Botany DNA barcoding of a complex genus, Aesculus L. (Sapindaceae) reveals lack of species-level resolution Journal: Botany Manuscript ID cjb-2019-0018.R2 Manuscript Type: Article Date Submitted by the 10-May-2019 Author: Complete List of Authors: Aygoren Uluer, Deniz; Ahi Evran Universitesi, Cicekdagi Vocational College, Department of Plant and Animal Production; Alshamrani, Rahma; King Abdulaziz University, Department of Biological Sciences Draft Keyword: Aesculus, DNA barcoding, ITS/ITS2, matK, phylogeny Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 28 Botany DNA barcoding of a complex genus, Aesculus L. (Sapindaceae) reveals lack of species-level resolution Deniz Aygoren Uluer1,3, Rahma Alshamrani 2 1 Ahi Evran University, Cicekdagi Vocational College, Department of Plant and Animal Production, Boyalık Mahallesi, Stadyum Caddesi, Turan Sok. No:18 40700 Cicekdagi, Kirşehir, Turkey. [email protected] 2 King Abdulaziz University, Department of Biological Sciences, PO Box 80206, 21589, Jeddah, Saudi Arabia, email: [email protected]. Draft 3Author for correspondence: Deniz Aygoren Uluer, Ahi Evran University, Cicekdagi Vocational College, Department of Plant and Animal Production, Boyalık Mahallesi, Stadyum Caddesi, Turan Sok. No:18 40700 Cicekdagi, Kirşehir, Turkey, email: [email protected], Work phone: +903862805500, Fax: +903862805528. 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 28 Abstract Aesculus L. is a small genus of horticulturally important trees and shrubs, comprising 13-19 species. Frequent hybridization among species, particularly in cultivation, has contributed to taxonomic confusion and difficulties in identification of plants. In this study, we evaluated three widely employed plant DNA barcode loci, matK, and the entire ITS region (ITS1+5.8S+ITS2) as well as subunit ITS2 for 50 individuals representing 13 species of Aesculus, excluding only A. wangii (=A. assamica). In contrast to the plastid matK region, both the ITS and ITS2 loci displayed low levels of species discrimination, especially in our “first hit” BLASTn searches. We also presented the phylogeny of Aesculus based on matK and the entire ITS region, with additional matK and ITS sequences from GenBank. Our results show that Aesculus chinensis, A. flava, A. glabra, A. pavia and A. sylvatica are probably not monophyletic.Draft Furthermore, with the widest taxon coverage until now, the current study highlights the importance of sampling multiple individuals, not only for DNA barcoding, but also for phylogenetic studies. Keywords: Aesculus, DNA barcoding, ITS/ITS2, matK, phylogeny Introduction Aesculus L. (Sapindaceae) is a small, but taxonomically difficult genus comprising 13-19 species of horticulturally important deciduous shrubs and trees. The genus is noticed with its palmately compound leaves, large and showy inflorescences and poisonous capsule fruits (buckeyes or horse- chesnuts) (Turland and Xia 2005; Zhang et al. 2010). Aesculus is distributed in North America, south- eastern Europe and Asia (Turland and Xia 2005). Frequent hybridization among species, particularly in cultivation, has contributed to taxonomic confusion and difficulties in identification of plants. Aesculus is traditionally divided into five sections: Aesculus, Calothyrsus, Macrothyrsus, Parryana and Pavia, according to flower colour, petals, bud viscidity, fruit exocarp morphology and geographic 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 28 Botany distribution (Hardin 1957; Hardin 1960). However, previous studies have shown that, these five traditionally accepted sections of Aesculus as well as species boundaries are problematic; the phylogenetic relationships of these sections change from one study to another, and recognition of some recently suggested species such as, A. assamica Griff. (=A. wangii Hu) and A. tsiangii Hu&Wang are still doubtful (Hardin 1957; Hardin 1960; Turland and Xia 2005; Xiang et al. 1998; Forest et al. 2001; Harris et al. 2009; Harris et al. 2016). DNA barcoding is a tool for quick identification of organisms by the use of a short gene sequence from short standardized gene region(s) and comparison with a barcode library (Hebert et al. 2003a, b). DNA barcoding, as a fast and inexpensive method, can be used where morphological identification is not possible, such as in the food industry, herbal medicines, conservation, forensic investigations, biodiversity inventories, animal diet, sterile material, seeds and seedlings (Cowan et al. 2006; Taberlet et al. 2006; Barcaccia etDraft al. 2015; Larranaga and Hormaza 2015; Ivanova et al. 2016). It may also help to identify new species in taxonomy (Hebert et al. 2004; Hajibabae et al. 2007). In this study, we explored the utility of DNA barcoding of Aesculus, since identification of Aesculus species heavily depends on floral and fruit characters, in which DNA barcoding could permit identification of these sterile materials. Therefore, we evaluated the use of one chloroplast coding region, namely matK, nuclear internal transcribed spacer (ITS) region and subunit ITS2 singly and in combination in 50 individuals of 13 Aesculus species. Intron Group II maturase matK, is one of the most rapidly evolving coding regions in plants (CBOL 2009), and many studies have reported its successful use (e.g., Clerc-Blain et al. 2010; Seberg and Petersen 2009) from family to even species level (Dong et al. 2012). However, not having universal primers, reported PCR problems and less discrimination power in some groups are the most important drawbacks of this region (Chase et al. 2007; Newmaster et al. 2008; CBOL 2009; Barcaccia et al. 2015). 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 28 In plants, the ITS region has been used in plant systematic studies frequently, especially in low-level taxonomic studies (Baldwin 1992; Baldwin et al. 1995; Sonnante et al. 2003). The success of this locus as a DNA barcode has also been reported by many studies (e.g., Ellison et al. 2006; Clement and Donoghue 2012) due to its ease of amplification and rapidly evolving nature (Baldwin et al. 1995; Baker et al. 2000; Kress and Erickson 2007; Huang et al. 2015). However, gene conversions, concerted evolution, requirement of different PCR conditions and additives, fungal contamination, cloning requirement in some cases and not being amplifiable with universal primers are some reasons that limit the use of this nuclear region as a DNA barcode (Alvarez and Wendel 2003; Wendel et al. 1995; Kress et al. 2005; Cowan et al. 2006; Chase et al. 2007). Therefore, CBOL (2009) regarded this region as a supplementary barcode. While the multiple copy problem still exists (Bailey et al. 2003), ITS2, on the other hand, as one of the two very variable internal transcribed spacers of the ITS region, has been reported as a novelDraft barcode across land plants by many studies, with a high level of discrimination rate with universal primers and ease of amplification compared to ITS, even with herbarium specimens or in challenging environments (Chen et al. 2010; Yao et al. 2010; Li et al. 2011; Xu et al. 2015; Braukmann et al. 2017; Ramalho et al. 2018). We evaluated both the entire ITS region (ITS1+5.8S+ITS2) and ITS2 alone as a DNA barcode for Aesculus, due to the ease of amplification and sequencing of the ITS2 region with available universal primers, the cases where obtaining the entire ITS locus is problematic (Li et al. 2011). Over the past years, several studies have assessed the phylogenetic relationships of Aesculus using morphological characters and markers. However, none of these studies have yielded a species-rich phylogeny of Aesculus, which may indicate monophyly problems of taxonomic groups. Therefore, the second aim of the current paper is to present the phylogeny of Aesculus based on these two DNA regions, namely matK and ITS. Materials and methods 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 28 Botany Taxon sampling, DNA extraction, amplification and sequencing Fieldwork in this study was conducted from 2012 to 2014. Specimens representing Aesculus were collected from the University of Reading Whiteknights campus, as well as from the Royal Horticultural Society Garden at Wisley. All the trees were previously identified in the field using morphological characters (i.e. available reproductive or vegetative characters) based on Sell and Murrell’s (2009) and Stace’s (2010) identification keys. Furthermore, we compared our specimens with the dry specimens at the Royal Botanic Gardens, Kew Herbarium and Royal Horticultural Society Garden at Wisley Herbarium. All corresponding voucher samples are curated in the King Abdulaziz University Herbarium. The genomic DNA was extracted from freshDraft material using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions and were held at the University of Reading herbarium. Except for Aesculus assamica (=A. wangii), all species of Aesculus were included to the current study. Polymerase chain reactions were performed in 25 µL reaction volumes, with 12,5 µL of Biomix (Bioline, 2x), 2 µL bovine serum albumin (Sigma-Aldrich, 10 mg/mL), 0.875 µL of each primer (20mM), and 1 µL of template DNA and made up to 25 µL with distilled water. For the nuclear regions, the DNA templates were diluted to 1:50. For reactions amplifying ITS2, if the first PCR attempt was unsuccessful, 1 µL of dimethyl sulfoxide (DMSO) was added to reduce the effect of secondary structures and increase efficiency of PCR. We followed Harris et al. (2009) and used the primers which are shown in Table 1. The PCR profiles for the matK and the ITS (ITS 1, ITS2 and the 5.8s ribosomal gene) regions were also shown in Table 1. All amplifications were performed on a GeneAmp PCR system 2700 (Applied Biosystems). Products were sent to Source BioScience (Nottingham, UK) for purification and sequencing. 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 28 The matK data matrix comprised 46 sequences, the entire ITS and ITS2 matrices contained 44 sequences and ITS+matK and ITS2+matK matrices contained 50 samples.