Identification of Kalidium species (Chenopodiaceae) by DNA barcoding

XiaoHui Liang, YuXia Wu

State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China

Identification of Kalidium species (Chenopodiaceae) by DNA barcoding

XiaoHui Liang, YuXia Wu*

State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China

DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification.Kalidiumspecies are distributed in saline soil habitat throughout Southeast Europe and Northwest Asia, and used mainly as forage grass in China. The discrimination ofKalidiumspecies was based only on morphology-based identification systems and limited to recognized species. Here, we tested four DNA candidate loci, one nuclear locus (ITS, internal transcribed spacer) and three plastid loci (rbcL,matKandycf1b), to select potential DNA barcodes for identifying differentKalidiumspecies. Results showed that the best DNA barcode was ITS locus, which displayed the highest species discrimination rate (100%), followed bymatK(33.3%),ycf1b(16.7%), andrbcL(16.7%). Meanwhile, four loci clearly identified the variant species,Kalidium cuspidatum(Ung.-Sternb.) Grub.var.sinicumA. J. Li, as a single species inKalidium.

DNA barcoding;Kalidium; species identification

1 Introduction

KalidiumMoq. (Chenopodiaceae), are identified as Euhalophytes and divided into five species,Kalidium caspicum(L.),Kalidium gracileFenzl,Kalidium cuspidatum(Ung.-Sternb.) Grub. var.cuspidatum,Kalidium foliatum(PALL.),Kalidium schrenkianumBunge. ex Ung. -Sternb and one variant species,Kalidium cuspidatum(Ung.-Sternb.) Grub. var.sinicumA. J. Li, mainly distributed in Southeast Europe and Northwest Asia as shrubs (Kong, 1979; Kreveret al., 1998). Previous studies have demonstrated thatKalidiumspecies play an important role in maintaining the balance of grassland ecosystems and preventing soil erosion (Zhaoet al., 2002). In comparative studies from three other Euhalophytes species (Suaeda sala,Atriplex centralasiatieaandNitrariasibirica),Kalidiumhave proven to possess strong tolerance to saline-alkali soil and drought, as a dominant species in desert areas (Zhaoet al., 2002; Zhouet al., 2009). In China,Kalidiumis a succulent salt plant mainly used as forage grass for camels, horses and sheep, excellent for grazing herds in the winter. Traditional taxonomic methods for identifyingKalidiumrelied on morphological and phenotypic characters, which have limits in differentiating species (Kong, 1979). Therefore, developing a common DNA barcoding for species identification ofKalidiumis required.

As an increasingly prevalent molecular technique to remedy the limitation of taxonomic research relying solely on morphological features, DNA barcoding has been used to facilitate accurate species-level identification using specific DNA regions (Kress and Erickson, 2007). A potential DNA barcode, CO1 (cyto-chrome c oxidase subunit 1), was successfully identified as a standard mitochondrial region to discriminate species in animal groups (Hogg and Hebert, 2004; Barrett and Hebert, 2005; Fuet al., 2011). Although previous studies have proposed many potential DNA barcodes, including a nuclear DNA locus (ITS) and several chloroplast DNA regions (matK,rbcL,trnH-psbA,atpF-atpH,et al.) has been widely applied in plant ecology and evolutionary studies (Kress and Erickson, 2007; Newmaster and Ragupathy, 2009; Moniz and Kaczmarska, 2010), universal DNA barcodes for plant species identification have not been very informative (Newmasteret al., 2006; Fordet al., 2009; Donget al., 2015).

In this study, four candidate DNA loci were selected, one nuclear gene (ITS) and two previously used chloroplast genes (matKandrbcL) and one recently developed candidate chloroplast gene (ycf1b) (Donget al., 2015), to screen their suitability as DNA barcodes forKalidium. The major purpose of this study is to address the following two questions: (1) determine ideal DNA markers for species-level identification ofKalidium, (2) verify identification of the variant species ofK. cuspidatumvar.sinicumis consistent with that of traditional taxonomy relying only on morphology.

2 Material and methods

2.1 Plant samples

Leaf tissue of five species and one variant species (Figure 1) were collected within the full range ofKalidiumin China. For each species, 20 to 30 individuals were sampled from each population; and all the samples were silica gel-dried. Three to four individuals were sampled from three to five populations for each species in order to avoid individual bias for this study. A total of 82 samples, representing geographic location and collection information are available in Table 1. We selectedSalsola laricifoliaTurcz. ex Litv andChenopodium albumL. (Chenopodiaceae) as outgroups.

Figure 1Morphological characteristics of differentKalidiumspecies. (a)K. schrenkianum, (b)K. cuspidatumvar.cuspidatum, (c)K. foliatum, (d)K. gracile, (e)K. caspicum, (f)K. cuspidatumvar.sinicum

Table 1Locations of sampled individuals ofKalidium

2.2 DNA extraction, amplification and sequencing

DNA was extracted from leaves by the slightly modified SDS method (Jiaet al., 2010). PCR amplification of four candidate barcodes was carried out on the CyclerTMThermal Cycler (Bio-Rad, USA). Detailed information on the amplification conditions of the tested four regions is available in Table 2. Direct sequencing PCR products were carried out by two directions. Five individuals of the ITS locus contained segregating indels that prevented direct sequencing, PCR fragments were purified and sub-cloned into the pMD19-T vector (Takara, China) and three to five clones were then directly sequenced.

2.3 Sequence analysis

Sequence chromatograms were edited and aligned using Aligner v.5.1.0 (Codon Code Corporation, Dedham, MA), with all posterior probabilities ＞ 0.8 and all polymorphic and heterozygous sites manually confirmed. We calculated K2P (Kimura 2-parameter) distances using MEGA 5 software to evaluate the intra-specific and inter-specific differences (Kumaret al., 2008). The distribution graphs of intra-specific and inter-specific K2P genetic distances of each DNA locus were made and compared to value barcode gaps using Origin 8.5 (Meieret al., 2006). The differentiation power of the four DNA barcodes was estimated using tree-based methods. Bootstrap support values were performed through 1,000 random replicates.

3 Results

3.1 PCR amplification, sequencing and alignmentAll four candidate loci exhibited high PCR success and sequencing success (100%) (Table 3). The 336 new sequences of the four DNA regions were obtained representing five species and one variant ofKalidium, which also included two outgroup taxa,S.laricifoliaandC. album, respectively (Table 3). The results showed that the tested primers possessed prominentuniversality (Table 3). GeneBank accession numbers of all the sequences are listed in Table 4.

For four DNA loci, aligned sequence lengths possessed a relatively great range 681 bp for ITS to 902 bp formatK(Table 3). The DNA barcode containing the most variable sites was ITS (119), followed byycf1b(35),rbcL(7) andmatK(7). The distribution graphs of the intra-specific and inter-specific distance for the four DNA regions displayed the highest mean inter-specific divergences of ITS region (0.0437), andmatKhad the lowest mean inter-specific divergences (0.003) (Table 3).

3.2 DNA barcoding gap

Graphing the distribution of K2P distances is to evaluate the barcoding divergence between intraspecific and interspecific genetic distances for all DNA barcodes tested (Figure 2). We found a large barcoding gap only in the ITS region and the other three DNA sequences tested proved to have no such barcoding gap (Figure 2).

Table 2PCR primers of four DNA barcodes

Table 3Variability comparisons of four DNA barcodes

Table 4GeneBank accession numbers of four DNA barcodes tested for allKalidiumspecies and two outgroups

Figure 2Relative distribution photographs of inter-specif i c and intra-specif i c distances for the four DNA barcodes ofKalidium. Thex-axes stands for K2P distances arranged in intervals and they-axes stands for the percentage of occurrences

3.3 Phylogenetic analyses

Neighbor-Joining (NJ) trees were constructed with the single-locus or combined DNA loci to evaluate the discrimination power for the four DNA barcodes (Figures 3, 4). As the single locus analyses, the species discrimination power of the ITS locus was highest, with a success rate of 100%, followed bymatK(33.3%),ycf1b(16.7%) andrbcL(16.7%) (Table 3). The tree-based method for theycf1blocus identified the same species as that of therbcLlocus (Figure 4). For possible combination of the other three DNA loci excluding ITS region analyses, the results showed that the possible combinations ofmatKwith the other two loci had the same performance as usingmatKalone (Figure 4). Due to the relatively low success of species identification for single locus or random combination of the tested three barcodes excluding ITS region, the confident tree obtained only from ITS (Figure 3).

NJ tree analysis of ITS sequences revealed that the 82 samples used in this study were significantly split into six clades (Figure 3). Single or any combination of all four DNA barcodes tested identifiedK. cuspidatumvar.sinicumas a distinctive clade (Figures 3, 4). The analysis of all NJ trees identifiedK. cuspidatumvar.sinicumas a single species with high bootstrap values.

Figure 3Neighbor-joining tree based on the ITS gene sequences with the Kimura 2-parameter distance model. Bootstrap values are available above the relevant branches and species are shown in the right column

Figure4Neighbor-joining phylogram based on the three DNA regions (matK,rbcL,ycf1b) with the Kimura 2-parameter distance model. Bootstrap values are available above or below the relevant branches and the values lower than 50% were covered

4 Discussion and conclusions

The success rate of PCR amplification and sequencing has long been treated as a significant index to estimate DNA barcodes. In this study, all four DNA barcodes tested were universal with PCR amplification and sequencing success. Through sequences analysis, the ITS region exhibited high resolution, as identification power has been showed inAlnus(Renet al., 2010) and Euphorbiaceae (Panget al., 2010). For species level identification, ITS showed great potential for being an ideal DNA barcode forKalidiumdue to its high inter-specific divergence and discrimination rate (100%) in the four DNA loci tested.rbcL, as one of the core DNA barcodes for plants, has performed well for mosses, ferns and angiosperms (Hollingsworthet al., 2009; Liuet al., 2011; Zhenget al., 2015). Also, previous studies have indicated that therbcLregion showed relatively low inter-specific K2P distances on determining closely related species (Hasebeet al., 1995; Newmasteret al., 2008; Gonget al., 2015). Our results showed thatrbcLhad relatively low inter-specific distance and the lowest species differentiation rate, and as such is not suitable as a DNA locus for discrimination inKalidium. Recently, Donget al. (2015) demonstrated thatycf1bis a plastid genome region with relatively high variable sites and proposedycf1bas a core DNA barcode for species identification of land plants. Our results showed thatycf1bhad the same discrimination rate (16.7%) asrbcLand wereunfit for potential DNA barcode inKalidium. ThematKregion has proven to possess a relatively significant inter-specific K2P distances and good generality in some land plants (Kellyet al., 2010; Gonget al., 2015). Lahayeet al. (2008) identifiedmatKgene showed prominent universality for identification of flower plants species. Asahinaet al. (2010) demonstrated thatmatKrather thanrbcLwas better suited in identifying medicinalDendrobiumspecies due to a high-level resolution. ThematKlocus showed relatively high level of discrimination rate inKalidiumspecies compared with the other two candidates (rbcLandycf1b, thematKlocus was proposed as a candidate DNA barcode rather than an ideal barcode owning to its lower species level identification than the ITS region inKalidium.

Numerous studies have demonstrated the limitation of species delimitation relying solely on morphological features. For example,Protoparmeliawas more diverse than what was expected from traditional taxonomy, consisting of several previous unknown depicted species, and cryptic species-lineages (Singhet al., 2015). According to morphological and biogeographic information, Wood (2006) treatedDendrobium officinaleandD. tosaenseas a common species, but by molecular technique identification results suggested that they should be identified as two different species (Asahinaet al., 2010). Compared toK. cuspidatumvar.cuspidatum,K. cuspidatumvar.sinicumwas identified as a variant species based solely on different morphological traits (Kong, 1979). According to our observations on morphological characteristics of these two species in the field, there were significant differences on growth of the shoots, the length and diameter of the infructescences. Also, the phylogenetic analyses of all four DNA barcodes tested in this study showed thatK. cuspidatumvar.sinicumclustered into a single clade with strong bootstrap values greatly separated from the other five species. Obviously, species identification ofKalidiumrelying solely on morphology has its limitations. Thus, we suggested thatK. cuspidatumvar.sinicumshould be identified as a single species inKalidium, rather than as a variant.

In conclusion, results showed that the ITS region was an ideal DNA barcode inKadilium, and the variant,K. cuspidatumvar.sinicum, should be treated as a single species.

Acknowledgments:

We are grateful to KuiBing Meng, DeCheng Liu and FengZhu Zhang for sample collections in the field. This work was supported by the Program for New Century Excellent Talents in the Ministry of Education in China (NCET-09-0446), and lzujbky-2012-k22 to YuXia Wu.

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:Liang XH, Wu YX, 2017. Identification ofKalidiumspecies (Chenopodiaceae) by DNA barcoding. Sciences in Cold and Arid Regions, 9(1)： 0089-0096.

10.3724/SP.J.1226.2017.00089.

Received: July 12, 2016 Accepted: October 19, 2016

*Correspondence to: Ph.D., YuXia Wu, School of Life Sciences, Lanzhou University. No. 222, South Tianshui Road, Lanzhou, Gansu 730000, China. E-mail: wuyx@lzu.edu.cn