Naturalised Hakea. What species are we actually talking about in Europe?

ABSTRACT

Hakea sericea Schrad. & J.C.Wendl. (Proteaceae) is a shrub or small tree native to south-eastern Australia. The species has been introduced to other countries; it has naturalised and become invasive in France, New Zealand, Portugal, and South Africa. During the review process of the Pest Risk Analysis for the European and Mediterranean Plant Protection Organization (EPPO) on Hakea sericea in 2017, the actual identity of the plant causing major negative biodiversity impact in Portugal was questioned. Flowering material from Portugal and additional collections from South Africa and New Zealand were collected in order to clarify the taxonomical status. To make the survey on the proper identity of the Hakea plants invasive in Portugal more concise, all Hakea species encountered in Portugal, France, South Africa and New Zealand were included. For morphological distinction of the species the length of the pistil and fruit characters were used. For molecular analysis Illumina sequencing in combination with de novo assembly and extraction of loci was used. Results show that invasive populations of Hakea in Portugal belong to H. decurrens R.Br a species that, although very similar and sometimes considered synonymous with H. sericea, can be distinguished both morphologically and on the basis of cpDNA. Both species occur in France and in New Zealand, while only H. sericea occurs in South Africa. Both species are invasive and seem to have similar impacts on biodiversity in invaded ranges. The morphologically similar species H. gibbosa (J.White) Cav. occurs in both New Zealand and South Africa. By adopting the H. sericea sensu lato concept for inclusion of the species on the List of Union Concern pursuant of Regulation (EU) No 1143/2014, a potential legislative controversy can be prevented.

KEYWORDS:

• Barcoding
• Regulation (EU) No 1143/2014
• invasive species
• Bushy Needlewood
• Silky Hakea

Introduction

The genus Hakea Schrad. (Proteaceae) comprises 154 species and is native to Australia. Some species have been introduced into other parts of the world as ornamental or for hedging and became naturalized in New Zealand, Southern Africa, Portugal, Spain and France. (https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:32921–1). Hakea sericea Schrad. & J.C.Wendl. has become invasive in New Zealand, South Africa, Portugal, Spain and France (EPPO 2017), H. gibbosa (J.White) Cav. has become invasive in New Zealand and South Africa (Henderson 2001; Popay et al. 2010), H. salicifolia (Vent.) B.L.Burtt has become naturalized and is considered potentially invasive in New Zealand, South Africa, Portugal, Spain and France (Henderson 2001; Popay et al. 2010; Terrin et al. 2022), and H. drupacea (C.F.Gaertn.) Roem. & Schult. has become naturalized in New Zealand and South Africa.

Hakea sericea is a shrub or small tree native to south-eastern Australia (Barker 1996). The species has been introduced into other countries; it has naturalised and become invasive in France, New Zealand, Portugal, and South Africa. There is also a small population in Spain which is likely to be established given its similar climatic conditions to Portugal. In South Africa, it has become particularly well known for its spread and impacts in the Mediterranean-type climate fynbos region, especially of the mountain catchments in the south western and southern regions of the Western Cape Province (Kluge and Neser 1991). In New Zealand H. sericea is similarly invasive on poor soils, gumland, scrub, open hillside and dunes, being included on the list of environmental weeds (Howell 2008). Although the species has been known from Europe for some time (e.g. Ball et al. 1964), concern over the invasiveness of H. sericea in its non-native ranges in France, Portugal, and adjacent parts of Spain, has been increasing (Brunel et al. 2010, 2010; Fried 2010; Martins et al. 2016; Morais et al. 2017; Terrin et al. 2022). In Portugal, H. sericea has been listed as invasive since 1999 (Decree-Law nº 565/99). When the legislation was revised, in 2019, it continued to be listed (Decree-Law nº 92/2019). The species is present in disturbed areas but increasingly found in semi-natural habitats and poses a threat to the EU Habitat Directive Annex I habitat types Thermo-Mediterranean and pre-desert scrub (5330) and European dry heaths (4030).

Management of Hakea can be mechanical by cutting of the trees and direct removal of the biomass or leaving the biomass on site, triggering seed release and germination, followed by a prescribed burning following seedling establishment. As an alternative, less impacting, method a biocontrol program was started in South Africa, introducing natural enemies from Australia that prey on the seed, thereby reducing the propagule pressure.

Hakea sericea was added to the EPPO Alert List in 2007 and transferred to the List of Invasive Alien Plants in 2012 following a prioritization assessment (Brunel et al. 2010). Brunel et al. (2010) also concluded that this species was a high priority for a full Pest Risk Analysis (PRA). In 2016, the species was prioritized for PRA within the EU funded LIFE project “Mitigating the threat of invasive alien plants to the EU through pest risk analysis to support the Regulation (EU) No 1143/2014’ (see www.iap-risk.eu). Hakea sericea was one of the 16 species identified as having a high priority for PRA (Tanner et al. 2017), and a PRA was conducted within the remit of the project.

The EPPO PRA (EPPO 2017) for H. sericea highlights the risk for the EPPO region, more precisely the Mediterranean, Atlantic, Black Sea and Macaronesian biogeographical regions, and resulted in listing the species in the EPPO A2 List of species recommended for regulation in the EPPO region. Additionally, the species was included in the list of invasive alien species of Union concern of the Regulation (EU) No1143/2014 in 2022.

During the review process of the EPPO PRA on Hakea sericea (EPPO 2017), the actual identity of the plant causing major negative biodiversity impact in Portugal became uncertain, since although this species is listed in the Flora Europaea and Flora Ibérica, another reference (Barker 1996) refers to H. decurrens instead of H. sericea. This lack of clarity resulted in an additional note addressing this issue in the final PRA version. Material collected by the first author in Portugal and New Zealand in 2017 and 2018 was fruiting, therefore the diagnostic floral characters could not be analysed to ascertain the identity. Hence, to solve this, new flowering material from Portugal and additional collections from South Africa and New Zealand were needed.

It is also noted here that the species H. decurrens R.Br., peripatric with H. sericea in its native range (Barker 1996), can be easily confused with H. sericea (Q-bank 2023). According to the key of Barker (1996), the length of the pistil (<9 mm = H. sericea; > 9 mm = H. decurrens) is the main diagnostic feature.

William Barker (1996, p. 198) states that the material of Hakea seen by him from Europe is H. decurrens; he goes on to note that “the published records of H. sericea from southern Europe (e.g. Ball 1964) may also be that species”. The reference to Ball et al. (1964) refers to the species entry in Flora Europaea Vol 1. (1st ed.), where it is stated that H. sericea is “Planted for reclamation of arid land in Spain and Portugal, and locally naturalized”. Indeed, under his entry for H. decurrens ssp. physocarpa W.R.Barker, Barker (1996) lists a specimen in BR (the National Botanic Garden of Belgium), giving the following details “E. Orey, D. Pereina & Reis 56, 15.xi.1966, Lusitania. Reg. Estremadura, Estrada da Picanceira, próximo de Mafra”. The situation remains to be clarified: Paiva et al. (1997) does not list H. decurrens for Portugal, and the Plant List, updated until 2017, considered H. decurrens to be a synonym of H. sericea (Plant List 2023, At present, a note is added on the website that it has been superseded by http://www.plantsoftheworldonline.org/taxon/urn:lsid:ipni.org:names:704178–1. Flora-On (https://flora-on.pt/#/1hakea+sericea; coordinated by the Portuguese Botanical Society) now has a note on H. sericea saying that “In fact, the taxon present in Portugal of this species complex is Hakea decurrens subsp. physocarpa…. Paulo Alves on 17/09/2019” To complicate taxonomical matters further, in Flora Gallica (Tison and de Foucault (coords) 2014), it is indicated that the individuals observed in France should be named H. lissosperma R.Br., a species considered by some to be a synonym of H. sericea. Robyn Barker (2010) gives the name H. sericea auct. non Schrad. & J.C.Wendl. in the section on synonymy for H. decurrens, indicating that specimens, now referable to H. decurrens, have been subsumed within H. sericea by some taxonomists (but not explicitly by Schrader and Wendland, whose taxonomic concept is considered to circumscribe the “true” H. sericea and exclude H. decurrens).

Given the fact that there is genetic evidence that South African populations of H. sericea may have originated from two separate source populations (Dyer and Richardson 1992), and that biocontrol programs for H. sericea have previously been complicated by confusion between H. sericea and H. decurrens in their native ranges (Kluge and Neser 1991; Hosking et al. 2000; Barker 2010), the possibility that Portugal (and/or Spain) harbours additional or misidentified species (sensu Barker 1996, 2010) was worth investigating. To make the survey more concise, all Hakea species encountered were included.

Materials and methods

Plant material was obtained by dedicated surveys of Hakea stands in Portugal and South Africa between 2019 and 2021. In each population, one herbarium voucher with silica-dried leaf sample for DNA analysis plus silica dried leaf samples of two additional trees, 5 meters apart, were collected. Populations in Portugal were at least 7 kilometres apart and in South Africa at least 10 kilometres apart. These surveys were complemented by herbarium collections with silica-dried leaf samples from New Zealand and France in 2021 (Table 1). Previously collected Hakea material (herbarium and silica dried leaf sample) by the first author in Portugal, New Zealand and South Africa and a fruiting H. decurrens collection from Portugal by E. Marchante were added to the analysis.

Table 1. Sampling localities. All sampling from naturalized populations (species: 1. H. decurrens; 2. H. drupacea; 3. H. gibbosa; 4. H. lissosperma; 5. H. salicifolia; 6. H. sericea).

	Location	Coordinates	Species
Portugal
	Mata do Sobral	40°09’34.2“N, 8°10’29.3“W	1
	Serpins	40°09’22.2“N, 8°14’11.2“W	1
	Coimbra	40°10’38.8“N, 8°27’38.5“W	1, 5
	Vale de Canas	40°12’38“N, 8°22’40“W	5
	Entroncamento	39°27’53“N, 8°25’55“W	1
South Africa
	Outenikwa Pass	33° 52 ‘31“S, 22° 22’ 57“E	6
	Joubertina Hoek	33° 52 ‘8“S, 23° 50’ 8“E	6
	Sandrif	33° 50 ‘23“S, 23° 47’ 53“E	6
	DeVlug draai	33° 45 ‘5“S, 22° 57’ 38“E	6
	Vergelegen	34° 3 ‘23“S, 18° 55’ 0“E	6
	Pniel	33° 53 ‘60“S, 18° 56’ 52“E	6
	Caledon	34° 13 ‘5“S, 19° 24’ 12“E	6
	Karwyderskraal	34° 20 ‘56“S, 19° 11’ 36“E	3, 6
	Glenthorpe farm	33° 21 ‘36“S, 26° 30’ 9“E	6
	Grahamstown	33° 17 ‘1“S, 26° 42’ 7“E	6
	Grahamstown	33° 20 ‘1“S, 26° 35’ 42“E	6
	Fisherhaven	34° 22 ‘ 1”S, 19° 7 ‘ 22”E	2
	Sir Lowry’s pass	34° 8 ‘59“S, 18° 55’ 40“E	5
	Slanghoek	33° 32 ‘24“S, 19° 12’ 36“E	6
New Zealand
	North Island, Whangarei	35° 42 ‘37“S, 174° 20’ 27“E	6
	Gumlands, Mangaroa Stream	35° 40 ‘59“S, 174° 1’ 13“E	6
	Kawakawa, Karetu Valley	35° 22 ‘38“S, 174° 11’ 5“E	6
	Mahurangi Peninsula	36° 27 ‘0“S, 174° 43’ 60“E	6
	Orewa	36° 34 ‘0“S, 174° 40’ 0“E	6
	Sweetwater, Lake Ngatu	35° 2 ‘17“S, 173° 11’ 57“E	6
France
	Théoule-sur-Mer	43° 29 ‘47“N, 6° 56’ 39“E	1
	Saint-Raphaël, Arboretum Plan Estérel	43° 29 ‘33“N, 6° 49’ 23“E	6
	Arboretum du Caneiret	43° 29 ‘47“N, 6° 49’ 55“E	1

To verify the identification and solve some taxonomical confusion, material originating from the native range and material annotated by Barker (1996) was consulted and sampled from the herbarium collection at Naturalis Leiden (L), as well as additional material from the Allan Herbarium at Lincoln New Zealand (CHR) (Table 2). Acronyms of herbaria follow the Index Herbariorum (Thiers continuously updated).

Table 2. Herbarium collections, yielding good quality DNA, from the native range or annotated by Barker at Naturalis Leiden (L) for verification and additional specimens from the Allan herbarium (CHR) for New Zealand.

	Subdivision	Species	Collector	Barcode
Australia
	Australian Capital Territory	H. decurrens ssp. decurrens	Taylor J 13	L.1634603
	New South Wales	H. decurrens ssp. decurrens	Coveny RG 4189	L.1634991
	New South Wales	H. lissosperma	Johnson LAS 19,281	L.1634614
	Tasmania	H. lissosperma	Eichler H 16,627	L.1634582
New Zealand
	North Auckland Land District	H. gibossa	Sykes WR 399/86	CHR 437,550
	North Auckland Land District	H. decurrens	Healy AJ s.n	CHR 70,931
	North Auckland Land District	H. salicifolia	Bell B s.n	CHR 671,880
	South Auckland Land District	H. decurrens	Inst. In Agr. s.n.	CHR 90,114
	Wellington Land District	H. decurrens	Healy AJ s.n.	CHR 33,544
	Wellington Land District	H. decurrens	Healy AJ s.n.	CHR 58,539
	Marlborough Land District	H. decurrens	Beggs JP s.n.	CHR 98,049
	Nelson Land District	H. salicifolia	Wright K s.n.	CHR 635,162
	Nelson Land District	H. decurrens	Ecroyd CE s.n.	CHR 670,269
Portugal
	Madeira	H. decurrens	Hegnauer R LEP 11,999	L.1634989

For all specimens, identification was verified both morphologically, with the herbarium collections at Leiden (L) and Lincoln (CHR), and molecularly, by comparing sequences with those in NCBI GenBank, NCBI GenBank contained 200 cpDNA Hakea sequences including 4 incomplete ones of our target species.

No duplicates of Barker annotated specimens of H. decurrens ssp. physocarpa could be found at Naturalis. This subspecies is mentioned by Barker as being present in Portugal.

DNA extraction

Genomic DNA was isolated from approx. 100 mg leaf material with the DNeasy plant mini kit (Qiagen, Venlo, The Netherlands) using the TissueLyser procedure and eluted with 50 μL preheated (65 ºC) AE buffer. DNA extracts were stored at − 20 °C until further use.

Illumina sequencing

DNA extracts were sent to GenomeScan (Leiden, the Netherlands) for Illumina 150PE (paired-end) sequencing using the NovaSeq with at least 2Gb output per sample.

Samples were processed using the NEBNext® Ultra II FS DNA module and the NEBNext® Ultra II Ligation module. Fragmentation, A-tailing and ligation of sequencing adapters and PCR using NEBNext® Ultra II Q5 master mix of the resulting product was performed according to the procedure described in the NEBNext Ultra II FS DNA module and NEBNext Ultra II Ligation module instruction manual.

After preparation, the quality and yield for all samples was measured with the Fragment Analyzer (Agilent Technologies, USA). The size of the resulting products was consistent with the expected size of approximately 500–700 bp.

De novo assembly and extraction of loci

Reads of samples were trimmed and de novo assembled in CLC Genomics Workbench 21.0.4 (Qiagen, Aarhus, Denmark) using default settings. Contigs of interest were selected with BLASTn (standard settings) from assembled contigs using a custom BLAST database in Geneious Prime® 2021.1.1 (Biomatters Auckland, New Zealand) containing the reference sequences mined from several NCBI accessions (Locus rbcL: JX572664, locus trnH-psbA: OL312364, and locus matK: JQ257182). Loci rbcL and trnH-psbA were extracted from the selected contigs with Primer3 2.3.7 (Primer3—new capabilities and interfaces - PMC (nih.gov)) using the primers mentioned in Table 3. Locus matK was extracted using the BLAST hit coordinates on the contig. For phylogenetic analysis, the loci were concatenated (matK – rbcL – trnH-psbA) and treated as a single sequence with a length between 2375 and 2425 base pairs.

Table 3. Primers used in this study.

Locus	Primer	Primer sequence	Reference
rbcL	Forward	5′-ATG TCA CCA CAA ACA GAG ACT AAA GC-3′	Kress et al. (2007)
	Reverse	5′-GTA AAA TCA AGT CCA CCR CG-3′	Kress et al. (2009)
trnH-psbA	Forward	5′-CGC GCA TGG TGG ATT CAC AAT CC-3′	Tate (2002)
	Reverse	5′-GTT ATG CAT GAA CGT AAT GCT C-3′	Sang et al. (1997)

Phylogenetic analyses

Sequences were aligned using MUSCLE version 3.8.425 (Edgar 2004) within Geneious Prime® 2021.1.1 (Biomatters Auckland, New Zealand) with standard settings. We performed model testing on the alignments to select the most appropriate method for constructing a maximum likelihood tree using the FastTree version 2.1.11 with standard settings. Specimen Valkenburg 4055 (NPPO-NL ref. 06145681; H. drupacea) was used to root the tree.

Results

Morphological identification

The flowering H. sericea plants from South Africa have a pistil length of 4.5–7.5 mm, whereas the H. decurrens plants from Portugal have a pistil length of 10 mm (Table 4). Fruiting plants of H. decurrens from Portugal and France have a pale wood width of 6–12 mm with an outer layer of 2–5 mm. This corresponds with the descriptions for H. decurrens ssp. physocarpa (Barker 1996, 2010). Flowering plants of H. decurrens from New Zealand have a pistil length of 8.5–11 mm. The measurements of the fruiting material of H. decurrens and H. sericea in the present study did not allow reliable distinction between the two taxa (Table 2). Hakea gibbosa plants in fruit can be easily identified by the seeds being oriented transversely to the direction of the fruit stalk (Figure 1). As could be expected, H. salicifolia can easily be distinguished by its linear to lanceolate leaf shape compared with the terete spiny leaves of the other species.

Figure 1. Flower and fruit of Hakea decurrens, H. sericea and H. gibbosa.

Table 4. Measurement of flower (10 per specimen) and fruit of Hakea decurrens, H. sericea and H. gibbosa.

		Length of pistil (mm)	Length Fruit (mm)	Width fruit (mm)	pale wood zone (mm)	outer layer (mm)
H. decurrens
	Marchante s.n. Coimbra WAG0452351	10	32	32	12	5
	Marchante s.n. Coimbra WAG0452210		32–34	25–26	10	4
	Marchante s.n. Serpins	10
	Marchante s.n. Mata do Sobral	10
	Beggs s.n. CHR 98,049	10.5
	Healy s.n. CHR 58,539	8.5
	Healy s.n. CHR 33,544	9.5–11
	Inst. in Agric. s.n. CHR 90,114	9
	Diadema s.n.		22–26	20–25	6.0–8.0	2.0–4.0
	Valkenburg 3860		24–29	20–25	7.0–8.0	3.0–4.0
H. sericea
	Kritzinger-Klopper 26	7
	Kritzinger-Klopper 25	4.5–6.5	24–29	25–26	8	4
	Valkenburg 4063		25–30	24–28	9	5
	Kinsler s.n.	5–7.5
H. gibbosa
	L.1634877; L.1634878	9–10.5
	Valkenburg 4056		32–35	32–36	14–16	5

Molecular identification

The 96 different Hakea specimens evaluated using molecular analysis formed distinct species-specific clusters for five of the six species present in the phylogenetic analysis. Only H. decurrens did not form a species-specific cluster forming two distinct groups in the phylogenetic tree (Figure S1). The main H. decurrens cluster comprises 35 specimens and 4 genotypes. 31 specimens have 100% similarity and form the largest subcluster. Two H. decurrens specimens (Taylor, J 13 and Coveny, RG 4189) fall outside the H. decurrens cluster and show 17 base pairs differences compared to the most prevalent genotype of H. decurrens. These specimens were labelled as H. decurrens ssp. decurrens.

Of the seven H. salicifolia specimens, six show 100% similarity. The only specimen (6143774) that has a different genotype shows an inversion of 24 nucleotides on locus trnH-psbA. This inversion is also found in all H. decurrens, H. sericea and H. gibbosa but not in H. drupacea and H. lissosperma.

Within the H. sericea species cluster, three distinct groups are formed (Figure 2). The main group consists of 35 specimens with 100% similarity originating from the Northland Region, North Island of New Zealand, France and the western part of South Africa. The other two groups with 100% similarity within the cluster comprises three and seven specimens and originate from the Auckland Region, North Island of New Zealand and eastern part of South Africa respectively. Although there is a distinct formation of several phylogenetic clusters, the actual differences on a sequence level are minimal and all specimens group together in a species-specific cluster.

Figure 2. Detail of phylogenetic tree based on concatenated matK, rbcL and trnH-psbA barcode sequences of 45 Hakea sericea specimens. A 2,425 (including gaps) alignment of 96 Hakea spp. specimens was used to construct the dendrogram with FastTree using a Jukes-Cantor substitution model and optimization of the Gamma20 likelihood. Fast global bootstrap values are shown on the internal nodes and H. drupacea (taxon level) was used to root the (taxon level) sequences. Origin of the sampled specimens is indicated with vertical bars. The full tree containing 96 Hakea spp. Is shown in S-Fig. 1.

It was not possible to establish a link between H. sericea and a sample from the native range, since four of the nine samples from the native range and the material annotated by Barker (1996) to confirm identification and resolve some taxonomic confusions (samples from the Naturalis Leiden (L) herbarium collection) did not yield good quality DNA.

Discussion

We acknowledge that not all Hakea populations in Portugal have been sampled. However, all populations sampled and flowering plants observed over the last years by botanists fit the criterion of a >9 mm long pistil, equating to H. decurrens. This is further supported by our molecular results.

Collections of H. lissosperma by Eichler and Johnson from Australia (Tasmania and New South Wales, respectively) are clearly distinct from any of the Hakea collections from France thereby “disqualifying” the claim for the French material to be this species according to Flora Gallica (Tison and de Foucault (coords) 2014).

The assumed different introductions of H. sericea in South Africa (Dyer and Richardson 1992) is supported by the findings in the present study where we observed a uniform pattern from Vergelegen to Joubertina Hoek, a mixed “provenance “in the site near Coleridge game reserve and Glenthorpe farm, and the other type from Thomas Baines Nature Reserve and probably eastwards (Figure 3).

Figure 3. Map of South Africa illustrating the distribution of the two haplotypes of Hakea sericea.

The variation observed in the collections from New Zealand matching a geographical pattern might also hint at a difference in provenance and multiple introductions of H. sericea. Barker (1996) mentions one collection of H. decurrens for New Zealand, apparently not in optimal condition as it could not be ascribed to a subspecies. Recent herbarium collections from New Zealand and a re-evaluation of the collection at CHR confirm the presence of the species both on the North and South Island.

The closely related H. gibbosa can be distinguished from both H. decurrens and H. sericea by the positioning of the seed at right-angles to the direction of the stalk, and our results confirm this separation. This species is naturalized in both South Africa and New Zealand but not reported for Europe.

As already reported by Ball et al. (1964), Paiva et al. (1997) and Marchante et al. (2014), H. salicifolia can be found naturalized in Portugal and Spain and was reported for South-eastern France (Terrin et al. 2022) (nature.silene.eu/Telebotanica/https://www.gbif.org/species/7287627), but has been subsequently eradicated in 2023 (K. Diadema pers. com.). The species is also naturalized in South Africa and New Zealand. Another easily distinguishable species by its upright seemingly pinnate terete leaves is H. drupacea, also naturalized in South Africa and New Zealand and with a single collection from 1904 in France (https://www.gbif.org/occurrence/694245702).

Inclusion of a species on the List of invasive alien species of Union Concern (‘the Union List’) pursuant of Regulation (EU) No 1143/2014 implies that a species shall not be intentionally brought into the territory of the Union, kept, bred or placed on the market, permitted to reproduce or released into the environment. In addition to that, Member States shall take all necessary steps to prevent the unintentional introduction or spread. So restrictive measures have to be taken. If there is confusion on the proper circumscription of the taxon that in on the Union list it may well result in the Hakea plants causing a problem in Portugal to be not included in the Union list. As a result, the restrictions as such would no longer apply. Prior to the revision of the genus Hakea sect. Hakea by Barker in Barker (1996), most authors referred to both species simply as H. sericea, including Paiva’s revision of the genus for the Flora Ibérica that was published in 1997. This broad species concept has been adopted for the EPPO PRA (2017) and was highlighted by the additional note in the taxonomy section including the citation of Barker (2010) who gives the name H. sericea auct. non Schrad. & J.C.Wendl. in the section on synonymy for H. decurrens, indicating that specimens, now referable to H. decurrens, have been subsumed within H. sericea by some taxonomists (but not explicitly by Schrader and Wendland, whose taxonomic concept is considered to circumscribe the “true” H. sericea and exclude H. decurrens). However, the listing of the species on the Union list as H. sericea Schrad. & J.C.Wendl. could potentially cause confusion. If the revision by Barker (1996) is followed it should be concluded that the invasive populations in Portugal and France that have now been confirmed to belong to H. decurrens are not part of the species Hakea sericea that is on the Union list. This could be solved by adopting the broad species concept for H. sericea as it was adopted prior to the revision by Barker (1996) including H. decurrens. EPPO (2017), on which the listing is based, also follows this Hakea sericea sensu lato concept. A simple change of the taxon name on the Union list to H. sericea Schrad. & J.C.Wendl. s.l. would be adequate. This is also justified by the observed similar impact both species, sensu Barker (1996), have in their introduced range (EPPO 2017).

It is advisable that the publications of “new records” of alien plant species should always be supported by careful identifications and specimens stored in one or more herbaria. This is once again highlighted by the confusion on the proper identity of the Hakea species in Portugal over the last decades. Natural History Collections and Herbaria are the basic building blocks for the discovery, understanding and management of diversity of alien species. Similarly, PRAs and legislation should be based on a solid taxonomical base.

Conclusion

The present study has shown that all sampled invasive populations of Hakea in Portugal belong to H. decurrens ssp. physocarpa, a species that, although very similar and sometimes considered synonymous with H. sericea, can be distinguished both morphologically and on the basis of cpDNA. Both species occur in France, where H. decurrens ssp. physocarpa has colonized the largest area of natural habitat. The invasive ability and impact of both species on the biodiversity in their introduced ranges is similar (EPPO 2017). Prior to the revision of the genus Hakea sect. Hakea by Barker in Barker (1996), most authors referred to both species simply as H. sericea, including Paiva’s revision of the genus for the Flora Ibérica that was published in 1997. By explicitly adopting the H. sericea sensu lato concept for inclusion of the species on the List of Union concern pursuant of Regulation (EU) No 1143/2014, by an amendment of the species name through an Annex of the next Commission Implementing Regulation, a potential legislative controversy can be prevented.

Author contributions

Johan van Valkenburg conceived and designed the experiment and collected the field data. Jesse Beyer managed the metadata of the botanical collections. Paul Champion coordinated the fieldwork in New Zealand. Julie Coetzee coordnated fieldwork in the Eastern Cape province. Katia Diadema coordinated the fieldwork in France. Suzaan Kritzinger-Klopper coordinated the fieldwork in the Eastern Cape province. Elizabete Marchante conceived and designed the experiment and collected the field data. Laurens Piet performed the analysis of the molecular data and drafted the manuscript. David Richardson conceived and designed the experiment. Ines Schönberger was instrumental in unearthing the additional Ha)kea collections from New Zealand. All authors drafted the manuscript.

Acknowledgements

Lisa Forester and Maureen Young are acknowledged for collecting flowering material of Hakea from New Zealand. Jan Wieringa has been instrumental for retrieving Hakea collections from the native range at Naturalis Leiden and Esmée Winkel has made the fine botanical drawings.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The annotated DNA sequences will be made available on NCBI GenBank and on EPPO Q-bank (//qbank.eppo.int/plants/). A complete list of Herbarium vouchers analysed (including NCBI codes) is included in Appendix 1.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/23818107.2024.2318761

Additional information

Notes on contributors

J.L.C.H. van Valkenburg

Johan van Valkenburg is senior botanist at the Netherlands Institute for Vectors, Invasive plants and Plant health. He focusses on invasive alien plants. He conceived and designed the experiment and collected the field data. He drafted the manuscript.

J. Beyer

Jesse Beyer is botanist at the Netherlands Institute for Vectors, Invasive plants and Plant health. He focusses on invasive alien plants. He managed the metadata of the botanical collections. He drafted the manuscript.

P. Champion

Paul Champion is principal scientist at the National Institute of Water & Atmospheric Research Ltd (NIWA). He coordinated the fieldwork in New Zealand. He drafted the manuscript.

J. Coetzee

Julie Coetzee is Professor of Botany and Deputy Director of the Centre for Biological Control, Rhodes University, She coordinated fieldwork in the Eastern Cape province. She drafted the manuscript.

K. Diadema

Katia Diadema is head of the conservation department at the Conservatoire botanique national méditerranéen de Porquerolles. She coordinated the fieldwork in France. She drafted the manuscript.

S. Kritzinger-Klopper

Suzaan Kritzinger-Klopper is chief technical officer at the Centre for Invasion Biology, Department of Botany and Zoology, Stellenbosch University. She coordinated the fieldwork in the Eastern Cape province. She drafted the manuscript.

E. Marchante

Elizabete Marchante is Professor at the Centre for Functional Ecology, Associate Laboratory TERRA, Department of Life Sciences, University of Coimbra. She conceived and designed the experiment and collected the field data. She drafted the manuscript.

L. Piet

Laurens Piet is a specialist molecular biology at the Netherlands Institute for Vectors, Invasive plants and Plant health. He performed the analysis of the molecular data and drafted the manuscript.

D. M. Richardson

David Richardson is Distinguished Professor at the Centre for Invasion Biology, Department of Botany and Zoology, Stellenbosch University, South Africa. Between July 2022 and June 2023 he was visiting scientist at the Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic. He conceived and designed the experiment. He drafted the manuscript.

I. Schönberger

Ines Schönberger is collection manager at Allan Herbarium (CHR), Manaaki Whenua – Landcare Research. She was instrumental in unearthing the additional Hakea collections from New Zealand. She drafted the manuscript.

Appendix 1

Hakea decurrens R.Br.

AUSTRALIA • Australian Capital Territory, Western slope near summit of Black Mountain; 35° 16 ‘31”S, 149° 5 ‘ 39”E; Herbarium specimen; 22 Jul. 1975; J. Taylor 13;J.J. Wieringa det.; L;NPPO-NL ref. no. 6164145. GenBank no.:;OQ446994 (matK); OQ446995 (rbcL); OQ446996 (trnH-psbA).

AUSTRALIA • New South Wales, The Mullions Range, 14 miles [22.5 km] NNE of Orange; 33° 4 ‘ 60”S, 149° 10 ‘ 60”E; Herbarium specimen; 16 Apr 1972; R.G. Coveney;–; L;NPPO-NL ref. no. 6164217. GenBank no.:;OQ447006 (matK); OQ447007 (rbcL); OQ447008 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Herbarium specimen; 30 Apr 2021; K. Diadema s.n. 33042021; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163986. GenBank no.:;OQ447024 (matK); OQ447025 (rbcL); OQ447026 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_1; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163978. GenBank no.:;OQ447027 (matK); OQ447028 (rbcL); OQ447029 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_2; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163961. GenBank no.:;OQ447030 (matK); OQ447031 (rbcL); OQ447032 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_3; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163951. GenBank no.:;OQ447033 (matK); OQ447034 (rbcL); OQ447035 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_4; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163943. GenBank no.:;OQ447036 (matK); OQ447037 (rbcL); OQ447038 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_5; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163935. GenBank no.:;OQ447039 (matK); OQ447040 (rbcL); OQ447041 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_6; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163927. GenBank no.:;OQ447060 (matK); OQ447061 (rbcL); OQ447062 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_7; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163919. GenBank no.:;OQ447042 (matK); OQ447043 (rbcL); OQ447044 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_8; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163900. GenBank no.:;OQ447045 (matK); OQ447046 (rbcL); OQ447047 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_9; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163898. GenBank no.:;OQ447078 (matK); OQ447049 (rbcL); OQ447050 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_10; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163881. GenBank no.:;OQ447051 (matK); OQ447052 (rbcL); OQ447053 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_11; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163871. GenBank no.:;OQ447054 (matK); OQ447055 (rbcL); OQ447056 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Alpes-Maritimes, Théoule-sur-Mer; 43° 29 ‘ 47”N, 6° 56 ‘ 39”E; Silica dried; 30 Apr 2021; K. Diadema s.n. 33042021_12; K. Diadema det.; WAGPD;NPPO-NL ref. no. 6163863. GenBank no.:;OQ447057 (matK); OQ447058 (rbcL); OQ447059 (trnH-psbA)

FRANCE • Provence-Alpes-Côte d’Azur, Arberetum du Caneiret; 43° 29 ‘ 47”N, 6° 49 ‘ 55”E; Herbarium specimen; 2 Jun 2021; R. Ardito s.n. Caneiret 1;–; WAGPD;NPPO-NL ref. no. 5377511. GenBank no.:;OQ447093 (matK); OQ447094 (rbcL); OQ447095 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arberetum du Caneiret; 43° 29 ‘ 47”N, 6° 49 ‘ 55”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Caneiret 2;–; WAGPD;NPPO-NL ref. no. 6163425. GenBank no.:;OQ447096 (matK); OQ447097 (rbcL); OQ447098 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arberetum du Caneiret; 43° 29 ‘ 47”N, 6° 49 ‘ 55”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Caneiret 3;–; WAGPD;NPPO-NL ref. no. 6163433. GenBank no.:;OQ447099 (matK); OQ447100 (rbcL); OQ447101 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arberetum du Caneiret; 43° 29 ‘ 47”N, 6° 49 ‘ 55”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Caneiret 4;–; WAGPD;NPPO-NL ref. no. 6163441. GenBank no.:;OQ447102 (matK); OQ447103 (rbcL); OQ447104 (trnH-psbA).

NEW ZEALAND • South Auckland Land District, Ngutunui near Te Awamutu; 38° 4 ‘ 44”S, 175° 6 ‘ 25”E; Herbarium specimen; 18 Jul 1955; Inst., in Agric. s.n.; I. Schoenberger; CHR;NPPO-NL ref. no. 6143741. GenBank no.:;OR352972 (matK); OR352981 (rbcL); OR352990 (trnH-psbA).

NEW ZEALAND • Wellington Land District, Makino; 40° 9 ‘ 50”S, 175° 36 ‘ 3”E; Herbarium specimen; 2 Okt 1934; A.J. Healy. s.n.; I. Schoenberger; CHR;NPPO-NL ref. no. 6143766. GenBank no.:;OR352970 (matK); OR352979 (rbcL); OR352988 (trnH-psbA).

NEW ZEALAND • Wellington Land District, Hills between Wallaceville and Mangaroa, Hutt Valley; 41° 8 ‘ 34”S, 175° 5 ‘ 6”E; Herbarium specimen;–; A.J. Healy. s.n.; I. Schoenberger; CHR;NPPO-NL ref. no. 6143758. GenBank no.:;OR352971 (matK); OR352980 (rbcL); OR352989 (trnH-psbA).

NEW ZEALAND • Rai Valley, Marlborough; 41° 13 ‘ 40”S, 173° 34 ‘ 43”E; Herbarium specimen; 8 Aug 1958; J.P. Beggs s.n.; I. Schoenberger; CHR;NPPO-NL ref. no. 6143723. GenBank no.:;OR352974 (matK); OR352983 (rbcL); OR352992 (trnH-psbA).

NEW ZEALAND • North Auckland Land District, Waipu Cove; ; Herbarium specimen;28 May 1950; A.J. Healy s.n.; W.R. Barker; CHR;NPPO-NL ref. no. 6143731. GenBank no.:;OR352973 (matK); OR352982 (rbcL); OR352991 (trnH-psbA).

NEW ZEALAND • Nelson Land District, Abel Tasman National Park, Awaroa Bay; 40° 51 ‘ 56”S, 173° 1 ‘ 52”E; Herbarium specimen;28 Sep 2021; C.E. Ecroyd s.n.; C.E. Ecroyd; CHR;NPPO-NL ref. no. 6143694. GenBank no.:;OR352975 (matK); OR352984 (rbcL); OR352993 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 38”W; Herbarium specimen, Silica dried; 7 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det.; WAGPD;NPPO-NL ref. no. 6145366. GenBank no.:;OQ446865 (matK); OQ446866 (rbcL); OQ446867 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 38”W; Silica dried; 7 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145374. GenBank no.:;OQ446862 (matK); OQ446863 (rbcL); OQ446864 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 38”W; Silica dried; 7 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145382. GenBank no.:;OQ446859 (matK); OQ446860 (rbcL); OQ446861 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 38”W; Herbarium specimen, Silica dried; 1 Jun 2020; E. Marchante s. n.;–; WAGPD;NPPO-NL ref. no. 6164399. GenBank no.:;OQ446868 (matK); OQ446869 (rbcL); OQ446870 (trnH-psbA).

PORTUGAL • Centro, East of Entroncamento; 39° 27 ‘ 53”N, 8° 25 ‘ 55”W; Herbarium specimen; 8 Sep 2017; Valkenburg 3860; J.L.C.H. van Valkenburg det; WAGPD, L;NPPO-NL ref. no. 6149842. GenBank no.:;OQ446988 (matK); OQ446989 (rbcL); OQ446990 (trnH-psbA).

PORTUGAL • Centro, Mata do Sobral; 40° 9 ‘ 34”N, 8° 10 ‘ 29”W; Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145421. GenBank no.:;OQ446871 (matK); OQ446872 (rbcL); OQ446873 (trnH-psbA).

PORTUGAL • Centro, Mata do Sobral; 40° 9 ‘ 34”N, 8° 10 ‘ 29”W; Herbarium specimen, Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145438. GenBank no.:;OQ446877 (matK); OQ446878 (rbcL); OQ446879 (trnH-psbA).

PORTUGAL • Centro, Mata do Sobral; 40° 9 ‘ 34”N, 8° 10 ‘ 29”W; Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145446. GenBank no.:;OQ446874 (matK); OQ446875 (rbcL); OQ446876 (trnH-psbA).

PORTUGAL • Centro, Serpins; 40° 9 ‘ 22”N, 8° 14 ‘ 11”W; Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145390. GenBank no.:;OQ446883 (matK); OQ446884 (rbcL); OQ446885 (trnH-psbA).

PORTUGAL • Centro, Serpins; 40° 9 ‘ 22”N, 8° 14 ‘ 11”W; Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145403. GenBank no.:;OQ446880 (matK); OQ446881 (rbcL); OQ446881 (trnH-psbA).

PORTUGAL • Centro, Serpins; 40° 9 ‘ 22”N, 8° 14 ‘ 11”W; Herbarium specimen, Silica dried; 6 Jan 2020; E. Marchante s. n.; J.L.C.H. van Valkenburg det; WAGPD;NPPO-NL ref. no. 6145411. GenBank no.:;OQ446886 (matK); OQ446887 (rbcL); OQ446888 (trnH-psbA).

PORTUGAL • Madeira Island, between Camacha and Poiso; 32° 41 ‘ 6”N, 16° 51 ‘ 44”W; Herbarium specimen; 11 May 1968; R. Hegnauer LEP 11999; W.R. Barker; L;NPPO-NL ref. no. 6164196. GenBank no.:;OQ447000 (matK); OQ447001 (rbcL); OQ447002 (trnH-psbA).

Hakea drupacea (C.F.Gaertn.) Roem. & Schult.

SOUTH AFRICA • Western Cape, Fisherhaven, Outspan Road; 34° 22 ‘ 1”S, 19° 7 ‘ 22”E; Herbarium specimen, Silica dried; 17 Feb 2020; Valkenburg 4055; J.L.C.H. van Valkenburg det; WAGPD, L;NPPO-NL ref. no. 6145681. GenBank no.:;OQ446961 (matK); OQ446962 (rbcL); OQ446963 (trnH-psbA).

Hakea gibbosa (Sm.) Cav.

NEW ZEALAND • Great Barrier Island, State Forest park, Forestry Rd, near Coffins Creek; 36° 12 ‘ 10”S, 175° 22 ‘ 45”E; Herbarium specimen; 30 Nov 1986; W.R. Sykes 399/86; W.R. Sykes; CHR;NPPO-NL ref. no. 6143715. GenBank no.:;OR352976 (matK); OR352985 (rbcL); OR352994 (trnH-psbA).

SOUTH AFRICA • Western Cape, Karwyderskraal; 34° 21 ‘ 0”S, 19° 11 ‘ 45”E; Herbarium specimen, Silica dried; 17 Feb 2020; Valkenburg 4056; J.L.C.H. van Valkenburg det; WAGPD, L;NPPO-NL ref. no. 6145673. GenBank no.:;OQ446964 (matK); OQ446965 (rbcL); OQ446966 (trnH-psbA).

Hakea lissosperma R.BR

AUSTRALIA • Tasmania, Lake St.Clair National Park, Lake St.Clair, southern end, Cynthia Bay, near camping area; 42° 6 ‘ 55”S, 146° 10 ‘ 33”E; Herbarium specimen; 14 Jan 1960; H. Eichler 16627;–; L;NPPO-NL ref. no. 6164209. GenBank no.:;OQ447003 (matK); OQ447004 (rbcL); OQ447005 (trnH-psbA).

AUSTRALIA • New South Wales, Piper’s Creek & Snowy River, Kosciusko district; ; Herbarium specimen; 23 Jan 1951; L.A.S. Johnson & E. F. Constable 19281;–; L;NPPO-NL ref. no. 6164188. GenBank no.:;OQ446997 (matK); OQ446998 (rbcL); OQ446999 (trnH-psbA).

Hakea salicifolia (Vent.) B.L. Burtt

NEW ZEALAND • Nelson Land District, Puramahoi, Golden Bay; 40° 48 ‘ 56”S, 172° 45 ‘ 52”E; Herbarium specimen; 23 Okt 2014; K. Wright s.n.; P.B. Heenan; CHR;NPPO-NL ref. no. 6143774. GenBank no.:;OR352969 (matK); OR352978 (rbcL); OR352987 (trnH-psbA).

NEW ZEALAND • North Auckland Land District, Bay of Islands, Kawakawa, Plot CB19; 35° 13 ‘ 59”S, 174° 2 ‘ 29”E; Herbarium specimen; 14 Apr 2013; B. Bell s.n.; A. Fergus; CHR;NPPO-NL ref. no. 6143782. GenBank no.:;OR352968 (matK); OR352977 (rbcL); OR352986 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 39”W; Herbarium specimen, Silica dried; 11 Jun 2021; E. Marchante s.n. COI;–; WAGPD;NPPO-NL ref. no. 6163548. GenBank no.:;OQ447072 (matK); OQ447073 (rbcL); OQ447074 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 39”W; Silica dried; 11 Jun 2021; E. Marchante s.n. COI;–; WAGPD;NPPO-NL ref. no. 6163556. GenBank no.:;OQ447075 (matK); OQ447076 (rbcL); OQ447077 (trnH-psbA).

PORTUGAL • Centro, Coimbra; 40° 10 ‘ 39”N, 8° 27 ‘ 39”W; Silica dried; 11 Jun 2021; E. Marchante s.n. COI;–; WAGPD;NPPO-NL ref. no. 6163564. GenBank no.:;OQ447078 (matK); OQ447079 (rbcL); OQ447080 (trnH-psbA).

PORTUGAL • Centro, Vale de Canas; 40° 12 ‘ 38”N, 8° 22 ‘ 40”W; Herbarium specimen, Silica dried; 11 Jun 2021; E. Marchante s.n. VDC;–; WAGPD;NPPO-NL ref. no. 6163513. GenBank no.:;OQ447063 (matK); OQ447064 (rbcL); OQ447065 (trnH-psbA).

PORTUGAL • Centro, Vale de Canas; 40° 12 ‘ 38”N, 8° 22 ‘ 40”W; Silica dried; 11 Jun 2021; E. Marchante s.n. VDC;–; WAGPD;NPPO-NL ref. no. 6163521. GenBank no.:;OQ447069 (matK); OQ447070 (rbcL); OQ447071 (trnH-psbA).

PORTUGAL • Centro, Vale de Canas; 40° 12 ‘ 38”N, 8° 22 ‘ 40”W; Silica dried; 11 Jun 2021; E. Marchante s.n. VDC;–; WAGPD;NPPO-NL ref. no. 6163531. GenBank no.:;OQ447066 (matK); OQ447067 (rbcL); OQ447068 (trnH-psbA).

SOUTH AFRICA • Western Cape, Sit Lowry’s pass look-out; 34° 8 ‘ 59”S, 18° 55 ‘ 40”E; Herbarium specimen, Silica dried; 17 Feb 2020; Valkenburg 4054; J.L.C.H. van Valkenburg det; WAGPD, L;NPPO-NL ref. no. 6145691. GenBank no.:;OQ446958 (matK); OQ446959 (rbcL); OQ446960 (trnH-psbA).

Hakea sericea Schrad. & J.C. Wendl.

FRANCE • Provence-Alpes-Côte d’Azur, Arboretum Plan Esterel; 43° 29 ‘ 33”N, 6° 49 ‘ 23”E; Herbarium specimen, Silica dried; 2 Jun 2021; R. Ardito s.n. Plan Esterel 1;–; WAGPD;NPPO-NL ref. no. 39738539. GenBank no.;: OQ447081 (matK); OQ447082 (rbcL); OQ447083 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arboretum Plan Esterel; 43° 29 ‘ 33”N, 6° 49 ‘ 23”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Plan Esterel 2;–; WAGPD;NPPO-NL ref. no. 6163451. GenBank no.:;OQ447084 (matK); OQ447085 (rbcL); OQ447086 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arboretum Plan Esterel; 43° 29 ‘ 33”N, 6° 49 ‘ 23”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Plan Esterel 3;–; WAGPD;NPPO-NL ref. no. 6163468. GenBank no.:;OQ447087 (matK); OQ447088 (rbcL); OQ447089 (trnH-psbA).

FRANCE • Provence-Alpes-Côte d’Azur, Arboretum Plan Esterel; 43° 29 ‘ 33”N, 6° 49 ‘ 23”E; Silica dried; 2 Jun 2021; R. Ardito s.n. Plan Esterel 4;–; WAGPD;NPPO-NL ref. no. 6163476. GenBank no.:;OQ447090 (matK); OQ447091 (rbcL); OQ447092 (trnH-psbA).

NEW ZEALAND • North Island, Auckland Ecologocial region, Rodney Ecological district, Mahurangi Peninsula, intersection of Mahurangi East and Scotts Landing Roads; 36° 27 ‘ 0”S, 174° 43 ‘ 60”E; Herbarium specimen; 19 Feb 2021; Young s.n. ; M.E. Young; WAGPD;NPPO-NL ref. no. 6163839. GenBank no.:;OQ447015 (matK); OQ447016 (rbcL); OQ447017 (trnH-psbA).

NEW ZEALAND • North Island, Auckland Ecologocial region, Rodney Ecological district, Orewa, Hillcrest Road; 36° 34 ‘ 0”S, 174° 40 ‘ 0”E; Herbarium specimen; 21 Feb 2021; Young s.n.; M. E. Young; WAGPD;NPPO-NL ref. no. 6163820. GenBank no.:;OQ447018 (matK); OQ447019 (rbcL); OQ447020 (trnH-psbA).

NEW ZEALAND • North Island, Gumlands, Macmillan Property, Mangaroa Strean, Moore Road to South; 35° 40 ‘ 59”S, 174° 1 ‘ 13”E; Herbarium specimen; 19 Feb 2021; Forester s.n.; L.J. Forester; WAGPD;NPPO-NL ref. no. 6163855. GenBank no.:;OQ447012 (matK); OQ447013 (rbcL); OQ447014 (trnH-psbA).

NEW ZEALAND • North Island, Kawakawa, Karetu Valley south of Karety Stream; 35° 22 ‘ 38”S, 174° 11 ‘ 5”E; Herbarium specimen; 27 Feb 2021; Forester s.n.; L.J. Forester; WAGPD;NPPO-NL ref. no. 6163847. GenBank no.:;OQ447009 (matK); OQ447010 (rbcL); OQ447011 (trnH-psbA).

NEW ZEALAND • North Island, Sweetwater, Lake Ngatu; 35° 2 ‘ 17”S, 173° 11 ‘ 57”E; Herbarium specimen; 30 Mar 2021; Young s.n.; M.E. Young; WAGPD;NPPO-NL ref. no. 6163812. GenBank no.:;OQ447021 (matK); OQ447022 (rbcL); OQ447023 (trnH-psbA).

NEW ZEALAND • North Island, Whangarei, Memorial Drive; 35° 42 ‘ 37”S, 174° 20 ‘ 27”E; Herbarium specimen; 26 Feb 2018; Valkenburg 3918; J.L.C.H. van Valkenburg det.; WAGPD, L;NPPO-NL ref. no. 6147812. GenBank no.:;OQ446985 (matK); OQ446986 (rbcL); OQ446987 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Glenthorpe Farm; 33° 21 ‘ 36”S, 26° 30 ‘ 9”E; Herbarium specimen; 21 Jun 2021; D. Kinsler s.n.;–; WAGPD;NPPO-NL ref. no. 6231790. GenBank no.:;OQ447111 (matK); OQ447112 (rbcL); OQ447113 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Glenthorpe Farm; 33° 21 ‘ 36”S, 26° 30 ‘ 9”E; Silica dried; 21 Jun 2021; D. Kinsler s.n.;–; WAGPD;NPPO-NL ref. no. 6231803. GenBank no.:;OQ447114 (matK); OQ447115 (rbcL); OQ447116 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Glenthorpe Farm; 33° 21 ‘ 36”S, 26° 30 ‘ 9”E; Silica dried; 21 Jun 2021; D. Kinsler s.n.;–; WAGPD;NPPO-NL ref. no. 6231811. GenBank no.:;OQ447117 (matK); OQ447118 (rbcL); OQ447119 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, 20 km east of Grahamstown along N2; 33° 17 ‘ 2”S, 26° 42 ‘ 8”E; Herbarium specimen, Silica dried; 21 Feb 2020; Valkenburg 4076; J.L.C.H. van Valkenburg; WAGPD, L;NPPO-NL ref. no. 6145489. GenBank no.:;OQ446976 (matK); OQ446977 (rbcL); OQ446978 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, 20 km east of Grahamstown along N2; 33° 17 ‘ 2”S, 26° 42 ‘ 8”E; Silica dried; 21 Feb 2020; Valkenburg 4077; J.L.C.H. van Valkenburg; WAGPD;NPPO-NL ref. no. 6145501. GenBank no.:;OQ446979 (matK); OQ446980 (rbcL); OQ446981 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, 20 km east of Grahamstown along N2; 33° 17 ‘ 2”S, 26° 42 ‘ 8”E; Silica dried; 21 Feb 2020; Valkenburg 4078; J.L.C.H. van Valkenburg; WAGPD;NPPO-NL ref. no. 6145470. GenBank no.:;OQ446982 (matK); OQ446983 (rbcL); OQ446984 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, road Port Alfred - Grahamstown; 33° 20 ‘ 1”S, 26° 35 ‘ 43”E; Herbarium specimen, Silica dried; 20 Feb 2020; Valkenburg 4063; J.L.C.H. van Valkenburg; WAGPD, L;NPPO-NL ref. no. 6145630. GenBank no.:;OQ446967 (matK); OQ446968 (rbcL); OQ446969 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, road Port Alfred - Grahamstown; 33° 20 ‘ 1”S, 26° 35 ‘ 43”E; Silica dried; 20 Feb 2020; Valkenburg 4064; J.L.C.H. van Valkenburg; WAGPD;NPPO-NL ref. no. 6145649. GenBank no.:;OQ446970 (matK); OQ446971 (rbcL); OQ446972 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Albany, Grahamstown, road Port Alfred - Grahamstown; 33° 20 ‘ 1”S, 26° 35 ‘ 43”E; Silica dried; 20 Feb 2020; Valkenburg 4065; J.L.C.H. van Valkenburg; WAGPD;NPPO-NL ref. no. 6145614. GenBank no.:;OQ446973 (matK); OQ446974 (rbcL); OQ446975 (trnH-psbA).

SOUTH AFRICA • Western Cape, Outenikwa Pass; 33° 52 ‘ 31”S, 22° 22 ‘ 57”E; Herbarium specimen, Silica dried; 25 Okt 2019; Kritzinger-Klopper 1; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145155. GenBank no.:;OQ446955 (matK); OQ446956 (rbcL); OQ446957 (trnH-psbA).

SOUTH AFRICA • Western Cape, Outenikwa Pass; 33° 52 ‘ 31”S, 22° 22 ‘ 57”E; Silica dried; 25 Okt 2019; Kritzinger-Klopper 2; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145147. GenBank no.:;OQ446952 (matK); OQ446953 (rbcL); OQ446954 (trnH-psbA).

SOUTH AFRICA • Western Cape, Outenikwa Pass; 33° 52 ‘ 31”S, 22° 22 ‘ 57”E; Silica dried; 25 Okt 2019; Kritzinger-Klopper 3; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145139. GenBank no.:;OQ446949 (matK); OQ446950 (rbcL); OQ446951 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Joubertina Hoek; 33° 52 ‘ 8”S, 23° 50 ‘ 8”E; Herbarium specimen, Silica dried; 26 Okt 2019; Kritzinger-Klopper 4; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145120. GenBank no.:;OQ446946 (matK); OQ446947 (rbcL); OQ446948 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Joubertina Hoek; 33° 52 ‘ 8”S, 23° 50 ‘ 8”E; Silica dried; 26 Okt 2019; Kritzinger-Klopper 5; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145112. GenBank no.:;OQ446943 (matK); OQ446944 (rbcL); OQ446945 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Joubertina Hoek; 33° 52 ‘ 8”S, 23° 50 ‘ 8”E; Silica dried; 26 Okt 2019; Kritzinger-Klopper 6; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145104. GenBank no.:;OQ446940 (matK); OQ446941 (rbcL); OQ446942 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Sandrif; 33° 50 ‘ 23”S, 23° 47 ‘ 53”E; Herbarium specimen, Silica dried; 26 Okt 2019; Kritzinger-Kloppe 7; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145091. GenBank no.:;OQ446937 (matK); OQ446938 (rbcL); OQ446938 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Sandrif; 33° 50 ‘ 23”S, 23° 47 ‘ 53”E; Silica dried; 26 Okt 2019; Kritzinger-Klopper 8; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145083. GenBank no.:;OQ446934 (matK); OQ446935 (rbcL); OQ446936 (trnH-psbA).

SOUTH AFRICA • Eastern Cape, Sandrif; 33° 50 ‘ 23”S, 23° 47 ‘ 53”E; Silica dried; 26 Okt 2019; Kritzinger-Klopper 9; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145075. GenBank no.:;OQ446931 (matK); OQ446932 (rbcL); OQ446933 (trnH-psbA).

SOUTH AFRICA • Western Cape, DeVlug draai; 33° 45 ‘ 5”S, 22° 57 ‘ 38”E; Herbarium specimen, Silica dried; 27 Okt 2019; Kritzinger-Klopper 10; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145067. GenBank no.:;OQ446928 (matK); OQ446929 (rbcL); OQ446930 (trnH-psbA).

SOUTH AFRICA • Western Cape, DeVlug draai; 33° 45 ‘ 5”S, 22° 57 ‘ 38”E; Silica dried; 27 Okt 2019; Kritzinger-Klopper 11; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145059. GenBank no.:;OQ446925 (matK); OQ446926 (rbcL); OQ446927 (trnH-psbA).

SOUTH AFRICA • Western Cape, DeVlug draai; 33° 45 ‘ 5”S, 22° 57 ‘ 38”E; Silica dried; 27 Okt 2019; Kritzinger-Klopper 12; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145040. GenBank no.:;OQ446922 (matK); OQ446923 (rbcL); OQ446924 (trnH-psbA).

SOUTH AFRICA • Western Cape, Vergelegen; 34° 3 ‘ 23”S, 18° 55 ‘ 0”E; Herbarium specimen, Silica dried; 28 Nov 2019; Kritzinger-Klopper 13; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145032. GenBank no.:;OQ446919 (matK); OQ446920 (rbcL); OQ446921 (trnH-psbA).

SOUTH AFRICA • Western Cape, Vergelegen; 34° 3 ‘ 23”S, 18° 55 ‘ 0”E; Silica dried; 28 Nov 2019; Kritzinger-Klopper 14; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145024. GenBank no.:;OQ446916 (matK); OQ446917 (rbcL); OQ446918 (trnH-psbA).

SOUTH AFRICA • Western Cape, Vergelegen; 34° 3 ‘ 23”S, 18° 55 ‘ 0”E; Silica dried; 28 Nov 2019; Kritzinger-Klopper 15; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6145016. GenBank no.:;OQ446913 (matK); OQ446914 (rbcL); OQ446915 (trnH-psbA).

SOUTH AFRICA • Western Cape, Pniel; 33° 53 ‘ 60”S, 18° 56 ‘ 52”E; Herbarium specimen, Silica dried; 4 Feb 2020; Kritzinger-Klopper 16; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164921. GenBank no.:;OQ446910 (matK); OQ446911 (rbcL); OQ446912 (trnH-psbA).

SOUTH AFRICA • Western Cape, Pniel; 33° 53 ‘ 60”S, 18° 56 ‘ 52”E; Silica dried; 4 Feb 2020; Kritzinger-Klopper 17; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164911. GenBank no.:;OQ446907 (matK); OQ446908 (rbcL); OQ446909 (trnH-psbA).

SOUTH AFRICA • Western Cape, Pniel; 33° 53 ‘ 60”S, 18° 56 ‘ 52”E; Silica dried; 4 Feb 2020; Kritzinger-Klopper 18; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164903. GenBank no.:;OQ446904 (matK); OQ446905 (rbcL); OQ446906 (trnH-psbA).

SOUTH AFRICA • Western Cape, Caledon; 34° 13 ‘ 5”S, 19° 24 ‘ 12”E; Herbarium specimen, Silica dried; 9 Feb 2020; Kritzinger-Klopper 19; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164890. GenBank no.:;OQ446901 (matK); OQ446902 (rbcL); OQ446903 (trnH-psbA).

SOUTH AFRICA • Western Cape, Caledon; 34° 13 ‘ 5”S, 19° 24 ‘ 12”E; Silica dried; 9 Feb 2020; Kritzinger-Klopper 20; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164882. GenBank no.:;OQ446898 (matK); OQ446899 (rbcL); OQ446900 (trnH-psbA).

SOUTH AFRICA • Western Cape, Caledon; 34° 13 ‘ 5”S, 19° 24 ‘ 12”E; Silica dried; 9 Feb 2020; Kritzinger-Klopper 21; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164874. GenBank no.:;OQ446991 (matK); OQ446992 (rbcL); OQ446993 (trnH-psbA).

SOUTH AFRICA • Western Cape, Karwyderskraal; 34° 20 ‘ 56”S, 19° 11 ‘ 36”E; Herbarium specimen, Silica dried; 17 Feb 2020; Kritzinger-Klopper 22; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164866. GenBank no.:;OQ446895 (matK); OQ446896 (rbcL); OQ446897 (trnH-psbA).

SOUTH AFRICA • Western Cape, Karwyderskraal; 34° 20 ‘ 56”S, 19° 11 ‘ 36”E; Silica dried; 17 Feb 2020; Kritzinger-Klopper 23; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164858. GenBank no.:;OQ446892 (matK); OQ446893 (rbcL); OQ446894 (trnH-psbA).

SOUTH AFRICA • Western Cape, Karwyderskraal; 34° 20 ‘ 56”S, 19° 11 ‘ 36”E; Silica dried; 17 Feb 2020; Kritzinger-Klopper 24; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6164841. GenBank no.:;OQ446889 (matK); OQ446890 (rbcL); OQ446891 (trnH-psbA).

SOUTH AFRICA • Western Cape, Cape Winelands; 33° 32 ‘ 24”S, 19° 12 ‘ 36”E; Herbarium specimen; 5 Jul 2021; Kritzinger-Klopper 25; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6161622. GenBank no.:;OQ447105 (matK); OQ447106 (rbcL); OQ447107 (trnH-psbA).

SOUTH AFRICA • Western Cape, Overberg; 34° 13 ‘ 12”S, 19° 23 ‘ 60”E; Herbarium specimen; 29 Jul 2021; Kritzinger-Klopper 26; S. Kritzinger-Klopper det.; WAGPD;NPPO-NL ref. no. 6161657. GenBank no.:;OQ447108 (matK); OQ447109 (rbcL); OQ447110 (trnH-psbA).