Pennisetum setaceum or Pennisetum advena cultivars, what ornamental do we have in our garden

Abstract Pennisetum Rich. or following recent taxonomic insights Cenchrus L. is a genus with some 120 species worldwide, especially in warm areas. The genus includes some crops, some ornamentals but mostly species that are considered weedy. The name of one of the weedy species Pennisetum setaceum (Forssk.) Chiov. is also found on labels of ornamental grasses as P. setaceum “Rubrum.” It has been debated to belong to a species on its own Pennisetum advena Wipff & Veldkamp or Cenchrus advena (Wipff & Veldkamp) Morrone, only known from cultivation, whereas others still adhere to a broader species concept of P. setaceum. The recent inclusion of P. setaceum on the EU List of Union concern has revitalized the discussion on this issue for commercial reasons. Based on a morphological and molecular comparison (ITS, rbcL, and the trnh‐psbA intergenic spacer sequences) of the type specimen of P. advena, five of its “cultivars” in trade and collections of P. setaceum from different regions of the world we conclude that plants currently in trade in Western Europe belong to a separate species P. advena. A drooping inflorescence is consistent as is the difference in width of the leaf blade, the leaf blade being flat or involute, the central vein being swollen or not, and the length of the stipe being 0.3–1.1 mm in P. advena and 1.1–3.1 mm in P. setaceum. On the chloroplast markers rbcL and trnH‐psbA, the species consistently differ in 2 and 4 base pairs, respectively. On the nuclear ITS sequence, there is only 90% overlap between the two species. This justifies these ornamentals to be excluded from the List of Union concern of EU regulation 1143/2014.

It has been debated that these cultivars belong to a species of its own, Pennisetum advena Wipff and Veldkamp (1999) or Cenchrus advena (Wipff & Veldkamp, 1999) Morrone (Chemisquy et al., 2010;Veldkamp, 2014), only known from cultivation. However, others still adhere to a broader species concept of Pennisetum setaceum (Groom et al., 2017). Finally, there are those that interpret a discussion on relatedness as a proxy for a hybrid status (Meyer, 2012;Padhye et al., 2008). The recent inclusion of P. setaceum (Figure 1) on the List of Union concern of EU regulation 1143/2014 has revitalized the discussion on this issue for commercial reasons (see, e.g., the recommendation by Val'hor, 2017). Listing of a species on the List of Union concern implies that all its lower taxa or hybrids are potentially subject to the same rules and prohibitions, thus resulting in an effective ban on sale of these popular cultivars traded under the name of either P. setaceum or P. advena (Figure 2).
Provisional molecular results pointing to a distinction in the aforementioned cultivars (Anonymous, 2017) have resulted in these taxa being provisionally excluded from the ban, while a more detailed morphological and molecular study is conducted. Results of this study are presented here.

| Acquisitions of plants
For a study of Pennisetum species and cultivars in trade in the Netherlands, a large number of living plants was acquired from commercial growers and garden centers and subsequently grown in a quarantine glasshouse (Costerus, 2018;Costerus & van Valkenburg, 2018). In addition, a total of 168 herbarium specimen from the National Herbarium at Leiden belonging to 10 Pennisetum species were consulted for descriptive purposes.

| Macromorphological approach
Of all living material height of the plant including inflorescence was measured, color of the stem, width, length and color of leaves; color and length of inflorescence; length of spikelet; texture of axis of inflorescence; length of stipe; number of spikelets per fascicle; and color, length and presence of long hairs on bristles. For all herbarium specimens, macromorphological measurements were similar.
All measurements are used to build an interactive image-driven key using LUCID software (Identic, Stafford Heights, Australia).

| DNA extractions
Genomic DNA was isolated from approximately 100 mg plant material with the DNeasy Plant Mini Kit (Qiagen, Venlo, the Netherlands) using the TissueLyser procedure and eluted with 50 μl prewarmed (65°C) AE buffer. DNA was stored at −20°C until use.

| PCR amplification and Sanger sequencing
PCRs for the chloroplast rbcL gene and trnH-psbA intergenic spacer and nuclear ITS (partial 18S, ITS1, 5.8S, ITS2, partial 28S) loci were performed in 25 μl reaction mixes containing 200 nmol/L of either primers rbcL-a F and rbcLa SI_Rev, trnH2, and psbAF or ITS5 and ITS4 (Table 2) the Netherlands) and sequenced using a 3500 Genetic Analyzer (Thermo Fisher Scientific, Bleiswijk, the Netherlands). Consensus sequences were generated from an assembly with trace files from both Sanger sequencing runs in Geneious R10 (Biomatters Auckland, New Zealand). Amplification primer sequences were trimmed in the assembly, and when needed, additional trimming was performed to obtain high-quality (PHRED >30) consensus sequences.

| Macromorphological differences observed
The analysis of all living material and the herbarium collections of the wild samples and cultivars belonging to the 10 species can be found in Costerus (2018) and Costerus and van Valkenburg (2018). It has been reworked to an interactive image-driven identification key using LUCID software and can be found at https://keys.lucid centr al.org/keys/v3/penni setum/ en/index.html.
For this paper, we focus on the distinguishing characters between P. advena and P. setaceum. Most obvious morphological characters when judging its ornamental value are the culm of P. setaceum being rarely branched as opposed to P. advena and the inflorescence of the latter being flexuous and drooping as opposed to rigid in P. setaceum. Both characters are added to the attractiveness of the species. Leaf characters in addition to the color of P. setaceum never being purplish are the consistently larger width and flatness of the blade in P. advena as well as the more prominent thickened central vein and involute leaf in P. setaceum. These character differences also apply to the ornamental P. setaceum collected in New Zealand. More detailed measurements on the inflorescence and leaves of P. setaceum. P. advena and cultivars are given in Table 3 with the values as published for both species (Veldkamp, 2014) for comparison.

| Sequence analysis
The results of the sequence analysis for the chloroplast rbcL gene and trnH-psbA intergenic spacer as well as for the nuclear ITS (partial 18S, ITS1, 5.8S, ITS2, partial 28S) sequence are summarized in Sanger sequencing of the ITS region revealed the presence of SNPs in both P. advena (n = 3) and P. setaceum (n = 2) specimens.
Illumina sequencing revealed that in each of the specimens, two variants of the ITS sequence were present. For P. advena, the ratio between these two variants was for each specimen approximately 55% for variant aI and 45% for variant aII. For P. setaceum sample "Simons 2006," the ratio between variants sI and sII was approximately 70% and 30%, while for P. setaceum sample "Mooney 9419," this was the other way around (30% sI and 70% sII). Nevertheless, P. advena and P. setaceum could be distinguished based on their ITS sequences as they are only 90% identical. The cultivars "Fireworks," "Rubrum," and "Summer Samba" contained both variants I and II of the P. advena ITS sequences. Based on all three loci (rbcL, trnH-psbA, and ITS), these three cultivars could therefore be matched to P. advena.
The ITS sequences of the cultivars "Cherry Sparkler" and "Sky Rocket" could not be obtained by Sanger sequencing. Illumina sequencing of these cultivars revealed that both cultivars contained ITS variants I and II of P. advena (aI and aII) and variant I of P. setaceum (sI). For "Cherry Sparkler," the ratio between the different variants was approx. 45% (aI), 15% (aII), and 40% (sI) and for "Sky Rocket" 20% (aI), 50% (aII), and 30% (sI). European (Verloove, 2012;Verloove et al., 2014), Southeast Asian (Veldkamp, 2014), Australian (Symon, 2010), and Pacific species (Tornabene & Wagner, 2013). However, as the ornamental plant trade is rather conservative, we have retained the old Pennisetum names for P. advena and P. setaceum and the other common ornamentals P. orientale and especially P. alopecuroides both represented by numerous cultivars in trade. Likewise, the name Pennisetum has been retained on the EU List of Union concern based on the risk assessment by Danas and Verloove (2015).

TA B L E 3
Although the exact origin of the ornamental plants commonly referred as P. setaceum "Rubrum" is unclear, apart from it being Old World, its introduction in the United States in 1916 is well documented as well as the quest for a valid name for the species. For an elaborate discussion, see Wipff and Veldkamp (1999).
Confusion on hybrid status goes back to Padhye et al. (2008) who somehow misinterpreted the Wipff and Veldkamp (1999) discussion on the origins of P. advena. This misinterpretation was later repeated by Meyer (2012) and has been adopted by numerous people who apparently failed to verify this in the original publication by Wipff and Veldkamp (1999). This view has been expressed already by Melanie Schori from USDA (pers. comm. 2017) in response to questions raised by European umbrella organizations of plant growers to the Community Plant Variety Office, following listing of P. setaceum on the List of Union concern.
Descriptions of some of the patented cultivars such as "Fireworks," "Cherry Sparkler," and "Sky Rocket" can be found online Distinguishing morphological characters for P. advena have been elaborately discussed by Wipff and Veldkamp (1999). Some From an European perspective, a difference in potential risk of invasiveness of P. advena compared with P. setaceum would be a major criterion to justify it not to be regulated. Simpson and Bashaw (1969) showed that P. advena (purple P. setaceum) rarely sets seed under field conditions and does not behave as an apomictic species To obtain more resolution for molecular identification, the sequence of the nuclear ITS locus was used. Interestingly, both P. advena and P. setaceum each contained two variants of the ITS sequences with 2 and 3 SNP, respectively. Most likely these variants are caused by their polyploid nature, as P. setaceum for instance is known to be hexaploid (Martel et al., 2004), although plants in general may contain multiple copies of ITS (Feliner & Rosselló, 2007). The ratio between the two variants was not the same in each of the specimens, but this could be caused by a sequence artifact or an uneven distribution of the variants among the different chromosome copies.
Using the ITS sequence as a proxy for species hybridization has been successfully used for the identification of Impatiens hybrids (van Valkenburg et al., 2019). The cultivars "Cherry Sparkler" and "Sky Rocket" contained the ITS sequences from both P. advena and P. setaceum, suggesting that these specimens have a hybrid status.
Interesting, they contain both P. advena ITS variants, but only one of the P. setaceum variants. This points to a hybrid origin, with P. advena being the seed plant.

| CON CLUS ION
Based on molecular and morphological characters, both species can be clearly separated and the popular ornamentals in trade in Europe belong to P. advena. This justifies these ornamentals to be excluded from the List of Union concern of EU regulation 1143/2014.