21st Australasian Weeds Conference Identification of eight Panicum species in Riverina region of NSW using DNA sequence analysis Yuchi Chen1,2, Xiaocheng Zhu2, David E. Albrecht3, Panayiotis Loukopoulos1,2, Jane C. Quinn1,2 and Leslie A. Weston2,4 1 School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia 2 Graham Centre for Agricultural Innovation; Charles Sturt University and NSW Department of Primary Industries, Wagga Wagga, New South Wales 2650, Australia 3 Australian National Herbarium, Centre for Australian National Biodiversity Research, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia 4 School of Agriculture and Wine Science, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia ([email protected]) Summary Australia has over 30 Panicum spp. followed, up to 500 species are recognised (Byng (panic grass) including several non-native species 2014). Major photosynthetic types (C3, C4, and C3/C4 that cause crop and pasture loss due to competi- intermediate) reported in the Poaceae are represented tion. To develop appropriate management strategies in the genus (Zuloaga et al. 2010). for each species, it is critical to correctly identify Panicum ssp. are widely distributed in Australia panic grass species encountered. Currently, panic (Figure 1) with 24 indigenous and 9 non-native species grass identification relies on microscopic examina- currently recognised (CHAH 2005 onwards). They tion of the inflorescence and spikelets, an approach are currently ranked as one of the most economically that is only useful for flowering specimens and re- important weeds of summer fallows in Australia quires significant taxonomic expertise. To overcome (Llewellyn et al. 2016). Their toxic profile in livestock this limitation, we applied both morphological and is suggested to relate to the presence of saponins or molecular techniques for identification of Panicum sapogenins (Miles et al. 1992). However, variable spp. in the Riverina region of New South Wales. We steroidal saponin profiles may exist in different identified three molecular markers: one nuclear gene Panicum spp., and could be associated with their region (ITS) and two chloroplast gene regions (matK differential ability to cause hepatogenous or secondary and trnL intron-trnF) capable of differentiating eight photosensitisation in livestock (Quinn et al. 2014). Panicum spp. Concatenation of sequences from ITS, Panicum is a taxonomically challenging group due matK and trnL intron-trnF gene regions provided clear to the wide range of variation in both morphological separation of eight species collected regionally and and physiological features (Zuloaga et al. 2010). identified a maximum intraspecific distance of 0.22% Subtle morphological differences required for species and minimum interspecific distance of 0.33%. Based differentiation have not been well documented (Zu- on comparison with verified voucher specimens, P. loaga et al. 2010). Existing identification keys to hillmanii Chase was prevalent and constituted 78.9% Panicum spp. in Australia are based primarily on of all samples collected and identified. DNA barcoding reproductive features and therefore are only effective represents an accurate and potentially cost effective for plants in their reproductive phase (Coissac et al. tool for distinguishing Panicum spp. at the species 2016). Furthermore, keys for identification can be level regardless of growth stage. Molecular markers difficult to use with confidence and even experienced may also be useful for accurate demographic analysis taxonomists find Panicum identification problematic of Panicum grass invasion in Australia and abroad. at the species level. Therefore, establishment of robust Keywords Panicum spp., Panicum hillmanii genotyping methods are desirable for unequivocal Chase, DNA barcoding, matK, trnL intron-trnF, ITS, plant species identification. Identification of Panicum phylogenetic tree. at an individual plant level may also be critical for development of targeted management strategies for INTRODUCTION invasive and potentially toxic Panicum spp. (Quinn The genus Panicum is one of the largest genera in et al. 2014). the Poaceae, and is globally distributed (Aliscioni DNA barcoding, a simplified DNA-based species et al. 2003). Depending on the taxonomic concepts identification approach (Hebert et al. 2003), has been 312 21st Australasian Weeds Conference Figure 1. Distribution of Panicum spp. in Australia based on herbarium specimens represented in major Australian herbaria. Collection data extracted from The Australasian Virtual Herbarium. utilised as an important and complementary method Panicum plants dispersed across multiple sites within to traditional morphological identification (Zhu et al. a 200 km radius range of Wagga Wagga, NSW (Figure 2014). DNA barcoding uses standardised DNA regions 2). Voucher specimens were lodged at the Australian (‘barcodes’) to identify different species (CBOL Plant National Herbarium (ANH). Fresh leaf samples were Working Group 2009). However, the optimal suite stored in silica gel prior to extraction. of barcoding loci for Panicum spp. identification in To supplement the field-collected materials, an Australia has not yet been established. Following on additional 19 dried leaf samples, representing seven from Hollingsworth et al. 2011, one nuclear locus Panicum spp., were sampled from specimens within (ITS) and two chloroplast loci (matK and trnL intron- the ANH collection. A total of 36 samples, representing trnF) were evaluated in this study for their ability eight species found in Riverina, were selected for to differentiate eight Panicum spp. occurring in the further analysis in this study (Table 1). Riverina region of New South Wales. DNA extraction, PCR and sequencing were per- formed as described (Zhu et al. 2014) using primer MATERIALS AND METHODS pairs ITS5A-ITSR, 390F-1326R and ucp_c-ucp_f for In early 2017, fresh leaf samples and accompanying ITS, matK and trnL intron-trnF regions, respectively. herbarium voucher specimens were collected from 90 Geneious version 11.0.5 (Kearse et al. 2012) was used 313 21st Australasian Weeds Conference to read, edit and align the sequences. Sequence align- RESULTS AND DISSCUSION ments were analysed using MEGA7.0.26 (Kumar et The majority of field-collected specimens were identi- al. 2016) to calculate intra- and interspecific distances fied as P. hillmanii (78.9%, 71/90) by comparison with with the Kimura 2-parameter (K2P) model. Sequences verified voucher specimens at the ANH (Figure 2). of all loci for each Panicum specimen were concate- Other species identified included P. gilvum (10.0%, nated and phylogenetic relationships among species 9/90), P. decompositum (5.6%, 5/90), P. effusum (4.4%, were inferred by MrBayers 3.2.6 (Huelsenbeck and 4/90) and P. capillare (1.1%, 1/90). The predominance Ronquist 2001) using default settings with GTR+R of P. hillmanii, a non-native species that was previ- substitution model as suggested by JModelTest 2.1.10 ously rarely reported in the Riverina region, suggests (Darriba et al. 2012). the species has spread considerably in recent years. Therefore, further investigation in regards to its inva- Table 1. Panicum specimens used for DNA extrac- sion ecology should be conducted for the development tion and sequencing. of a more holistic management strategy. Dried Successful PCR amplification and sequencing was Fresh leaf achieved in three selected gene loci (ITS, matK and Species identification leaf (ANH) trnL intron-trnF) for all specimens. Alignments were P. capillare L. 1 2 then truncated to 641, 730, and 750 bps for ITS, matK P. decompositum R.Br. 3 5 and trnL intron-trnF, respectively. In addition, concate- nated data over all three gene loci were evaluated for P. effusum R.Br. 4 1 phylogenetic analysis. Intraspecific and interspecific P. gilvum Launert 5 0 genetic distances were calculated for the eight species P. hillmanii Chase 4 2 identified (Table 2). P. laevinode Lindl. 0 3 The efficacy of DNA barcoding depends on the P. miliaceum L. 0 3 extent of intraspecific and interspecific divergence in a P. queenslandicum Domin 0 3 selected locus or combined loci, and the ideal situation Figure 2. Location and identification of 90 field-collected Panicum spp. in a 200 km radius around Wagga Wagga, New South Wales. 314 21st Australasian Weeds Conference Table 2. Intraspecific and interspecific K2P distances (%) of eight Panicum spp. (MinInterD: Minimum interspecific distance; MaxID: Maximum intraspecific distance; UAA: trnL intron-trnF; PC: P. capillare; PD: P. decompositum; PE: P. effusum; PG: P. gilvum; PH: P. hillmanii Chase; PL: P. laevinode Lindl; PM: P. miliaceum; PQ: P. queenslandicum Domin). ITS- matK- ITS matK UAA UAA MinInterD % 0.71 0.07 0 0.33 MaxID in PC% 0.33 0.09 0 0.22 MaxID in PD% 0.03 0.06 0 0.03 MaxID in PE% 0.34 0 0 0.15 MaxID in PG% 0 0 0 0 MaxID in PH% 0 0 0 0 MaxID in PL% 0.07 0.09 0 0.06 MaxID in PM% 0 0.09 0 0.03 MaxID in PQ% 0.14 0.14 0.27 0.16 is a lack of overlap between these two distance values global Panicum spp. specimens are required to further (Aliabadian et al. 2009). For Panicum spp., ITS explore this observation. showed the highest minimum interspecific distance In conclusion, this study indicates that a genetic (0.71%), which was over 2-fold higher than the highest approach using three concatenated loci provided an maximum intraspecific distance (P. effusum, 0.34%). accurate and potentially cost effective tool for distin- This result confirmed that the ITS locus was used as an guishing Panicum spp. at the species level, regardless efficient barcode for separation of the eight Panicum of growth stage. Using this method, we confirmed that spp. in this study. Conversely, the overlap between the majority of Panicum specimens collected across intraspecific and interspecific values of either the the Riverina in 2017 were P. hillmanii. This was matK or the trnL intron-trnF regions suggests that somewhat unexpected since very few (6) herbarium use of these barcodes alone may prove problematic collections had been made in NSW prior to our study, for differentiation of some Panicum spp.