DNA Barcoding of Two Narrow Endemic Plants; Astragalus Argaeus and Astragalus Stenosemioides from Mount Erciyes, Turkey

doi:10.21203/rs.3.rs-357931/v1

The genetic resources and biological diversity of countries are very important. Biodiversity and genetic resources should be protected, especially as endemic species. In this concept, DNA barcoding studies are an effective way to identify an unknown taxon and protected the biodiversity of a country. Astragalus argaeus and A. stenosemioides are narrow endemic species from Mt. Erciyes, Turkey. To determine its phylogenetic relationships and DNA barcoding, sequence data from the chloroplast DNA (matK region) were analyzed by parsimony, maximum likelihood, and Bayesian methods. In this study, A. argaeus, A. stenosemioides samples, and 23 sequences from GenBank, including a closely related species were performed. The phylogenetic analysis showed that the matK gene region could clearly distinguish A. argaeus and A. stenosemioides from its closely related species. DNA barcoding surveys can contribute to taxonomic and biodiversity research, various molecular ecology, and population genetics studies. Also, it is possible to determine the species by separating the matK DNA gene region, which is one of the molecular characters, and A. argaeus and A. stenosemioides have been successfully barcoded for the first time; therefore, it has been shown that this gene region can be used for barcoding.

Molecular Biology

Plant Molecular Biology and Genetics

Biodiversity and genetic resources

A. stenosemioides

phylogenetic analysis

molecular characters

barcoding.

Many molecular markers are used for plant and animal genetic studies. Recently, the most commonly used methods to determine the genetic variation of DNA-based methods such as DNA barcoding, phylogenetic studies (Carew et al. 2005). It is very important for taxonomy and biodiversity studies by using DNA barcode methods to fast and correctly make taxa identification. In particular, DNA barcoding has been provided a great utility in biodiversity surveys. Moreover, the Conservation of biodiversity and natural plant resources is very important for the ecosystem (Kress & Horvitz 2005; Hosein et al. 2017). DNA barcoding has the potential to support species identification and discovery, vegetation, and floristic species surveys, in addition to studies on ecological forensics, all of which are critical to biodiversity management (Hollingsworth et al. 2016; Valentini et al. 2008; von Crautlein et al. 2011; Hosein et. al. 2017). Especially, DNA Barcoding and phylogenetic survey of the endemic plants are especially critical. DNA barcoding is a useful tool to quickly and accurately identify species. Also, DNA barcoding has the potential to prompt the discovery of new species.

Turkey has a significant and rich flora because of its geographical location and its paleogeography and ecological features. Turkey is the only country covered almost entirely by three of the world's 34 global biodiversity hotspots (the Caucasus, Iran-Anatolian, and the Mediterranean) (Sekercioglu et al. 2011). Mountain Erciyes is the highest mountain in Inner Anatolia. Moreover, mountain Erciyes in Turkey's Central Anatolia region due to rare species of plants in that area is one of Turkey's most important floristic area and has a different significance. (Byfield et al. 2003; Özhatay et al. 2009; Vural & Şapcı 2012). The Mount Erciyes has 89 families, 433 genera, and 1170 plant taxa (1116 species and 54 intraspecific taxa) (Vural & Aytaç 2005). While 44 of these taxa are endemic taxa from Turkey, of which 12 of them are known only from Mount Erciyes. One of these points endemic species is Astragalus argaeus Boiss. & Balansa and A. stenosemioides D.F. Chamb and V.A. Matthews (Vural & Aytaç 2005; Vural & Şapcı 2012). In this study, the ability of DNA barcoding to confirm the identities of Critically Endangered endemic plant taxon (Astragalus argaeus) in Mountain Erciyes was assessed using a DNA barcode (matK).

Taxon collection

Research materials were having gathered from Mount Erciyes, Kayseri, Turkey, and deposited at Herbarium of Erciyes University, Turkey (ERCH).

DNA isolation, amplification, and PCR product sequencing

Genomic DNA (gDNA) from leaf tissues was extracted by using a commercial DNA extraction kit (DNeasy Plant Mini Kit, Qiagen), following the manufacturer's protocol. Agarose gel electrophoresis of 1.5% was used to detect the presence of gDNA with ethidium bromide. The obtained genomic DNA was used in PCR for the amplification of the matK gene region using specific primer pairs 1R_KIM and 3F_KIM (Fazekas et al. 2008).

The PCR products were sequenced in both directions (Forward and Reverse) using an automatic sequencer (ABI 3100 Genetic Analyzer, RefGen Biotechnology, Ankara, Turkey). Reaction mixture used for the PCR amplifications contained a volume of 50 µl; 10 × Taq Buffer with (NH4)2SO4: 5 µl, 10 mM dNTP mix: 1.3 µl, 5u/ul Taq DNA polymerase (Thermo Scientific): 0.25 µl, 25 mM MgCl2: 3 µl, 10 mg/ml BSA: 3 µl, 10 uM each primer: 1 µl, and genomic DNA extract: 1 µl, dH2O: 34.45 µl). The following cycling parameters were used for matK, A pre-denaturation procedure consisting of 5 min at 95°C by 1 cycle, a denaturation step of 1 min at 94°C by 35 cycles, an annealing step of 1.30 min at 48°C by 35 cycles, an extension step of 1 min at 72°C by 35 cycles and an ending step of 10 min by 1 cycle.

Phylogenetic analyses

Sequences were aligned used for the multiple sequence alignment with Geneious Software 6R (available from http://www.geneious.com). Phylogenetic analyses were performed by using the sequences of matK gene regions obtained from GenBank (NCBI: The National Center for Biotechnology Information) and this study (Table 1). The related matK sequences of Astragalus species (23) were retrieved from the GenBank database. Taraxacum alpinum K.Koch. were used as the outgroup for the phylogenetic analysis. The best fit model for nucleotide substitution for sequences of matK gene regions was chosen using JModelTest 2.1 based on the corrected Akaike Information Criterion (AICc) and Bayesian Information Criterion (BIC) (Darriba et al. 2012). The BI analysis was performed by using BEAST v.1.8 (Drummond et al. 2012). All parameters were the effective sample sizes (ESS) > 200. Bayesian analysis was performed using four simultaneous runs of Metropolis-coupled Markov Chain Monte Carlo (MCMC) sampling for 10 million generations and under the uncorrelated lognormal relaxed clock model (Drummond et al. 2006) and also selected to Yule process for speciation as tree prior (Yule 1925). Checking of the Quality analysis was done by using likelihood parameters and values, which were estimated by using different runs in Tracer V.1.5 (Rambaut & Drummond 2009).

Table 1

Accession numbers are available in GenBank database.
Taxon	Accession number
Astragalus boeticus	KX955075.1
Astragalus capito	KX955086.1
Astragalus caraganae	AB741312.1
Astragalus caspicus	MK958484.1
Astragalus cicer	KM387646.1
Astragalus commixtus	KX955090.1
Astragalus cornutus	KM387661.1
Astragalus corrugatus	AY920442.1
Astragalus dactylocarpus	AB741315.1
Astragalus daenensis	KX955095.1
Astragalus echinatus	JQ619978.1
Astragalus epiglottis	AB854563.1
Astragalus gaeobotrys	KX955113.1
Astragalus glycyphyllos	JN894914.1
Astragalus guttatus	KX955119.1
Astragalus hamosus	KX955121.1
Astragalus incertus	KX955127.1
Astragalus macrostachys	KX955143.1
Astragalus microcephalus	KX955147.1
Astragalus schizopterus	KM387635.1
Astragalus siliquosus	KX955182.1
Astragalus suberosus subsp. suberosus	KX955223.1
Astragalus tribuloides	KX955200.1
Astragalus argaeus	---
Astragalus stenosemioides	---

In this study, narrow endemic A. argaeus and A. stenosemioides were evaluated in the molecular data. Sequences of plastid DNA (matK gene) were successfully amplified from A. argaeus and A. stenosemioides. These sequences were analyzed together with sequences from GenBank and constructed phylogenetic tree. The phylogenetic tree was obtained by the Bayesian method using the BEAST program (Fig. 1). The phylogenetic tree constructed from matK sequences was clustered into the two main groups. Astragalus caragnae and A. dactylocarpus were grouped in the first lineage. Other species were classified in the second lineage. When we evaluated the DNA barcode-based tree, it shows that consistent with classical taxonomic classification. In this concept, the matK gene region sequences of the genus were useful to solve taxonomic problems. DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. An ideal DNA barcode should be routinely retrievable with a single primer pair. Additionally, it should be amenable to bidirectional sequencing with little requirement for manual editing of sequence traces, and also provide maximal discrimination among species. In this concept, there are several candidate barcode regions. One of these candidate barcode gene regions is the matK gene region (Ford et al. 2009). The matK gene region has a high rate of substitution, allowing substitutions to occur at almost the same rate in all triple codons (Hilu & Liang 1997, Hochbach et al. 2018). Besides, the excessive substitution rate of this gene is very effective in the formation of regions with parsimony information content and in obtaining strong phylogenetic predictions. Also, the rate of substitution of the matK gene provides an important guide in getting strong phylogenetic predictions and evaluating how the evolutionary past developed at different taxon levels (Johnson & Soltis 1994). However, some authors suggested that the matK gene region alone is insufficient for some plants (Quercus sp., Salix sp.) groups for determining DNA barcode. They stated that it is appropriate to use matK + rbcL gene regions together in such cases (Piredda et al. 2010; Ren et al. 2010; Von Crautlein et al. 2011). According to our study results, sequences of the matK gene region were enough variability for genus Astragalus ssp. Also, the matK gene region for DNA barcoding studies in genus Astragalus is a good candidate for DNA barcoding studies.

It is very important to protect and evaluate plant genetic resources. Hence, A. argaeus and A. stenosemioides are very important for endemic plants. These species have a very limited living area. Threat categories of A. argaeus and A. stenosemioides are Critically Endangered (CR). Especially, correct identification of endangered species is crucial for the future conservation of the species. The DNA barcoding is also proved valuable for accurate species identification as the important first step in conservation plans for threatened species (Kim et al. 2014). Barcoding studies can help on-going conservation prevention of the taxa in different ways. Species identification is the first important step in a correct evaluation of distribution, population abundance, and threats of target species (Hartvig et al. 2015). The present study was analyzed matK sequence data to determine if DNA barcoding could accurately identify genus Astragalus. Additionally, DNA sequences of endemic plants A. argaeus and A. stenosemioides were firstly determined in this study. The current study results show that matK gene region sequences can be used as potential a DNA barcoding region.

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DNA Barcoding of Two Narrow Endemic Plants; Astragalus Argaeus and Astragalus Stenosemioides from Mount Erciyes, Turkey

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Abstract

Figures

Introduction

Material And Methods

Taxon collection

DNA isolation, amplification, and PCR product sequencing

Phylogenetic analyses

Results And Discussion

References

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