2011) is followed, and the invasive water primrose in Britain is called Ludwigia hexapetala, then it is not covered by Schedule 9 of the Wildlife and Countryside Act (1981) as amended in 2010. However, if the invasive plant is referred to as L. grandiflora subsp. hexapetala, as this study recommends, then its infraspecific status within L. grandiflora means that it is covered by that legislation. We consider this situation highly unsatisfactory and strongly recommend, to improve clarity in the legislation, that the name Ludwigia hexapetala be added to Schedule 9, though it is important to understand that plants correctly attributable to this taxon may also be retailed and grown under other names. Further, it should be noted that though L. peploides is included in Schedule 9, our findings suggest that it is neither in cultivation nor present in the wider environment in the UK.

Acknowledgements We gratefully acknowledge the kind assistance of Brenda Grewell, Exotic and Invasive Weeds Research Ecologist with the United States Department of Agriculture, for providing verified material for the DNA analysis; Johan van Valkenburg of the Plant Protection Service (NL) for providing material of L. peploides and passing on his observations of characters to distinguish it from L. grandiflora subsp. hexapetala; Trevor Renals, Senior Technical Advisor for invasive species at the UK Environment Agency for providing data concerning occurrences of Ludwigia in the UK; and Steven Heaton and Joanna Heisse, Conservation

Armitage et al.

Officer, Thames South East Area for collecting material from wild populations in the UK.


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