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.
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. The National Center for
Biotechnology Information (NCBI/GenBank) accession numbers for these newly produced DNA sequences are provided in Supplementary data1.
Sequence editing, alignment and phylogenetic analyses
Sequences were assembled and aligned using the Geneious alignment option in Geneious Pro 4.8.4
(Kearse et al. 2012) with the automatic pairwise alignment tool and subsequently edited manually.
Equivocal base calling at the beginning and end of assembled complementary strands were trimmed. All indels were scored as missing data. WeDraft also extracted the ITS2 region from the entire ITS alignment to see whether this 195 bp region is as useful as the entire ITS region.
The substitution models for each of the individual genes were estimated using jModelTest 2.1.10
(Guindon and Gascuel 2003; Darriba et al. 2012).
ML analyses were performed using RAxML version 7.0.4 as implemented on http://embnet.vital- it.ch/raxml-bb/web-server (Stamatakis et al. 2008). The GTRGAMMA model was applied to each partition individually and default maximum likelihood search options were selected with 100 bootstrap replicates. The best scoring trees with bootstrap values were saved. We used a cutoff of
50% to define support for “successful” resolution of monophyletic species.
For the phylogeny analyses, 40 each of matK, entire ITS and ITS2 sequences of Aesculus and outgroups were obtained from GenBank; accession numbers for these previously published DNA
1 Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjb- 2019-0018.
6 https://mc06.manuscriptcentral.com/botany-pubs Page 7 of 28 Botany
sequences are also provided in Supplementary data. In some cases, ITS1, 5.8S and ITS2 sequences
were downloaded separately and concatenated. GenBank accession numbers for these sequences
are also provided in Supplementary data. To reduce the problems associated with LBA (Felsenstein
1978) by breaking long branches between the ingroup and outgroup, we chose our 10 outgroup taxa
to root the ingroup representing the remaining four genera of the Hippocastanoideae subfamily,
other than Aesculus. Other than outgroup sequences, we used the same sample (i.e., same voucher
numbered sample) for our matK, ITS and ITS2 phylogenetic analyses. We also sequenced matK and
ITS loci for Billia hippocastanum Peyr. and deposited them in Genbank.
BLAST analysis Draft Since similarity-based approaches such as BLASTn are the most commonly used for DNA barcode
data analysis (Huang et al. 2015), all barcodes were tested singly and in combination by running a
BLASTn search in the GenBank global database to evaluate the taxonomic resolution. We used a
cutoff of 98% species identity for the BLASTn similarity approach. Additionally, we ‘BLASTed’ our
sequences again to see whether the first hit on the BLASTn results represented the correct species
names (i.e., unambiguous identification) (Vences et al. 2005; Brock et al. 2009).
Results
DNA barcoding of Aesculus
In total, we obtained 46 matK and 44 ITS sequences from tissue samples of 50 specimens,
representing 11 and 12 species, respectively (Table 2). While the outgroup-excluded alignment of
7 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 8 of 28
matK was 1,726 bp long, outgroup-excluded alignments of ITS and ITS2 were 605 and 195 bp, respectively. Of these, 94 characters were variable for both matK and ITS, while 36 were variable for the short ITS2 region. In terms of parsimony-informative characters, compared to the region’s length, ITS2 showed the highest percentage (12.8%), followed by ITS (11.7%) and matK (2.6%), revealing matK to have the lowest phylogenetic informativeness despite its length (1,726 bp).
However, between matK and ITS, matK showed the highest PCR and sequencing success (Table 2).
On the other hand, the entire ITS+matK had 187 variable characters, of which 115 were parsimony- informative. For the ITS2+matK region, among 132 total variable characters, 71 were parsimony- informative. All these alignment details for all datasets are summarized in Table 2.
The length of ITS is well suited for DNA barcoding (605 bp). In terms of primer pairs used, ITS required fewer primer pairs (i.e., three primerDraft pairs) compared to the matK region (i.e., four primer pairs). None of the loci could be successfully amplified for all taxa; while PCR and sequencing success for matK was 92%, it was 88% for the ITS region (Please note that we were able to sequence all PCR products successfully).
BLAST delivered a very high species resolution for matK, ITS2 and matK+ITS2 (98% to 100%) (Table
3). However, of the single barcodes (ITS, ITS2 and matK), most of our Aesculus ITS sequences were misidentified, even if the “discontinuous megablast” and “blastn” options were used instead of the
“megablast” option. Therefore, we did not include our ITS BLASTn search results in Table 3. On the other hand, ITS2 had a slightly better discriminatory power compared to matK. Combining matK with
ITS2 did not improve the BLASTn search results in general (Table 3). In contrast to the high species resolution percentages for matK, ITS2 and matK+ITS2, when we BLASTed our ITS2 sequences, the
“first hit” referred as the unambiguous identification for only four of the Aesculus species (A. californica, A. indica, A. parryi and A. parviflora) (Table 3). There were five species for the matK region (A. californica, A. indica, A. parryi, A. parviflora and A. tsiangii). Furthermore, for four
Aesculus species (A. turbinata, A. pavia, most of A. sylvatica and some of A. flava), we correctly
8 https://mc06.manuscriptcentral.com/botany-pubs Page 9 of 28 Botany
identified the species almost always in the “first hit”. Combining matK with ITS2 did not improve the
the “first hit” BLASTn search results.
Our ML results have shown that, while there was no difference between matK, ITS and ITS2 in terms
of retrieving monophyletic species (five for each region), genus Aesculus was monophyletic only in
the matK analysis (Table 3). However, combining ITS and ITS2 with matK definitely resulted in better
resolution, with eight monophyletic species for both regions.
Relationships within Aesculus
In this section, the results of the ITS2 analysesDraft are excluded. Only bootstrap support values above 50% are discussed.
The outgroup-included alignment of matK was 1,726 bp long; alignments of ITS and ITS2 were 657
and 209 bp, respectively. While Aesculus was found to be monophyletic in most of the analyses (77-
88%), three Billia Peyr. samples plus Handeliodendron bodinieri (H. Lév.) Rehder were grouped
within Aesculus, in the ITS tree. In general, among 13 Aesculus species, only A. californica Nutt. (84
to 100%), A. parviflora Walter (95 to 100%) and A. parryi A. Gray (100%) were found to be
monophyletic in all analyses (Table 4).
Species borders within section Pavia and the situation of A. californica and A. chinensis also seemed
to be problematic in all analyses (Figure 1, Table 3 and 4). However, there was noteworthy close
relationship between section Parryana and section Pavia (note that section Parryana was embedded
in the section Pavia in the matK tree). Section Macrothyrsus was monophyletic in all analyses.
Section Aesculus was monophyletic in ITS and matK+ITS analyses (Figure 1); however, one of the A.
chinensis samples was embedded in section Aesculus in the matK tree. Section Calothyrsus was not
9 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 10 of 28
monophyletic in any of the analyses, because, either all A. californica samples or one of the A. chinensis samples were embedded in other sections.
Discussion
DNA barcoding of Aesculus
Identifying phylogenetically problematic and horticulturally important Aesculus species using short
DNA sequences would be remarkably practical, especially for sterile Aesculus samples. Indeed, the genus proved to be a challenging phylogeny and barcoding test case, due to the low levels of DNA sequence variation. Our data showed that morphological species borders do not correlate with the phylogeny of Aesculus, particularly withinDraft section Pavia. Ancient-rapid radiation of the genus, which involves short internal branches (Harris et al. 2009), frequent hybridization, reticulation and continuous gene flow, gene duplication, polyploidy, long generation times and possible polyphyletic taxa could be the reason (Yan et al. 2015; Zarrei et al. 2015).
Unfortunately, our DNA barcoding study showed that both ITS and ITS2 loci have advantages with respect to length (i.e., relatively short as a DNA barcode), being amplifiable with universal primers
(note that we did not test this for the ITS2 locus) and retrieving monophyletic species in our ML trees; neither the ITS nor ITS2 loci yielded unambiguous species discrimination in most of our
BLASTn “first hit” searches (Table 3). Furthermore, most of our Aesculus ITS sequences were misidentified in our BLASTn searches when we used a cutoff of 98% species identity. However, like
Harris et al. (2009), our ITS sequencing results did not show significant double peaks in chromatograms; this would not rule out the possibility of paralogous copies and/or pseudogenes due to gene duplication and incomplete concerted evolution (Álvarez and Wendel 2003; Pelser et al.
2010). Furthermore, when employing either ITS or ITS2 regions as a DNA barcode for Aesculus,
10 https://mc06.manuscriptcentral.com/botany-pubs Page 11 of 28 Botany
extensive cloning of PCR products may be necessary, which is not a desired feature of a DNA
barcode. Further work is required in order to clarify this point. On the other hand, verified voucher
numbers and possible unreliable specimen identifications are one of the major drawbacks of not
only DNA barcoding (Kress and Erickson 2007; Deichmann et al. 2017) but also other botanical
studies (Eisenman et al. 2012; Culley 2013). Additionally, the amount of inaccurate identifications in
GenBank records could also be extremely high (e.g. Nilsson et al. 2006). Nevertheless, we certainly
believe that this was not the case for our study. First, after a detailed identification step using the
appropriate identification keys (Sell and Murrell 2009; Stace 2010), we compared our specimens
with several dry specimens deposited at the Royal Botanic Gardens, Kew and the Royal Horticultural
Society Garden at Wisley Herbariums. Second, almost all ITS sequences that we included in our study
were identified, sequenced and deposited to GenBank by Aesculus experts (i.e. Harris et al. 2009). Thus, we deduced that incorrect identificationDraft was not the cause for these mismatches. The rbcL+matK combination has been adopted as a standard DNA barcode for plants (CBOL 2009).
However, unsatisfactory results from closely related groups, especially woody plant groups, because
of their long generation times, have been reported (Clement and Donoghue 2012). From these
results, researchers concluded that standard DNA barcodes are not useful in every case, and
addition or substitution of other regions (e.g., ITS, ITS2, trnH-psbA) is necessary (e.g., Huang et al.
2015; Braukmann et al. 2017). In the case of Aesculus, our preliminary results have shown that
sequencing the rbcL coding region would be both time and money-consuming. However, in contrast
to having fewer parsimony-informative characters (2.6%) compared to ITS (11.7%) and ITS2 (12.8%),
and requiring more primer pairs compared to the ITS region, the matK region did well in both our
BLASTn and ML analyses (Table 3). In contrast to previous studies, which reported its problematic
performance in broad floristic experiments, the matK region also displayed the highest PCR and
sequencing success (92%), when compared to the ITS nuclear region (88%) (Tables 2 and 3).
Furthermore, the matK region was mostly successful in yielding the correct species identification,
other than for all A. glabra samples, almost all A. hippocastanum samples, some A. flava samples,
11 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 12 of 28
one A. sylvatica sample and one A. turbinata sample (note that we could not sequence A. californica and A. parryi for this region) (Table 3). Interestingly, our “first hit” searches for A. flava for example, resulted in A. pavia, while our A. glabra searches resulted in either A. pavia or A. flava, in which the monophyly of the above-mentioned species was also problematic in our ML analyses. On the other hand, while combining matK with ITS or ITS2 clearly improved results in terms of phylogenetic analyses, it did not improve the results of BLASTn searches.
In summary, even our preliminary results (unpublished data) have shown that sequencing the rbcL coding region would be both time and money-consuming; nevertheless, we recommend using at least the matK region as a DNA barcode for Aesculus. In the case of Aesculus hybrids, however, adding ITS or ITS2 may be helpful, because compared to nuclear genes, chloroplast loci do not present many problems like multiple copyDraft and fungal contamination; yet using only chloroplast loci cannot help to detect the paternal parent (Kress et al. 2005; Cowan et al. 2006). Therefore, comparing the ITS/ITS2 phylogeny with the chloroplast phylogeny can identify Aesculus hybrids
(Sang et al. 1997). However, particular caution must still be exercised when working with hybrids
(note that we did not include any hybrids in the current study), because ITS can easily have paralogous sequences as a result of gene duplication and incomplete concerted evolution (Alvarez and Wendel 2003).
Phylogeny of Aesculus
Despite its popularity and horticultural importance with a complex history of hybridization ex situ, the taxonomy and systematics of Aesculus are confused. While we did not include any hybrids, our study is still the first attempt to reveal phylogenetic relationships within the genus Aesculus, with the widest taxon coverage to date. We were able to include most of the Aesculus species in our
12 https://mc06.manuscriptcentral.com/botany-pubs Page 13 of 28 Botany
phylogenetic study (in total, 91 taxa in 13 species) except for A. assamica. Even though we recovered
all five sections of Aesculus, the taxonomy of the genus is still poorly resolved, even with the highly
informative matK and ITS regions and the wider taxon coverage that we employed. However,
whether this problem is caused by incomplete lineage sorting, frequent hybridization (Jakob and
Blattner 2006; Zarrei et al. 2015), low levels of genetic variation among Aesculus species or another
potential reason is still unkown, and indicates that further work is required.
On the other hand, increasing number of species sampling has clearly showed that many Aesculus
species are not monophyletic (e.g., A. chinensis, A. flava, A. glabra, A. pavia, A. sylvatica W. Bartram)
(Table 3 and Figure 1). Indeed, the results obtained by Harris et al. (2009) and Harris et al. (2016)
also support the notion that A. chinensis (including A. chinensis var. wilsonii (Rehder) Turland & N.H.Xia), A. glabra, A. pavia and A. sylvaticaDraft may not be monophyletic species. Our study has shown that monophyly of almost half of the species of Aesculus (i.e., A. chinensis, A. flava, A. glabra, A.
pavia, A. sylvatica) is doubtful; therefore, in contrast to sparsely sampled previous studies (Table 4),
a wide species sampling is clearly necessary, not only for DNA barcoding, but also for phylogenetic
studies (Clement and Donoghue 2012). Our results have also shown the following:
1) while sections Macrothyrsus, Parryana, Pavia and Aesculus are monophyletic, the monophyly of
section Calothyrsus is doubtful. Furthermore, the phylogenetic relationships inferred by previous
studies within five sections of Aesculus are still controversial, changing from one study to another
(e.g., Hardin 1957 and 1960; Xiang et al. 1998; Forest et al. 2001; Harris et al. 2009; Harris et al.
2016) (Table 4). Therefore, we believe that, there is an urgent need for further studies on the
placement of A. californica and the phylogenetic relationships within five sections of Aesculus.
2) Monophyly of species within section Pavia should be revised using wider species sampling. This is
because our phylogenetic analyses as well as our field trips have shown that the species borders
13 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 14 of 28
among four taxa of section Pavia are not clear. For example, during our field trips, we observed A. flava samples with characteristics similar to A. sylvatica, such as plants with 18 mm petioles and +22 mm stamens. On the contrary, it is possible that frequent hybridization within these four species may indeed constitute a species complex (Chase et al. 2005). However, this needs more work to be confirmed.
3) The situation of A. chinensis should be revised using wider species sampling. Interestingly, while one A. chinensis sequence downloaded from GenBank (GenBank# AY724267.1) was grouped with A. hippocastanum samples in our matK tree, another A. chinensis (A. chinensis var. wilsonii, Alshamrani
103 (H)) was grouped within section Pavia in our ITS tree.
4) Finally, our ML analyses of ITS yielded mixedDraft results for the monophyly Aesculus; similar results were also reported by Buerki et al. (2010) and Harris et al. (2016). However, we believe that Aesculus is monophyletic, and this is also supported not only by previous studies (e.g., Harris et al. 2009;
Harris et al. 2016), but also morphological characters (e.g., Forest et al. 2001). Therefore, it only seems that species boundaries in Aesculus are especially difficult and further studies are definitely needed.
Conclusions
The current study is the first attempt to barcode genus Aesculus. Our BLASTn cutoff of 98% species identity, BLASTn first hit and ML analyses showed that matK is the most useful DNA barcode for
Aesculus. Second, our phylogenetic analyses with the largest taxon sampling to date have also shown that monophyly of many species of Aesculus is doubtful; therefore, further studies with a wide taxon coverage are definitely needed. We think that inclusion of more samples from non-
14 https://mc06.manuscriptcentral.com/botany-pubs Page 15 of 28 Botany
monophyletic species of Aesculus is particularly necessary. On the contrary, we are confident that
the inclusion of more samples from already monophyletic taxa (e.g., A. californica) would not affect
the results. For some taxa, we were able to include only a few individuals (i.e., A. parryi, A. polyneura
Hu&Fang. and A. tsiangii) or no sequences at all (i.e., A. assamica) in our study; thus, we could not
properly assess their resolvability. Future studies should definitely include samples from these taxa.
Alternatively, employing more informative (i.e., variable) nuclear or chloroplast regions or whole
chloroplast genomes would yield better resolution for the taxonomically complex genus Aesculus.
Acknowledgments
We are grateful to the anonymous reviewerDraft for helpful comments.
Reference List
Álvarez, I., and Wendel, J.F. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Mol.
Phylogenet. Evol. 29(3): 417-434.
Baldwin, B.G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA
in plants: an example from the Compositae. Mol. Phylogenet. Evol. 1(1): 3-16.
Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S., and Donoghue,
M.J. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm
phylogeny. Ann. Mo. Bot. Gard. 82(2): 247-277.
Bailey, C.D., Carr, T.G., Harris, S.A. and Hughes, C.E. 2003. Characterization of angiosperm nrDNA
polymorphism, paralogy, and pseudogenes. Mol. Phylogenet. Evol. 29(3): 435-455.
15 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 16 of 28
Baker, W.J., Hedderson, T.A., and Dransfield, J. 2000. Molecular phylogenetics of subfamily
Calamoideae (Palmae) based on nrDNA ITS and cpDNA rps16 intron sequence data. Mol. Phylogenet.
Evol. 14(2): 195-217.
Barcaccia, G., Lucchin, M., and Cassandro, M. 2015. DNA barcoding as a molecular tool to track down mislabeling and food piracy. Diversity, 8(1): 2.
Braukmann, T.W., Kuzmina, M.L., Sills, J., Zakharov, E.V., and Hebert, P.D. 2017. Testing the efficacy of DNA barcodes for identifying the vascular plants of Canada. PloS One, 12(1): e0169515.
Brock, P.M., Döring, H. and Bidartondo, M.I. 2009. How to know unknown fungi: the role of a herbarium. New Phytol. 181(3): 719-724.
Buerki, S., Lowry, I.I., Porter, P., Alvarez, N., Razafimandimbison, S.G., Küpfer, P., and Callmander,
M.W. 2010. Phylogeny and circumscriptionDraft of Sapindaceae revisited: molecular sequence data, morphology and biogeography support recognition of a new family, Xanthoceraceae. Plant Ecol.
Evol. 143(2): 148-159.
CBOL Plant Working Group 2009. A DNA barcode for land plants. Proc. Natl. Acad. Sci. U.S.A.
106(31): 12794-12797.
Chase, M.W., Salamin, N., Wilkinson, M., Dunwell, J.M., Kesanakurthi, R.P., Haidar, N., and
Savolainen, V. 2005. Land plants and DNA barcodes: short-term and long-term goals. Philos. Trans. R.
Soc. Lond. B Biol. Sci. No. 360(1462): 1889-1895.
Chase, M.W., Cowan, R.S., Hollingsworth, P.M., van den Berg, C., Madriñán, S., Petersen, G., et al.
2007. A proposal for a standardised protocol to barcode all land plants. Taxon, 56(2): 295-299.
Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., et al. 2010. Validation of the ITS2 region as a novel
DNA barcode for identifying medicinal plant species. PloS One, 5(1): e8613.
16 https://mc06.manuscriptcentral.com/botany-pubs Page 17 of 28 Botany
Clement, W.L., and Donoghue, M.J. 2012. Barcoding success as a function of phylogenetic
relatedness in Viburnum, a clade of woody angiosperms. BMC Evol. Biol. 12(1): 73.
Clerc-Blain, J.L., Starr, J.R., Bull, R.D., and Saarela, J.M. 2010. A regional approach to plant DNA
barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the
Canadian Arctic Archipelago. Mol. Ecol. Resour. 10(1): 69-91.
Cowan, R.S., Chase, M.W., Kress, W.J., and Savolainen, V. 2006. 300,000 species to identify:
problems, progress, and prospects in DNA barcoding of land plants. Taxon, 55(3): 611-616.
Culley, T.M. 2013. Why vouchers matter in botanical research. Appl. Plant Sci. 1(11), p.1300076.
Darriba, D., Taboada, G.L., Doallo, R., and Posada, D. 2012. jModelTest 2: more models, new
heuristics and parallel computing. Nature Methods, 9: 772-772.
Deichmann, J.L., Mulcahy, D.G., Vanthomme,Draft H., Tobi, E., Wynn, A.H., Zimkus, B.M., Mcdiarmid, R.W.
2017. How many species and under what names? Using DNA barcoding and GenBank data for west
Central African amphibian conservation. PloS One, 12(11): p.e0187283.
Dong, W., Liu, J., Yu, J., Wang, L., and Zhou, S. 2012. Highly variable chloroplast markers for
evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PloS One, 7(4): e35071.
Eisenman, S.W., Tucker, A.O., and Struwe, L. 2012. Voucher specimens are essential for documenting
source material used in medicinal plant investigations. Journal of Medicinally Active Plants, 1(1): 30-
43.
Ellison, N.W., Liston, A., Steiner, J.J., Williams, W.M., and Taylor, N.L. 2006. Molecular phylogenetics
of the clover genus (Trifolium—Leguminosae). Mol. Phylogenet. Evol. 39(3): 688-705.
Felsenstein, J. 1978. Cases in which parsimony or compatibility methods will be positively
misleading. Syst. Biol. 27: 401-410.
Forest, F., Drouin, J.N., Charest, R., Brouillet, L., and Bruneau, A. 2001. A morphological phylogenetic
analysis of Aesculus L. and Billia Peyr. (Sapindaceae). Can. J. Bot. 79(2): 154-169.
17 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 18 of 28
Hajibabaei, M., Singer, G.A., Hebert, P.D., and Hickey, D.A. 2007. DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet. 23(4):
167-172.
Hardin, J.W. 1957. A revision of the American Hippocastanaceae. Brittonia, 9(3): 145-171.
Hardin, J.W. 1960. Studies in the Hippocastanaceae, V. Species of the old world. Brittonia, 12(1): 26-
38.
Harris, A.J., Xiang, Q.Y., and Thomas, D.T. 2009. Phylogeny, origin, and biogeographic history of
Aesculus L. (Sapindales)–an update from combined analysis of DNA sequences, morphology, and fossils. Taxon, 58(1): 108-126.
Harris, A.J., Fu, C., Xiang, Q.Y.J., Holland, L., and Wen, J. 2016. Testing the monophyly of Aesculus L. and Billia Peyr., Woody genera of tribe HippocastaneaeDraft of the Sapindaceae. Mol. Phylogenet. Evol.
102: 145-151.
Hebert, P.D., Ratnasingham, S., and de Waard, J.R. 2003a. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B Biol. Sci. 270
(Suppl 1), S96-S99.
Hebert, P.D., Cywinska, A., and Ball, S.L. 2003b. Biological identifications through DNA barcodes.
Proc. R. Soc. Lond. B Biol. Sci. 270(1512): 313-321.
Hebert, P.D., Penton, E.H., Burns, J.M., Janzen, D.H., and Hallwachs, W. 2004. Ten species in one:
DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc.
Natl. Acad. Sci. U.S.A. 101(41): 14812-14817.
Huang, X.C., Ci, X.Q., Conran, J.G., and Li, J. 2015. Application of DNA barcodes in Asian tropical trees–a case study from Xishuangbanna Nature Reserve, Southwest China. PLoS One, 10(6): e0129295.
18 https://mc06.manuscriptcentral.com/botany-pubs Page 19 of 28 Botany
Ivanova, N.V., Kuzmina, M.L., Braukmann, T.W., Borisenko, A.V. and Zakharov, E.V. 2016.
Authentication of herbal supplements using next-generation sequencing. PLoS One, 11(5):
p.e0156426.
Jakob, S.S. and Blattner, F.R. 2006. A chloroplast genealogy of Hordeum (Poaceae): long-term
persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for
phylogenetic inference. Mol. Biol. Evol. 23(8): 1602-1612.
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al. 2012. Geneious
Basic: an integrated and extendable desktop software platform for the organization and analysis of
sequence data. Bioinformatics, 28(12): 1647-1649.
Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A., and Janzen, D.H. 2005. Use of DNA barcodes to identify flowering plants. Proc. Natl. Acad.Draft Sci. U.S.A. 102(23): 8369-8374.
Kress, W.J., and Erickson, D.L. 2007. A two-locus global DNA barcode for land plants: the coding rbcL
gene complements the non-coding trnH-psbA spacer region. PLoS One, 2(6): e508.
Larranaga, N., and Hormaza, J.I. 2015. DNA barcoding of perennial fruit tree species of agronomic
interest in the genus Annona (Annonaceae). Front. Plant Sci. 6: 589.
Li, D.Z., Gao, L.M., Li, H.T., Wang, H., Ge, X.J., Liu, J.Q. et al. 2011. Comparative analysis of a large
dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode
for seed plants. Proc. Natl. Acad. Sci. U.S.A. 108(49): 19641-19646.
Miadlikowska, J., Lutzoni, F., Goward, T., Zoller, S., and Posada, D. 2003. New approach to an old
problem: Incorporating signal from gap-rich regions of ITS and rDNA large subunit into phylogenetic
analyses to resolve the Peltigera canina species complex. Mycologia, 95(6): 1181-1203.
Modliszewski, J.L., Thomas, D.T., Fan, C., Crawford, D.J., de Pamphilis, C.W., and Xiang, Q.Y. 2006.
Ancestral chloroplast polymorphism and historical secondary contact in a broad hybrid zone of
Aesculus (Sapindaceae). Am. J. Bot. 93(3): 377-388.
19 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 20 of 28
Newmaster, S.G., Fazekas, A.J., and Ragupathy, S. 2006. DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Botany, 84(3): 335-341.
Newmaster, S.G., Fazekas, A.J., Steeves, R.A.D., and Janovec, J. 2008. Testing candidate plant barcode regions in the Myristicaceae. Mol. Ecol. Resour. 8(3): 480-490.
Nilsson, R.H., Ryberg, M., Kristiansson, E., Abarenkov, K., Larsson, K.H., Kõljalg, U. 2006. Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective. PloS One, 1(1): p.e59.
Pelser, P.B., Kennedy, A.H., Tepe, E.J., Shidler, J.B., Nordenstam, B., Kadereit, J.W. and Watson, L.E.
2010. Patterns and causes of incongruence between plastid and nuclear Senecioneae (Asteraceae) phylogenies. Am. J. Bot. 97(5): 856-873.
Ramalho, A.J., Zappi, D.C., Nunes, G.L., Watanabe,Draft M.T., Vasconcelos, S., Dias, M.C., et al. 2018. Blind Testing: DNA Barcoding Sheds Light Upon the Identity of Plant Fragments as a Subsidy for Cave
Conservation. Front. Plant Sci. 9:1052.
Sang, T., Crawford, D.J. and Stuessy, T.F. 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am. J. Bot. 84(8): 1120-1136.
Seberg, O., and Petersen, G. 2009. How many loci does it take to DNA barcode a crocus? PloS One,
4(2): e4598.
Sell, P., Murrell, G. 2009. Flora of Great Britain and Ireland, Cambridge, Cambridge University Press.
Sonnante, G., Galasso, I., and Pignone, D., 2003. ITS sequence analysis and phylogenetic inference in the genus Lens mill. Ann. Bot. 91(1): 49-54.
Stace, C. 2010. New Flora of the British Isles, Cambridge, Cambridge University Press.
Stamatakis, A., Hoover, P., and Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol. 57(5): 758-771.
20 https://mc06.manuscriptcentral.com/botany-pubs Page 21 of 28 Botany
Sun, Y., Skinner, D.Z., Liang, G.H., and Hulbert, S.H. 1994. Phylogenetic analysis of Sorghum and
related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor. Appl. Genet. 89(1):
26-32.
Taberlet, P., Coissac, E., Pompanon, F., Gielly, L., Miquel, C., Valentini, A., Vermat, T., Corthier, G.,
Brochmann, C., and Willerslev, E. 2006. Power and limitations of the chloroplast trnL (UAA) intron
for plant DNA barcoding. Nucleic Acids Res. 35(3): e14-e14.
Turland, N.J., and Xia, N. 2005. A new combination in Chinese Aesculus (Hippocastanaceae). Novon,
15(3): 488-489.
Vences, M., Thomas, M., Bonett, R.M. and Vieites, D.R. 2005. Deciphering amphibian diversity
through DNA barcoding: chances and challenges. Philos. Trans. R. Soc. Lond. B Biol. Sci. No. 360(1462): 1859-1868. Draft
Wendel, J.F., Schnabel, A., and Seelanan, T. 1995. An unusual ribosomal DNA sequence from
Gossypium gossypioides reveals ancient, cryptic, intergenomic introgression. Mol. Phylogenet. Evol.
4(3): 298-313.
Xiang, Q.Y., Crawford, D.J., Wolfe, A.D., Tang, Y.C., and DePamphilis, C.W. 1998. Origin and
biogeography of Aesculus L. (Hippocastanaceae): a molecular phylogenetic perspective. Evolution,
52(4): 988-997.
Xu, S., Li, D., Li, J., Xiang, X., Jin, W., Huang, W., Jin, X., and Huang, L. 2015. Evaluation of the DNA
barcodes in Dendrobium (Orchidaceae) from mainland Asia. PloS One, 10(1): e0115168.
Yan, L.J., Liu, J., Möller, M., Zhang, L., Zhang, X.M., Li, D.Z., and Gao, L.M. 2015. DNA barcoding of
Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya–
Hengduan Mountains. Mol. Ecol. Resour. 15(4): 932-944.
Yao, H., Song, J., Liu, C., Luo, K., Han, J., Li, Y., Pang, X., Xu, H., Zhu, Y., Xiao, P., and Chen, S. 2010. Use
of ITS2 region as the universal DNA barcode for plants and animals. PloS One, 5(10): e13102.
21 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 22 of 28
Zarrei, M., Talent, N., Kuzmina, M., Lee, J., Lund, J., Shipley, P.R., Stefanović, S. and Dickinson, T.A.
2015. DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus
Crataegus. AoB Plants, 7: plv045.
Zhang, Z., Li, S., and Lian, X.Y. 2010. An overview of genus Aesculus L.: ethnobotany, phytochemistry, and pharmacological activities. Pharm. Crop. 1: 24-51.
Draft
22 https://mc06.manuscriptcentral.com/botany-pubs Page 23 of 28 Botany
Table 1: PCR profiles and primers used for amplifying and sequencing.
Name of Region Primer sequence Source the primer 1F ACT GTA TCG CAC TAT GTA TCA Modliszewski et al. 2006; Harris et al. 2009 300F GGG ATT TGC AGT CAT TGT GG Xiang Lab (unpub.); Harris et al. 2009 500R TGG ACR GGR TRG GGT ATT AG Xiang Lab (unpub.); Harris et al. 2009 3F AAG ATG CCT CTT CTT TGC AT Modliszewski et al. 2006; Harris et al. 2009 matK 799F TTC TGG ACT CCT TCT TGA GCA Harris et al. 2009 1288F TAT TAT CGA CCG GTT TGT GC Harris et al. 2009 1416R CGC GCA CAG TAC TTT TGT GT Harris et al. 2009 1R GAA CTA GTC GGA TGG AGT AG Modliszewski et al. 2006; Harris et al. 2009 17SE ACG AAT TCA TGG TCC GGT GAA GTG TTCG Sun et al. 1995 ITS5f GGA AGG AGA AGT CGT AAC AAG G Xiang et al. 1998; Harris et al. 2009 ITS2r GCT GCG TTC TTC ATC GAT GC Xiang et al. 1998; Harris et al. 2009 ITS ITS3f GCA TCG ATG AAG AAC GCA GC Xiang et al. 1998; Harris et al. 2009 ITS4r TCC TCC GCT TAT TGA TAT GC Xiang et al. 1998; Harris et al. 2009 26SE TAG AAT TCC CCG GTT CGC TCG CCG TTAC Sun et al. 1995 Region PCR profile matK ITS Initial denaturation 94 °C for 2 minutes Draft 94 °C for 2 minutes denaturationDenaturation 94 °C for 30 seconds 94 °C for 1 minute Annealing 52 °C for 1 minute 48 °C for 1 minute Extension 72 °C for 1.5 minutes 72 °C for 1.5 minutes Final extension 72 °C for 7 minutes 72 °C for 7 minutes Number of cycles 35 cycles 35 cycles
23 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 24 of 28
Table 2: PCR amplification and sequencing success, alignment length or ingroup taxa, total variable characters, parsimony-informative characters, number of total individuals and number of species sampled for 50 Aesculus samples in total for matK, ITS, ITS2, ITS+matK and ITS2+matK.
Region PCR amplification Aligned ingroup Total variable Parsimony- Total Species and sequencing length (bp) characters informative individuals sampled success (50 samples characters in total) matK 92% 1,726 94 45 (2.6%) 46 11 ITS 88% 605 94 71 (11.7%) 44 12 ITS2 88% 195 36 25 (12.8%) 44 12 ITS+ matK 100% 2,331 72 115 50 13 ITS2+ matK 100% 1,921 132 71 50 13
Draft
24 https://mc06.manuscriptcentral.com/botany-pubs Page 25 of 28 Botany
Table 3: Identification success of ITS, ITS2, matK, ITS+matK and ITS2+matK using ML and BLAST methods. Crosses (X) indicate the species was not monophyletic in the ML tree, and check marks () indicate monophyletic species. One asterisk (*) indicates that the “first hit” on the BLASTn results was correctly identified (i.e., unambiguous identifications), and two asterisks (**) indicate that at least more than half of the species were correctly identified. Empty cells indicate that data is not available.
Monophyletic species in the ML trees Percentage of BLASTn identification success
ITS ITS2 matK ITS+matK ITS2+matk ITS2 matK ITS2+matK
Aesculus X X (88%) (77%) (88%)
A. californica (100%) (100%) (84%) (100%) (100%) 100%* A. chinensis (including A. X X X X X 98% to 100% 100%* 100%* chinensis var. wilsonii) A. flava+A glabra+A. X X X (80%) X 98% to 100% 98% to 100%** 98% to 100%** sylvatica+A. pavia A. hippocastanum (67%) X X (98%) (90%) 100% 98% to 100% 99% to 100% A. indica (100%) (83%) X (99%) (93%) 100%* 99% to 100%* 99%* A. parryi (100%) (100%) Draft (100%) (100%) 100%* A. parviflora (100%) (95%) (98%) (100%) (100%) 100%* 100%* 100%* A. polyneura (100%) (98%) (95%) 100%* A. tsiangii X (55%) (99%) (88%) (98%) 100% 100%* 100%*
A. turbinata X X (63%) (56%) (59%) 99% to 100% 99% to 100%** 99% to 100%**
25 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 26 of 28
Table 4: Summary of the results of the total evidence tree from the current study and previous studies. The studies and phylogenetic trees, which sampled only one taxon from each species, were not included. The species sampling of the current study includes both newly created sequences and sequences downloaded from GenBank. The dark grey cells indicate that the species was not monophyletic in the study, and the light grey cells indicate a monophyletic species. Crosses (X) indicate this taxon was not included in the study. Asterisks (*) indicate only one taxon was sampled.
Study Name of the species Xiang et al. (1998) (matK) Harris et al. (2009) Harris et al. (2016) Current study (matK+ITS) (cpDNA) (cpDNA+nDNA) Aesculus californica monophyletic (2 spp) monophyletic (5 spp) * monophyletic (6 spp) Aesculus chinensis (including * not monophyletic (6 spp) not monophyletic (4 spp) not monophyletic (5 spp) Aesculus chinensis var. wilsonii) Aesculus flava not monophyletic (2 spp) not monophyletic (2 spp) monophyletic (2 spp) not monophyletic (9 spp) Aesculus glabra monophyletic (2 spp) * not monophyletic (3 spp) not monophyletic (4 spp) Aesculus hippocastanum * * * monophyletic (17 spp)
Aesculus indica * not monophyletic (2 spp) * monophyletic (7 spp) Aesculus pavia not monophyletic (2 spp) not monophyletic (2 spp) not monophyletic (4 spp) not monophyletic (7 spp) Aesculus parviflora * * monophyletic (2spp) monophyletic (6 spp) Aesculus parryi Draft * * * monophyletic (2 spp) Aesculus polyneura * X monophyletic (2 spp) Aesculus sylvatica not monophyletic (2 spp) * not monophyletic (2 spp) not monophyletic (6 spp) Aesculus turbinata * not monophyletic (2 spp) * monophyletic ( 8 spp) Aesculus tsiangii X * X monophyletic (2 spp) A. wangii (=A. assamica) * not monophyletic (2 spp) * X
26 https://mc06.manuscriptcentral.com/botany-pubs Page 27 of 28 Botany
Figure 1: Phylogenetic relationships within Aesculus inferred from ML analysis of matK+ITS. Outgroups and five Aesculus sections (section Aesculus, section Calothyrsus, section Macrothyrsus, section Pavia and section Parryana) are indicated. Bootstrap values are indicated below branches.
Draft
27 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 28 of 28
Draft
Figure 1: Phylogenetic relationships within Aesculus inferred from ML analysis of matK+ITS. Outgroups and five Aesculus sections (section Aesculus, section Calothyrsus, section Macrothyrsus, section Pavia and section Parryana) are indicated. Bootstrap values are indicated below branches.
https://mc06.manuscriptcentral.com/botany-pubs