Artemisia (Asteraceae) in Western Europe

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BioInvasions Records (2020) Volume 9, Issue 4: 685–701

CORRECTED PROOF

Research Article Morphological, genome-size and molecular evidence for the presence of another invasive East Asian Artemisia (Asteraceae) in Western Europe

Filip Verloove1,*, Steven B. Janssens1, Remko Andeweg2, Ben J.M. Zonneveld3 and Iris Van der Beeten1 1Meise Botanic Garden, Nieuwelaan 38, BE-1860, Meise, Belgium 2Bureau Stadsnatuur Rotterdam, Westzeedijk 345 (Museumpark), 3015 AA Rotterdam, the Netherlands 3Naturalis Biodiversity Center, Postbus 9517, 2300 RA Leiden, the Netherlands Author e-mails: [email protected] (FV), [email protected] (SBJ), [email protected] (RA), [email protected] (BJMZ), [email protected] (IVDB) *Corresponding author

Citation: Verloove F, Janssens SB, Andeweg R, Zonneveld BJM, Van der Abstract Beeten I (2020) Morphological, genomesize and molecular evidence for the Based on morphological characteristics, genome size and molecular phylogenetics presence of another invasive East Asian the identity of introduced taxa of Artemisia section Artemisia in Belgium and the Artemisia (Asteraceae) in Western Europe. Netherlands was critically re-assessed. It was shown that several populations of alleged BioInvasions Records 9(4): 685–701, A. verlotiorum belong to A. princeps instead. This species is native to Eastern Asia https://doi.org/10.3391/bir.2020.9.4.03 (China, Japan and Korea) and has not been reported before from Europe. Since the Received: 6 April 2020 time of its arrival in Belgium and the Netherlands, more than two decades ago, Accepted: 15 July 2020 A. princeps was able to locally grow as massive stands in mainly non-natural Published: 11 November 2020 habitats. Morphologically, A. princeps is in many respects intermediate between the Handling editor: Margarita Arianoutsou native species A. vulgaris and the Chinese species A. verlotiorum. Morphology, Thematic editor: Stelios Katsanevakis genome size and phylogenetic analyses of these three species and A. ×wurzellii— which is a hybrid of A. vulgaris × verlotiorum parentage that spontaneously arose Copyright: © Verloove et al. in the British Isles—are thoroughly discussed. Contrary to A. verlotiorum, A. princeps This is an open access article distributed under terms of the Creative Commons Attribution License produces viable seed in Western Europe. Hence, it is able to reproduce both clonally (Attribution 4.0 International - CC BY 4.0). and sexually and potentially is a much bigger threat to native biodiversity than OPEN ACCESS. A. verlotiorum. Key words: DNA weight, invasive species, molecular analysis, morphology, mugwort

Introduction Human-mediated dispersal of plants has led to unprecedented changes in global biodiversity. Although only a small proportion of the non-indigenous plants are considered invasive species, they have a disproportionate economic and ecological impact (Vilà et al. 2011; Senator and Rozenberg 2017). Invasions by non-native plants are associated with biological homogenization (McKinney 2004), loss of cropland, pasture and forest (Duncan et al. 2004) and huge economic costs resulting from chemical and mechanical control measures (Hoffmann and Broadhurst 2016). Besides, introduced plants are usually not impacted by natural enemies that are often present in their native distribution range (Blumenthal 2006). It is generally admitted that taxonomic resources are essential for the effective management of invasive plants and incorrect identifications can

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 685 Another invasive Artemisia in Europe

impede ecological studies (Pyšek et al. 2013). Advances in molecular methods provide great opportunities to study taxonomically complex genera or species complexes. They are essential in case traditional identification tools, based on macro- or micromorphological characteristics, do not allow accurate species identification. In some cases, invasive species may resemble an indigenous species or a non-invasive alien species and therefore pass unnoticed, as so-called “invaders in disguise” (Verloove 2010). Artemisia L. section Artemisia (Anthemideae – Asteraceae) is a good example of a taxonomically challenging complex in which several species have shown to be invasive. The genus is mainly distributed in the Northern hemisphere with only a few species in Africa and South America. Central Asia is the main centre of diversification, secondary ones being the Mediterranean region and Northwest America (Sanz et al. 2011). Only five species are native or archaeophytic to Belgium and the Netherlands: A. absinthium L., A. alba Turra, A. campestris L., A. maritima L. and A. vulgaris L. (Lambinon and Verloove 2012; Duistermaat 2020) with only the latter being a common and widespread species. The ecological and economic importance of Artemisia is high. Numerous species are cultivated as medicinal plants (anthelmintics), stimulants and culinary herbs or are used as ornamentals (e.g. Wright 2002; Mabberley 2008). Conversely, several species are known as reputed environmental or agricultural weeds. Holm et al. (1979) list more than 20 species that are classified as “world weeds”. In certain areas of Southern Europe, A. verlotiorum Lamotte is considered as an invasive species (e.g. Sanz et al. 2004; Boršić et al. 2008; Celesti-Grapow et al. 2010; Gassó et al. 2012; Bouvet 2013; etc.), while A. vulgaris is an aggressive invader in North and South America (e.g. Barney and DiTommaso 2002), although at least part of these populations certainly refers to morphologically similar introduced species (Mosyakin et al. 2019; Mosyakin et al. in prep.). Furthermore, Artemisia is a taxonomically complex genus with uncertain generic boundaries. A molecular phylogeny for the subtribe Artemisiinae using the internal transcribed spacers (ITS) of nuclear ribosomal DNA analyzed with parsimony, likelihood, and Bayesian criteria supported a broad concept of Artemisia s.l. that includes segregates like Neopallasia Poljakov, Crossostephium Less., Filifolium Kitam., Seriphidium (Besser ex Less.) Fourr. and Sphaeromeria Nutt. (Watson et al. 2002). However, some important contemporary accounts still accept many of these smaller genera (e.g. Ling et al. 2011). Therefore, additional revision studies are needed to correctly delineate the genus Artemisia. Likewise, species delimitation is also controversial within Artemisia with species numbers ranging from 522 (Oberprieler et al. 2007) to ca. 400 (Mabberley 2008) or even less (e.g. Bremer and Humphries 1993; Shultz 2006). This numeric uncertainty is especially present within section Artemisia to which A. vulgaris and its allies belong. Several species from this section are poorly understood and often overlooked. For instance, A. verlotiorum—a close

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 686 Another invasive Artemisia in Europe

relative of A. vulgaris from Eastern Asia—was neglected for quite a long time in Belgium and the Netherlands. Although first recorded in Belgium in 1937 already (Matagne 1938) it long remained unclear whether or not this species had truly been observed and, if so, what status should be assigned to it, where the species exactly occurred and—most importantly— how it could be best distinguished from the native A. vulgaris. Once these inquiries were addressed and its presence finally confirmed (Verloove 2003; Andeweg 2007) it became clear that some populations of Artemisia in Belgium and the Netherlands were yet different and could not be unequivocally attributed to either A. verlotiorum or A. vulgaris. Recently, a first assessment on the identity of these specimens, based on classic morphological methods, was carried out by Verloove and Andeweg (2020). It turned out that they belong to A. princeps Pamp., another East Asian weed species. In the present paper, we used additional methods such as molecular phylogenetics, genome size measurements and pollen staining to verify our earlier results. A. princeps is well-established in Belgium and the Netherlands and locally grows in massive, monospecific stands, although to date always in anthropogenic habitats. A map showing its actual distribution in the study area is presented in Figure 1 (see also Supplementary material Table S4).

Materials and methods Molecular protocols and sequence analyses Plant material Several dozen of accessions of Artemisia from various European and extra- European countries were analyzed (Table S1). Taxa relevant for this study included, among others, A. princeps, A. verlotiorum, A. vulgaris, A. ×wurzellii as well as putative hybrids of these species that spontaneously arose, either in our collections or in the wild. Voucher material was deposited in the herbaria of Meise Botanic Garden (Belgium, BR), Naturalis in Leiden (the Netherlands, L) and the Natural History Museum in Rotterdam (the Netherlands) (herbarium acronyms follow Thiers 2019+).

Protocols and analyses Total genomic DNA was extracted following a modified CTAB protocol (Tel-Zur et al. 1999). An initial step in which secondary metabolites were removed was carried out by washing pulverized leaf material with extraction buffer (100 mM TrisHCl pH 8, 5 mM EDTA pH 8, 0.35 M sorbitol). Additionally, 575 μl CTAB lysis buffer (incl. 3% PVP-40) was added after which the samples were incubated for 1.5 hours at a constant temperature of 60 °C. The extraction was performed twice with chloroform- isoamylalcohol (24/1 v/v). Subsequently, isopropanol precipitation was carried out and left overnight at −32 °C. During the next step, the samples were

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 687 Another invasive Artemisia in Europe

Figure 1. Distribution map of Artemisia princeps in Belgium and the Netherlands.

centrifuged after which the pellet was washed twice with 70% ethanol. Finally, the DNA pellet obtained was air-dried, and dissolved in 100 μl TE buffer (10 mM TrisHCl pH 8, 1 mM EDTA pH 8). PCR reactions of trnL-F and ITS were carried out using standard PCR conditions containing 1 μl DNA, 2 × 1 μl oligonucleotide primer (100 ng/μl), 2.5 μl of 10 mM dNTPs, 2.5 μl Taq Buffer, 0.2 μl KAPA Taq DNA polymerase and 16.8 μl MilliQ water. Amplification reactions were run on a Gene Amp PCR system 9700 (Applied Biosystems) and started with a 3 minute heating at 95 °C followed by 30 cycles consisting of a denaturation phase of 30 s at 95 °C, an annealing phase of 60 s, and an extension phase of 60 s at 72 °C. ITS primers were derived from White et al. (1990), whereas chloroplast

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 688 Another invasive Artemisia in Europe

trnL-F primers were obtained from Taberlet et al. (1991). After the purification of the PCR products using an ExoSap protocol (Wallis and Morrel 2011), samples were sent for sequencing to Macrogen, Inc. (Seoul, South Korea). Newly obtained sequences were deposited in the online DNA sequence repository GenBank and assembled with Geneious v11.2 (Biomatters, New Zealand). Automatic alignment was performed with MAFFT (Katoh et al. 2002) using an E-INS-i algorithm, a 100PAM/k = 2 scoring matrix, a gap open penalty of 1.3 and an offset value of 0.123, after which the dataset was manually optimized in Geneious v11.2. Putative incongruence between the nuclear (nr) ITS and plastid (pl) trnL-F dataset was assessed using the incongruence length difference test (ILD; Farris et al. 1995) as implemented in PAUP* v.4.0b10 (Swofford 2002). Due to some sensitivity issues with the ILD test (Barker and Lutzoni 2002) we compared the results of this test according to the overall resolution and support obtained for the different topologies (chloroplast vs. nuclear). Therefore, a putative conflict between the cp and nr data matrices was visually searched for (hard vs. soft incongruence; Johnson and Soltis 1998). The best-fit nucleotide substitution (under AIC criterion) model for the plastid and nuclear dataset was estimated with jModelTest 2.1.4. (Posada 2008). F81+I+G and GTR+I+G were found as the best models for trnL-F and ITS respectively. Maximum Likelihood analyses were carried out using the RaxML search algorithm (Stamatakis et al. 2005) under the GTRGAMMA+I approximation of rate heterogeneity for each gene (Stamatakis 2006). Five hundred bootstrap trees were inferred using the RaxML Rapid bootstrap algorithm (ML-BS) to provide support values for the best-scoring ML tree.

Genome size measurement Plant material Genome size was assessed for numerous accessions of Artemisia from various European and extra-European countries (Table S2). Taxa relevant for this study included, among others, A. princeps, A. verlotiorum, A. vulgaris, A. ×wurzellii as well as putative hybrids of these species that spontaneously arose, either in our collections or in the wild.

Flow-cytometric measurement of nuclear DNA content For the isolation of nuclei, c. 0.5 cm² of a young leaf was chopped together with a small slice (± 1 × 15 mm) of Agave americana “Aureomarginata” as an internal standard (Galbraith et al. 1983). Chopping of leaflets was done at room temperature with a new razor

blade in a Petri dish in 0.25 ml nuclei isolation buffer [per liter: MgCl2 ×

6H2O 9.15 g, tri-sodium citrate 8.8 g, MOPS 4.15 g, Triton X-100 1 ml, polyvinylpyrrolidone 10 000 25 g, dithiothreitol 1.55 g, 0.01% RNAse, pH 7

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(c. 1.12 g KOH)] (changed after Bharathan et al. 1994). After adding 1.50 ml propidium iodide solution (50 mg PI/l) in isolation buffer, the nuclei suspension was filtered through a 20 μm nylon filter. Fluorescence of the nuclei was measured 30 and 60 minutes after addition of PI using a BDA Accuri C6 flow cytometer. The more DNA present in a nucleus, the higher is the intensity of the fluorescence. The 2C DNA content of the sample was calculated as the sample peak mean, divided by the Agave peak mean, and multiplied by the amount of DNA of the Agave standard. For each clone, two to four different runs (determinations with around 3000–5000 nuclei) were measured. Nuclear DNA content (2C-value) of 15.9 picograms (pg) per nucleus for Agave americana “Aureomarginata” was determined with human leukocytes (2C = 7 pg; Tiersch et al. 1989) as the standard. In the case of Artemisia verlotiorum also Agave attenuata with 7.9 pg was used as a standard. Based on published male human genome size of 6.294 × 109 base pairs, the nucleus was calculated to contain 6.436 pg (Doležel et al. 2003). However, this is based on a human sequence for which the size of the very large repeat sequences could not be accurately determined, so the genome size could be closer to 7 pg than now envisioned. The Animal Genome Size Database (http://www.genomesize.com, release 2.0) also gives a haploid size (C-value) of 3.5 pg.

Morphological data Observations were made from the field in Belgium and the Netherlands, mostly between 2011 and 2018. In addition, individuals of Artemisia princeps and similar-looking species were grown ex situ to better understand the species’ morphological peculiarities. For their identification, numerous relevant floras (mainly covering the former USSR, Japan and China), as well as other literature references, were consulted (e.g. Mosyakin 1990; Sȋrbu and Oprea 2011). Also, several experts of the genus Artemisia were queried to assure species identification.

Pollen and seed viability Chemical pollen staining techniques were performed (following Peterson et al. 2010) for Western European populations of A. princeps and related taxa. Taxa included were, among others, A. princeps, A. verlotiorum, A. ×wurzellii and another putative hybrid (A. princeps × vulgaris) (see Table S3). Also, the seed viability of an A. princeps accession from Rotterdam (the Netherlands) was tested under controlled laboratory conditions.

Results and discussion Molecular characterization The chloroplast trnL-F dataset consists of 68 specimens (incl. two outgroup taxa) and 753 analyzed characters (8 variable characters), whereas the nuclear

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 690 Another invasive Artemisia in Europe

Figure 2. trnL-F maximum likelihood phylogram. Values on the branches indicate maximum likelihood bootstrap support values.

ribosomal ITS dataset contains 68 specimens (incl. two outgroup taxa) and 628 analyzed characters (84 variable characters). In general, no incongruency was found between the different datasets, except for the position of the two hybrids (A. mongolica × princeps and A. princeps × vulgaris), which clearly differs in position between the chloroplast and the nuclear topology (Figures 2 and 3). This incongruency was also detected by the partition homogeneity test (P < 0.05). After removing the hybrids both nuclear and plastid dataset were congruent (P > 0.05). As such, the combined plastid- nuclear phylogeny contains all specimens of the separated analyses except for the hybrids (Figure 4). When comparing resolution and support values of both plastid and nuclear trees produced by the ML analyses, it is clear that the plastid topology is very poorly resolved with only a few supported clades. The ingroup of the trnL-F topology forms a well-supported polytomy

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 691 Another invasive Artemisia in Europe

Figure 3. ITS maximum likelihood phylogram. Values on the branches indicate maximum likelihood bootstrap support values.

(ML-BS: 100) containing all specimens of A. princeps, both A. ×wurzellii hybrids, and A. momiyamae [syn.: A. princeps var. momiyamae (Kitam.) H. Hara] and A. ×wurzellii as well as two well-supported clades. Of these clades, one consists of all A. verlotiorum specimens (ML-BS: 100) whereas the other one contains all specimens of A. vulgaris, the A. princeps × vulgaris hybrid, and the two A. mongolica × princeps hybrids (ML-BS: 100). Within the latter clade, the two A. mongolica × princeps hybrids form a well- supported clade (ML-BS: 100). For the nuclear ITS topology, two large clades can be delineated (ML-BS: 84): one clade containing all accessions of A. verlotiorum (ML-BS: 61) and a clade consisting of accessions of A. princeps, A. momiyamae, A. vulgaris, A ×wurzellii and the hybrid taxa of A. princeps

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 692 Another invasive Artemisia in Europe

Figure 4. Maximum likelihood phylogram of the combined dataset. Values on the branches indicate maximum likelihood bootstrap support values.

× vulgaris and A. mongolica × princeps. Within this latter clade, all specimens of A. momiyamae, A. vulgaris, A ×wurzellii and the hybrid species A. princeps × vulgaris and A. mongolica × princeps (M3098) are included (ML-BS: 60). For A. vulgaris the situation is less clear as its infraspecific relationships are

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 693 Another invasive Artemisia in Europe

not monophyletically organized, yet either without or with only low support (except for the relationship between A. vulgaris (M3120) and A. vulgaris (M3116) which is highly supported (ML-BS: 93)). The combined topology looks similar to that of the separated analyses with the majority of the clades being better resolved and supported. Note that the two hybrid taxa A. mongolica × princeps, as well as A. princeps × vulgaris have been removed from the concatenated dataset as both specimens had a distinct position in the chloroplast and nuclear topology. In the combined topology, a large group of solely A. verlotiorum specimens (ML-BS: 93) is sister to the extant taxa in the analysis (ML-BS: 74). The latter clade is split into two lineages (ML-BS: 68) of which one contains all A. vulgaris accessions (ML-BS: 58), whereas the other is characterized by all A. princeps specimens as well as the three A. ×wurzellii accessions (ML-BS: 69).

Genome size measurement Artemisia vulgaris (4 accessions) had a mean genome size that ranged between 2C = 6.45 and 6.66 pg and an inferred chromosome size of 2n = 16. Artemisia verlotiorum (13 accessions) had a mean genome size that ranged between 2C = 13.3 and 14.2 pg and an inferred chromosome size of 2n = 52. A. princeps (10 accessions) had a mean genome size that ranged between 2C = 9.99 and 10.4 pg and an inferred chromosome size of 2n = 34. Artemisia ×wurzellii (3 accessions) had a mean genome size that ranged between 2C = 9.95 and 10.1 pg and an inferred chromosome size of 2n = 34. A spontaneous hybrid of putative A. princeps × vulgaris parentage had a mean genome size of 2C = 8.31 pg, whereas one of A. princeps × mongolica (A. vulgaris var. mongolica) ranged between 2C = 7.94 and 8.36 pg. The genome size of Artemisia vulgaris and A. verlotiorum on the one hand as compared with that of A. princeps and A. ×wurzellii on the other proved to be quite different. A mean (# = 4) genome size of 2C = 6.57 pg for A. vulgaris is in line with data previously published for this species (Garcia et al. 2013). An aberrant accession of A. vulgaris with 2C = 12.15, as reported by Garcia et al. (2004), originated in Tibet and most likely represents a similar-looking but different species. Mean measurements obtained for A. verlotiorum (# = 13) are 2C = 13.78 pg. A. vulgaris and A. verlotiorum have an inferred chromosome number of 2n = 16 and 2n = 52 respectively which corresponds well with previous counts (e.g. Vallès Xirau 1987; James et al. 2000). A. princeps and A. ×wurzellii are intermediate between these two species, both in genome size and in inferred chromosome number. The former has a mean (# = 10) genome size of 2C = 10.11 pg and an inferred chromosome number of 2n = 34. Pellicer et al. (2010) obtained a slightly lower genome size (2C = 9.48 pg). The inferred chromosome number agrees well with previous counts (e.g. Nishikawa 1986). Our measurements for A. ×wurzellii (including an accession from the type locality) are very similar: a mean (# = 3) genome size of 2C = 10.02 pg and an inferred chromosome number of 2n = 34. Garcia et al. (2004) obtained

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 694 Another invasive Artemisia in Europe

an identical chromosome count (see also James et al. 2000) but a markedly lower genome size (2C = 8.6 pg), although plants were probably collected from the same locality. Plants found to be intermediate in morphology and thought to be hybrids indeed had a genome size that was intermediate between that of the alleged parental species. A putative hybrid of A. princeps and A. vulgaris from Rotterdam, the Netherlands, had a mean genome size of 2C = 8.31 pg. Similarly, plants that spontaneously arose in our collection and of alleged A. mongolica (A. vulgaris var. mongolica) × princeps parentage indeed had a similar mean (# = 4) genome size, 2C = 8.16 pg. Thus, invasive populations recently found in Belgium and the Netherlands that are here assigned to Artemisia princeps differ in genome size and chromosome number from native A. vulgaris and introduced A. verlotiorum, the species with which it had been confused up to present. However, based on genome size and chromosome number its separation from A. ×wurzellii, a sterile hybrid of the latter two species, is problematic. Both have a very similar genome size and an identical chromosome number. This is likely a coincidence. In London material A. verlotiorum had 2n = 52, A. vulgaris 2n = 16 and their hybrid (A. ×wurzellii) 2n = 34 as could be expected (James et al. 2000). Alternative chromosome numbers for the parental species are known (2n = 18 for A. vulgaris, 2n = 48, 50 or 54 for A. verlotiorum). Perhaps A. princeps is derived from an alternative cross (e.g. 2n = (18+50)/2 = 2n = 34) which could explain why it is fertile and A. ×wurzellii sterile.

Pollen and seed viability Pollen viability was assessed for ten accessions of Artemisia (Table S3). All “pure” species showed a degree of pollen viability that ranged between 99.44 and 91.7%. The viability of pollen of three accessions of A. princeps ranged between 99.44 and 96.11%. In the hybrid A. ×wurzellii (A. verlotiorum × vulgaris), in contrast, most pollen was aborted (pollen viability ranging between 9.52% and 48.92%). Similarly, pollen of a putative hybrid A. princeps × vulgaris was also mostly aborted (11.23% viable pollen). While molecular and cytological data are not conclusive for the distinction between Artemisia princeps and A. ×wurzellii, both taxa are easily separated based on pollen viability: the former is a fertile species whereas the latter is largely sterile. This was also shown in a seed viability test. Germination tests demonstrated almost 100% seed germination for an Artemisia princeps accession from Rotterdam (the Netherlands) within one week. The sterile hybrid A. ×wurzellii did not produce any seeds.

Morphological data The results of our morphological study are discussed below in the taxonomical part of this paper (see also Verloove and Andeweg 2020 where Artemisia princeps and similar-looking species are copiously illustrated).

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 695 Another invasive Artemisia in Europe

Figure 5. Artemisia princeps in anthropogenic habitats in Belgium. A, B, C (Antwerp port area), D (Zeebrugge port area), all September-October 2019. Photographs by Filip Verloove.

Taxonomy Artemisia princeps Pamp., Nuovo Giorn. Bot. Ital., n.s. 36(4): 444–446 (1930) (Figure 5) Artemisia princeps shares characters with both A. verlotiorum and A. vulgaris and is not easily distinguished from these two species. Especially the identification of herbarium material is challenging. A. princeps differs from A. vulgaris in always having long rhizomes and hence ultimately forms large stands. From A. verlotiorum it differs in usually being much less aromatic (but see below). White glands are always absent from the upper leaf surface of A. princeps whereas the presence of such glands is characteristic for A. verlotiorum (Ling et al. 2011). A complicating factor is, however, that these glands are usually absent from or very sparse and/or early deciduous in A. verlotiorum populations that are naturalized in Europe. Artemisia princeps shares the long rhizomes with A. verlotiorum, but in A. princeps the rhizomes tend to be slightly shorter having the buds more closely spaced. As a result, the root system of A. princeps is more

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 696 Another invasive Artemisia in Europe

compact and massive and the plant grows in even denser stands, leaving no space for the pre-existing vegetation. A. princeps differs from both species in its flowering period. This period starts at the end of August / early September and lasts until the end of October or early November. It flowers later than A. vulgaris and markedly earlier than A. verlotiorum. The inflorescence of A. princeps has usually widely divaricate branches and heads of ca. 1.5–2 mm across and is usually somewhat larger than the inflorescence of A. vulgaris and larger than that of A. verlotiorum. The seed coat is remarkably loose in A. princeps as compared with the tight seed coat of the other two species. Finally, the number of tubular florets usually is lower. Verloove and Andeweg (2020) discuss and copiously illustrated the morphological traits of A. princeps and similar-looking species. Artemisia princeps is a variable species and its taxonomy is not uncontested. Some authors—mainly in Japan—prefer to treat it as a synonym of the very variable A. indica, or consider it as a mere variety of the latter, var. maximowiczii (e.g. Iwatsuki et al. 1995; Shimono et al. 2013). Despite being accommodated in the same series by Ling (1992) and being morphologically similar, A. princeps is not very closely related to A. vulgaris (Tkach et al. 2008; Vallès et al. 2011). Most of the varieties and forms of A. princeps formerly described by Pampanini (1930) are now subsumed under related species such as A. igniaria Maxim., A. indica (s.str.) and A. verbenacea (Kom.) Kitag. (Ling et al. 2011). The populations recently discovered in Belgium and the Netherlands also show some degree of variation, especially concerning the leaf shape, inflorescence shape, as well as to odour. In A. princeps mid stem leaves are pinnatipartite with usually a short apical segment but in some populations, the apical segment is much longer, somehow approaching that of A. verlotiorum. As a rule, inflorescences ultimately have widely divaricate, straight branches in A. princeps. Sometimes, however, emerging inflorescences can have slightly but distinctly arcuate branches, a feature also seen in A. verlotiorum. Based on panicle characteristics Pampanini (1930) divided all species into two major informal groups, Rectae and Flexuosae for species with straight and arcuate inflorescence branches respectively. He overestimated the weight of this feature. Odour also often is accepted as a useful feature, especially to distinguish between A. verlotiorum and A. vulgaris (e.g. Bini Maleci and Bagni Marchi 1983; James et al. 2000). The former usually is strikingly aromatic, its smell resembling that of citrus or florist’s daisy. The other taxa here concerned, in contrast, are almost odourless (in reality, these species have a slightly spicy smell of varying intensity). However, exceptions to this rule occur. One population of A. princeps (from the A12 motorway in Antwerp, Belgium) is more aromatic. Inversely, one population of A. verlotiorum (from the Weststation in Brussels, Belgium) was observed

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 697 Another invasive Artemisia in Europe

to be almost odourless. This can relate to the extent to which glands are present on the upper leaf surface of this species. The variability of A. princeps in Western Europe seems to point at separate introductions and/or a much broader genetic diversity than observed in the morphologically homomorphous A. verlotiorum. Artemisia ×wurzellii is—like A. princeps—more or less intermediate morphologically, although not in every diagnostic character (James et al. 2000). As shown above, both are diploids with a chromosome number of 2n = 34. They also have a similar genome size and form a cluster in a phylogenetic tree based on sequences from nuclear ribosomal ITS and chloroplast trnL-F region. Yet, they are considered to be two distinct entities that are morphologically quite dissimilar, although most of the characters are difficult to quantify precisely. First of all, A. ×wurzellii is a sterile hybrid in which stamens are absent in most flowers. In a few, stamens may be detected but these will contain a very high proportion of abortive pollen. In A. princeps, on the other hand, all flowers have stamens (usually five in number) that contain perfect pollen and viable seed is produced, also in Western Europe. In addition, A. ×wurzellii has a leafier inflorescence with narrow, not divaricately branched and contracted panicles. The most reliable feature for an accurate identification of the British specimens of A. ×wurzellii are the group of pectinate appendages at the junction of the leaf blade and petiole (“petiolar pectinations”): these are broader, shorter, more abruptly acute and more numerous than in either of the parent species. This character separates the hybrid from its parents (James et al. 2000) as well as from A. princeps (Verloove and Andeweg 2020). While this feature characterizes A. ×wurzellii well, the petiolar pectinations in A. princeps are more or less intermediate between those of A. vulgaris and A. verlotiorum.

Conclusion Based on classical gross morphology and subsequently confirmed by modern techniques like genome size measurements and DNA sequencing it was demonstrated that a recently naturalized, rather expansive species of Artemisia in Belgium and the Netherlands, with features more or less intermediate between A. vulgaris and A. verlotiorum, belongs to the East Asian weed A. princeps. This species has been present in the study area for at least two decades but its presence went unnoticed as a result of confusion with the two aforementioned species. At present it is still a rare species but in several localities, it occurs in massive, monospecific stands. Although all populations are at present confined to disturbed man-made habitats, this species is a potential threat to native biodiversity in Western Europe.

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 698 Another invasive Artemisia in Europe

Acknowledgements The authors are much indebted to Koji Yonekura (Tohoku University, Japan) for his help with the identification of Artemisia princeps. Prof. Clive Stace (University of Leicester, British Isles) is thanked for providing useful data about Artemisia ×wurzellii. Quentin Groom (Meise Botanic Garden, Belgium) accompanied the first author to the type locality of A. ×wurzellii in London. Henry Engledow and Sven Bellanger (both Meise Botanic Garden, Belgium) are thanked for preparing the distribution map and the plate with illustrations respectively. The authors wish to thank the following botanists for providing silica gel dried samples for species of Artemisia relevant to this study: J. Amigo (Spain), C. Argenti (Italy), T. Denters (the Netherlands), F. Hoffer-Massard (Switzerland), Q. Groom (Great Britain), M. Hohla (Austria), I. Hoste (Belgium), A. Mesterházy (Hungary), C. Peña (Spain), G. Pflugbeil (Austria), F. Portel (France), A. Sebastián (Spain), J.-M. Tison (France), J. Twibell (Great Britain) and N. Wysmantel (Belgium). Finally, two anonymous reviewers are acknowledged for providing useful comments on an earlier version of this paper.

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Supplementary material The following supplementary material is available for this article: Table S1. Accessions of Artemisia included in the molecular analysis. Table S2. Accessions of Artemisia used for flow-cytometric measurement of nuclear DNA content. Table S3. Accessions of Artemisia for which pollen viability was assessed. Table S4. List of georeferenced localities. This material is available as part of online article from: http://www.reabic.net/journals/bir/2020/Supplements/BIR_2020_Verloove_etal_SupplementaryMaterial.xlsx

Verloove et al. (2020), BioInvasions Records 9(4): 685–701, https://doi.org/10.3391/bir.2020.9.4.03 701