Volume 31 Issue 3
Mar.  2018
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AN Cui Hong, CHEN Bao Bao, FAN Suo Ping, SUN Yang Xin, LYU Wen, SHE Jian Jun. The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China[J]. Biomedical and Environmental Sciences, 2018, 31(3): 238-241. doi: 10.3967/bes2018.030
Citation: AN Cui Hong, CHEN Bao Bao, FAN Suo Ping, SUN Yang Xin, LYU Wen, SHE Jian Jun. The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China[J]. Biomedical and Environmental Sciences, 2018, 31(3): 238-241. doi: 10.3967/bes2018.030

The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China

doi: 10.3967/bes2018.030
Funds:

the grant of the Science and Technology Research and Development of Shaanxi Province 2012K16-12-03

More Information
  • Author Bio:

    AN Cui Hong, female, born in 1972, deputy director technician, majoring in plague, brucellosis, and etiologic biological vector control

  • Corresponding author: SUN Yang Xin, Tel: 86-29-68655600, E-mail: sxpco@126.com
  • Received Date: 2017-04-16
  • Accepted Date: 2018-01-23
  • This study was conducted to define the taxonomic status of Spermophilus in the plague area of Dingbian County in Shaanxi Province, China, through the two-factor variance analysis of morphological characteristics, DNA barcoding, and chromosome karyotype analysis. The Spermophilus samples collected from Dingbian and Zhengxiang Baiqi Counties exhibited significant differences in their morphological measurements. All Spermophilus samples form two distinct branches in neighbor-joining (NJ) tree. One branch included the Spermophilus samples collected from Inner Mongolia, and the other branch included samples collected from the plague foci of Shaanxi Province and the Ningxia Region. The Spermophilus samples collected from Dingbian County had a chromosome number of 2n = 38 in 84.40% of all their cells. The Spermophilus species collected from the plague area of Dingbian County was categorized as Spermophilus alashanicus (S.alashamicus). The findings reported in this study are epidemiologically significant for monitoring plague in this region of west-central China.
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  • [1] LIU J. Study on the important medical animals in Shaanxi province and its prevention and control, Xi'an, China. Publishing House of Northwest University. 2001; 3-20. (In Chinese)
    [2] Smith MA, Fisher BL, Hebert PDN. DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group:the ants of Madagascar. Philos Trans R Soc Lond B Biol Sci, 2005; 360, 1825-34. doi:  10.1098/rstb.2005.1714
    [3] Hebert PDN, Stoeckle MY, Zemlak TS, et al. Identification of Birds through DNA Barcodes. Plos Biolog, 2004; 2, 312. doi:  10.1371/journal.pbio.0020312
    [4] Ilves KL, Taylor EB. Molecular resolution of the systematics of a problematic group of fishes (Teleostei:Osmeridae) and evidence for morphological homoplasy. Mol Phylogenet Evol, 2009; 50, 163-75. doi:  10.1016/j.ympev.2008.10.021
    [5] Tsvirka MV, Chelomina GN, Korablev VP. Genetic evidence of hybridization between paletailed Spermophilus pallidicauda Satunin, 1903 and alashanic S. alaschanicus Büchner, 1888 ground squirrels in Mongolia. Genetika, 2006; 42, 530-7. doi:  10.1134/S1022795406040090.pdf
    [6] Ivanova NV, Zemlak TS, Hanner RH, et al. Universal primer cocktails for fish DNA barcoding. Molecular Ecology Not, 2007; 7, 544-8. doi:  10.1111/j.1471-8286.2007.01748.x
    [7] Irwin DM, Kocher TD, Wilson AC. Evolution of the cytochrome b gene of mammals. J Mol Evol, 1991, 32; 128-44. doi:  10.1007/BF02515385
    [8] Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol, 1980, 16, 111-20. doi:  10.1007/BF01731581
    [9] Saito N, Nei M. The neighbor-joining method:a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987; 4, 406-25. http://www.its.caltech.edu/~matilde/NeighborJoinMethod.pdf
    [10] Sun Y, Chen B, Lu Y, et al. Karyotype of Spermophilus alashanicus in Wei-Luo Delta of Shaanxi province, China. Chinese Journal of Vector Biology and Control, 2014; 25, 225-6. (In Chinese) http://med.wanfangdata.com.cn/Paper/Detail/PeriodicalPaper_zgmjswxjkzzz201403008
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The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China

doi: 10.3967/bes2018.030
Funds:

the grant of the Science and Technology Research and Development of Shaanxi Province 2012K16-12-03

  • Author Bio:

  • Corresponding author: SUN Yang Xin, Tel: 86-29-68655600, E-mail: sxpco@126.com

Abstract: This study was conducted to define the taxonomic status of Spermophilus in the plague area of Dingbian County in Shaanxi Province, China, through the two-factor variance analysis of morphological characteristics, DNA barcoding, and chromosome karyotype analysis. The Spermophilus samples collected from Dingbian and Zhengxiang Baiqi Counties exhibited significant differences in their morphological measurements. All Spermophilus samples form two distinct branches in neighbor-joining (NJ) tree. One branch included the Spermophilus samples collected from Inner Mongolia, and the other branch included samples collected from the plague foci of Shaanxi Province and the Ningxia Region. The Spermophilus samples collected from Dingbian County had a chromosome number of 2n = 38 in 84.40% of all their cells. The Spermophilus species collected from the plague area of Dingbian County was categorized as Spermophilus alashanicus (S.alashamicus). The findings reported in this study are epidemiologically significant for monitoring plague in this region of west-central China.

AN Cui Hong, CHEN Bao Bao, FAN Suo Ping, SUN Yang Xin, LYU Wen, SHE Jian Jun. The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China[J]. Biomedical and Environmental Sciences, 2018, 31(3): 238-241. doi: 10.3967/bes2018.030
Citation: AN Cui Hong, CHEN Bao Bao, FAN Suo Ping, SUN Yang Xin, LYU Wen, SHE Jian Jun. The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China[J]. Biomedical and Environmental Sciences, 2018, 31(3): 238-241. doi: 10.3967/bes2018.030
  • All the species of Spermophilus are important host animals in an area affected by plague. Rodents are important host animals for several zoonoses that threaten public health worldwide. The plague area of Shaanxi Province is located in Dingbian County, Yulin City, China, where the species of Spermophilus has been categorized as Spermophilus dauricus (S.dauricus) over the years[1].

    Traditionally, host animals are identified based on their morphological characteristics, assisted with chromosome karyotype technology. DNA barcoding, a taxonomic method that has been extensively used in recent years[2], also helps in the identification of morphologically conservative 'cryptic species'[3]. Currently, the most commonly used gene sequences for the classification of animals are the mitochondrial cytochrome C oxidase subunit Ⅰ (COI) sequence[2] and the cytochrome b (Cyt-b) gene sequence[4].

    Chromosome karyotype analysis is a key index that can be used to discriminate between S. dauricus and S. alashanicus. A previous study based on karyotype analysis from Russia reported that S. dauricus has a chromosome number of 2n = 36, while S. alashanicus in the southwest of Mongolia has a chromosome number of 2n = 38. The authors of that study insisted that S. alashanicus in the southwest of Mongolia is an independent species[5].

    The plague area of Shaanxi Province has been affected three times by animal plague from 1987 to 2016. During the treatment and monitoring of the epidemic, it is often difficult to identify some host animals based on their morphological characters. To further define the taxonomic status of Spermophilus in the plague area of Shaanxi and the distribution of S. alashanicus and S. dauricus in Shaanxi, Inner Mongolia, and Ningxia, we collected S. dauricus samples from Keyou Zhongqi and Zhengxiang Baiqi in Inner Mongolia and S. alashanicus samples from Haiyuan County and analyzed them using morphological characteristics, DNA barcoding technology, and chromosome karyotype analysis.

    The bodies and the organs of Spermophilus were collected from Dingbian County in Shaanxi Province, Haiyuan County, in the Ningxia Region, and from Keyou Zhongqi and Zhengxiang Baiqi in Inner Mongolia during the period from 2012 to 2016. The sample DB01-15 was obtained from Dingbian County, while the sample NX01-7 was collected from Haiyuan County. The samples NM49, NM50, NM52, and NM53 were collected from Keyou Zhongqi, while ZB01-17 was acquired from Zhengxiang Baiqi in Inner Mongolia. This study was approved by the Ethics Committee of Shaanxi Provincial Center for Disease Control and Prevention (No. SXCDC-2012SF001).

    For the Spermophilus samples that were collected from Dingbian and Zhengxiang Baiqi, measurements of the external morphology (weight, body length, tail length, ear length, rear foot length, greatest length of skull, interorbital width, upper cheek teeth length, diastema length, length of auditory bulla, and width of auditory bulla) and those of the skulls were recorded (Supplementary Table S1 available in www.besjournal.com). The two-factor variance analysis of morphological characteristics was performed for 15 Spermophilus samples collected from Dingbian County (skull characters of 7 samples were measured) and for 17 Spermophilus samples collected from Zhengxiang Baiqi.

    Gender and region were considered as independent variables, and morphological indexes were considered as dependent variables. No significant difference was observed in gender among the Spermophilus samples (Table 1). The Spermophilus samples collected from Dingbian and Zhengxiang Baiqi showed significant differences in body weight, body length, tail length, rear foot length, the ratio of tail length to body length, the ratio of interorbital width to greatest length of skull, upper cheek teeth length, and the ratio of upper cheek teeth length to diastema length (P < 0.05, Table 1). For individuals, we cannot judge accurately the types of Spermophilus based on morphological characters in Dingbian. However, in general, the Spermophilus samples collected from Dingbian and Zhengxiang Baiqi were significantly different.

    Independent Variable Dependent Variable Ⅲ Sum of Squares df Mean Square F P
    Gender Weight (g) 879.007 1 879.007 0.416 0.526
    Body length (mm) 0.827 1 0.827 0.003 0.959
    Tail length (mm) 119.422 1 119.422 2.449 0.133
    Ear length (mm) 0.024 1 0.024 0.018 0.894
    Rear foot length (mm) 3.124 1 3.124 0.792 0.383
    Tail length/Body length 0.003 1 0.003 1.333 0.261
    Greatest length of skull (mm) 2.490 1 2.490 0.986 0.332
    Interorbital width/Greatest length of skull 0.000 1 0.000 0.515 0.481
    Interorbital width (mm) 0.754 1 0.754 0.926 0.347
    Upper cheek teeth length (mm) 0.278 1 0.278 1.161 0.294
    Diastema Length (mm) 0.358 1 0.358 0.393 0.537
    Upper cheek teeth length/Diastema Length 0.000 1 0.000 0.031 0.863
    Length of auditory bulla (mm) 0.031 1 0.031 0.075 0.787
    Width of auditory bulla (mm) 0.000 1 0.000 0.000 0.999
    Length of auditory bulla/Width of auditory bulla 0.001 1 0.001 0.081 0.778
    Region Weight (g) 12831.570 1 12831.570 6.071 0.022
    Body length (mm) 2425.937 1 2425.937 7.821 0.011
    Tail length (mm) 1074.895 1 1074.895 22.043 0.000
    Ear length (mm) 0.164 1 0.164 0.125 0.727
    Rear foot length (mm) 17.445 1 17.445 4.425 0.048
    Tail length/Body length 0.074 1 0.074 34.936 0.000
    Greatest length of skull (mm) 6.070 1 6.070 2.404 0.136
    Interorbital width/Greatest length of skull 0.003 1 0.003 9.403 0.006
    Interorbital width (mm) 3.236 1 3.236 3.974 0.059
    Upper cheek teeth length (mm) 2.282 1 2.282 9.531 0.006
    Diastema Length (mm) 3.204 1 3.204 3.514 0.075
    Upper cheek teeth length/Diastema Length 0.117 1 0.117 11.278 0.003
    Length of auditory bulla (mm) 0.066 1 0.066 0.162 0.691
    Width of auditory bulla (mm) 0.241 1 0.241 0.647 0.430
    Length of auditory bulla/Width of auditory bulla 0.011 1 0.011 0.741 0.399

    Table 1.  Two-factor Variance Analysis of Morphological Index Data

    Liver samples were collected and stored in 100% ethanol. Total genomic DNA of Spermophilus was obtained using the DNeasy Blood & Tissue Kits (Qiagen, Pudong, Shanghai, China) according to the manufacturer's instructions. The COI gene was amplified using the cocktail primer sets[6] under the following conditions: 94 ℃ for 5 min, 94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 1 min. The cycle was repeated 35 times, with a final extension at 72 ℃ for 10 min. The Cyt-b genes were also amplified using the primers L14724 and H15915 as described by Irwin et al.[7]. Each polymerase chain reaction (PCR) cycle was carried out under the following conditions: 94 ℃ for 3 min, 94 ℃ for 30 s, 51 ℃ for 30 s, and 72 ℃ for 1 min. The cycle was repeated 30 times, with a final extension at 72 ℃ for 10 min. The cocktail primers and the Cyt-b primers are listed in Supplementary Table S2 (available in www.besjournal.com). All the amplified genes were directly sequenced in both directions.

    The quality of the sequencing peak pattern was observed and evaluated using the Chromos software. When the quality was not good enough for an accurate determination of the bases, the samples were re-amplified and sequenced. The determined sequences were run on the Blast program of NCBI for comparing the sequence homology. The base composition and the mutations of gene sequences were compared using the Mega 6 software. The unaligned bases at the end of the sequence were deleted. The genetic distance was calculated based on the Kimura-2-parameter (K2P) model[8]. Phylogenetic trees of COI and Cyt-b gene sequences were constructed using the neighbor-joining (NJ) method, and the NJ trees were analyzed using 1, 000 bootstraps to determine the confidence level of each branch[9].

    A total of 27 Spermophilus samples were evaluated using DNA barcoding technology, and the gene sequences of COI and Cyt-b were obtained from all these 27 samples.

    The genetic distance of the COI gene sequence obtained from the Spermophilus samples in the plague area of Dingbian County and from S. alashanicus samples in Ningxia was found to be ≤ 0.5%. When compared with S. dauricus samples collected from Inner Mongolia, the genetic distance was found to be between 7.9% and 9.3%. The genetic distance of the Cyt-b gene sequence obtained from the Spermophilus samples in the plague area of Dingbian County and from S. alashanicus samples in Ningxia was found to be ≤ 2.2%. When compared with S. dauricus samples collected from Inner Mongolia, the genetic distance was found to be between 8.9% and 11.3%.

    NJ trees were constructed using the Mega 6 software (Figure 1A: COI gene, Figure 1B: Cyt-b gene). The samples form two distinct branches with high support in each NJ tree. One branch included the Spermophilus samples collected from Inner Mongolia, and the other branch included the samples collected from the plague foci of Shaanxi and Ningxia.

    Figure 1.  Phylogenetic tree analysis based on gene sequences of COI (A) and Cyt-b (B).

    Hebert et al.[3] analyzed the COI gene sequence of the same genus from GenBank and reported that the degree of intraspecific difference of the COI gene sequence was < 2% and the average interspecific genetic distance was 11.3%.

    By analyzing the genetic distance and NJ phylogenetic trees, we found that the Spermophilus collected from the plague area of Shaanxi and the S.dauricus collected from Inner Mongolia were different species, whereas S. alashanicus collected from Ningxia were the same species.

    Five live Spermophilus species collected from the plague area of Dingbian County were investigated using chromosome karyotype analysis[10]. The chromosomes obtained from a total of 218 cells isolated from these five Spermophilus species were observed under the microscope with an oil-immersion lens (Supplementary Table S3 available in www.besjournal.com) and counted according to their morphology and type. Results showed that the chromosome number 2n = 38 accounted for 84.4% of all cells, whereas 2n = 37 accounted for 5.96% of all cells. However, the karyotype 2n = 36 accounted for 9.63% of all cells. These results indicate that the Spermophilus species collected from the plague area of Shaanxi must be classified as S. alashanicus.

    Taken together, based on the morphological characteristics, chromosome karyotype, and the results of DNA barcoding technology, the Spermophilus species collected from the plague area of Dingbian County is categorized as S. alashanicus.

    We express our deepest gratitude to LU Liang for his assistance with the experiments.

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