The study was designed to test the hypothesis, variations in nucleotide frequency distribution in cytochrome c oxidase subunit I (COI) in spiders used as a marker to delimit species. Based on morphological identification and sequencing of CO1 gene, a total of 22 species of spiders belonging to five families were investigated and compared within each species and among families for nucleotide frequency distribution of all species collected. We had found non-significant difference in frequency of nucleotides in COI within the members of species and within families (thymine 41.30-44.30%, adenine 24.96-27.19%, guanine 17.18-19.48% and cytosine 12.28-13.40% while GC 29.53-32.84%). Therefore, it was concluded that variation in nucleotide frequency distribution could not to bee used as a criterion for species delimitation in spiders.
Research Article · Volume 34, Issue 1, 5 July 2019 · Open Access
Variations in Nucleotide Frequency Distribution in Spider Families
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Affiliations & Notes ⌄
- 1Department of Zoology, Government College University, Lahore, Pakistan. 2Punjab Agriculture Research Board, Imam Hussain Road, Gulberg III, Lahore, Pakistan. , Corresponding author Shaukat Ali shuakatali134@yahoo.com
Article Info ⌄
- Journal:
- Punjab University Journal of Zoology
- Published:
- 5 July 2019
From the article
- Results showed that the frequency of nucleotides among members of same species, within different species of same family and even among different families differed non-significantly (P > 0.05).
- When different species of family Araneidae were compared for nucleotide frequency distribution significant difference was observed in G% distribution only (P =0.04).
- However, the variations in other nucleotides among different species were statistically non-significant (P> 0.05; Table 2).
- It is concluded that the variation in nucleotide frequency distribution in CO1 gene may not be useful for identification of spiders in the study area.
Selected from the article's own text.
Abstract
Introduction
Modern molecular techniques are efficient, affordable (Padial and De La Riva, 2007) and have been greatly accepted for identification and delimitation of species ;; . DNA barcoding is being widely used to identify and discover new species in a wide range of taxa . Cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA) was used to separate the insect species . Species delimitation is a necessary part of evolution, conservation and molecular ecology. Although there are several molecular markers (COI, CYTB, 16S, 18S, 28S, ITS1, and ITS2) available for species delimitation but no universal marker has been accepted yet . A molecular marker becomes ideal if it has a few properties like presence of single copy in haploid genome, easy to align, all sites equally free to vary, equal base composition, and high substitution rate . Recently, a handful number of universal markers are reported but finding all properties in a single marker is difficult . However, there are different molecular markers to delimit distinct species i.e., ITS1, ITS2 (second internal transcribed spacer regions) of the nuclear ribosomal gene cluster (18S rDNA, 5.8S rDNA and 28S rDNA), mitochondrial genes, 16S rDNA, 12S rDNA, CO1, COIII, nuclear ribosomal genes, 18S rDNA, nuclear protein-coding genes, 28S rDNA and others .
Table 1: Frequency distribution of nucleotide of cytochrome c oxidase subunit I among different families of spiders
| Spider Families | Frequency distribution (%) | ||||
| G | C | A | T | GC | |
| Araneidae | 19.48±0.64 | 13.4±0.05 | 24.96±0.05 | 41.30±0.14 | 32.84±0.1 |
| Lycosidae | 17.31±0.06 | 12.60±0.02 | 25.88±0.06 | 44.25±0.0.2 | 32.84±0.1 |
| Salticidae | 18.68±0.04 | 12.28±0.02 | 25.74±0.04 | 43.3±0.03 | 30.95±0.05 |
| Clubionidae | 18.01±0.06 | 12.3±0.06 | 26.32±0.06 | 42.73±0.045 | 30.95±0.08 |
| Oxyopidae | 17.18±0.09 | 12.34±0.055 | 27.19±0.13 | 43.28±0.04 | 29.53±0.12 |
| P- Value | > 0.05 | > 0.05 | > 0.05 | > 0.05 | > 0.05 |
Each value in the table represents the mean of 5 replicates and SEM (Standard Error of Mean).
Table 2: Frequency distribution of nucleotide of cytochrome c oxidase subunit I among different species of spider
| Frequency distribution (%) | ||||||||||
| Species of family Araneidae | G | C | A | T | GC | |||||
| Araneus mitficans | 19.58 ±0.05 | 13.16 ±0.05 | 24.98 ±0.07 | 42.28 ±0.04 | 32.74 ±0.09 | |||||
| Argiope pulchella | 19.07± 0.04 | 13.41 ±0.1 | 26.29 ± 0.06 | 41.22± 0.16 | 32.48 ± 0.1 | |||||
| Argiope trifasciata | 18.06±0.1 | 12.84±0.11 | 26.29±0.23 | 42.92±0.09 | 30.8± 0.2 | |||||
| Cheiracanthium inornatum | 17.2±0.15 | 12.44±0.1 | 27±0.07 | 43.36±0.14 | 29.64±0.2 | |||||
| Cyclosa chichawatnensis | 21.95±0.15 | 15.21±0.1 | 24.7± 0.07 | 38.14±0.14 | 37.16±0.2 | |||||
| Cyrtophora citricola | 18.33±0.04 | 14.44±0.07 | 27.29±0.05 | 39.67±0.06 | 33.04±0.1 | |||||
| Eriovixia exceisa | 23.59±0.09 | 14.42±0.03 | 21.32±0.03 | 40.67±0.1 | 38.01±0.09 | |||||
| Neoscona thesi | 18.3±0.04 | 12.19±0.02 | 28.18±0.05 | 41.32±0.04 | 30.5±0.03 | |||||
| Neoscona vigilans | 18.75±0.04 | 12.52±0.03 | 26.57±0.04 | 42.16±0.04 | 31.27±0.07 | |||||
| P- Value | 0.04 | >0.05 | >0.05 | >0.05 | >0.05 | |||||
| Species of family Lycosidae | ||||||||||
| Wadicosa fedalis | 17.79±0.05 | 12.65±0.03 | 25.52±0.05 | 44.04±0.04 | 30.44±0.06 | |||||
| Trocosa sp. | 17.33±0.09 | 12.77±0 | 25.84±0.09 | 43.92±0 | 29.94±0.09 | |||||
| Trocosa aquatic | 16.7 ±0.03 | 12.58±0.02 | 25.85±0.03 | 44.86±0.03 | 29.29±0.02 | |||||
| Draposa oakleyi | 17.34±0.07 | 12.82±0.04 | 26.15±0.07 | 43.7±0.05 | 30.15±0.07 | |||||
| Lycosa Terrestris | 17.26±0.04 | 12.77±0.01 | 25.45±0.04 | 44.52±0.02 | 30.03±0.04 | |||||
| Hippasa pisaurina | 17.48±0.08 | 12.06±0.03 | 25.99±0.08 | 44.47±0.03 | 29.54±0.1 | |||||
| P- Value | > 0.05 | > 0.05 | > 0.05 | > 0.05 | > 0.05 | |||||
| Species of family Salticidae | ||||||||||
| Trite sp. | 19.36±0.05 | 12.29±0.02 | 24.13±0.04 | 44.23±0.03 | 31.64±0.06 | |||||
| Thynes imparialis | 19.12±0.03 | 12.68±0.03 | 25.93±0.04 | 42.28±0.03 | 31.8±0.05 | |||||
| Talemonia dimidiate | 17.56±0.05 | 11.87±0.03 | 27.17±0.04 | 43.4±0.04 | 29.43±0.04 | |||||
| P- Value | > 0.05 | > 0.05 | > 0.05 | > 0.05 | > 0.05 | |||||
| Species of family Clubionidae | ||||||||||
| Clubiona drassodes | 17.77±0.05 | 13.21±0.09 | 26.55±0.04 | 42.48±0.12 | 30.98±0.12 | |||||
| Clubiona filicata | 18.25±0.07 | 12.66±0.03 | 26.09±0.05 | 42.99±0.04 | 30.92±0.08 | |||||
| P- Value | > 0.05 | > 0.05 | > 0.05 | > 0.05 | > 0.05 | |||||
| Species of family Oxyopidae | ||||||||||
| Oxyopes hindastanicus | 17.38±0.08 | 12.32±0.05 | 26.93±0.1 | 43.37±0.04 | 29.7±0.1 | |||||
| Oxypes ozyrae | 16.99±0.1 | 12.37±0.06 | 27.45±0.16 | 43.19±0.04 | 29.36±0.14 | |||||
| P- Value | > 0.05 | > 0.05 | > 0.05 | > 0.05 | > 0.05 | |||||
Each value in the table represents the mean of 5 replicates and SEM (Standard Error of Mean).
CO1 is the most popular marker used in DNA barcoding for delimiting species in the whole animal kingdom ; . This genetic region is important and widely used due to some distinguishing characteristics like presence across the animal kingdom, rare mutations (insertions and deletions) and enough divergence in sequence adequate to differentiate closely related species ; .
Despite of presence of many markers for species identification and delimitation, still there is unmet need to find more validated markers. For this purpose, the current study was designed to test whether variation in frequency distribution of nucleotide in CO1 in spiders could be used as a marker to delimit species or not.
Methodology
For the study, 658 base pair sequences of COI from 25 species of spiders belonging to five families were used. The study was conducted at Government College University Lahore in 2017. Spiders were collected by using pitfall traps and handpicking method. All the specimens were preserved in 70% alcohol for morphological study. The specimens used for molecular study were preserved in 95% alcohol and kept at -20oC in the refrigerator of Molecular Laboratory of Zoology Department, Government College University Lahore. Morphological identification of spiders was completed following the keys provided by Barrion and Litsinger (1995). Each species was represented by at least five individuals. Tissues for DNA extraction was taken from first leg of left side from each individual and after completing the necessary formalities tissue samples were sent to Centre for Biodiversity Genomics, University of Guelph, Canada for sequencing. A total of 110 samples belonging to 22 species and five families were processed for sequencing of CO1 gene. The nucleotide frequency distribution was computed using online sequence analysis tool available on Barcode of Life Data System (www.boldsystems.org). Nucleotide frequency distribution of CO1 gene among all members of same species and between different families was compared using one-way Analysis of variance (SPSS 16). GC composition within members of same species and between different families was also compared. The differences were considered significant if P < 0.05.
Results and Discussion
Results showed that the frequency of nucleotides among members of same species, within different species of same family and even among different families differed non-significantly (P > 0.05). The frequency of thymine nucleotide among members of all families was highest (about 41%), followed by adenine, guanine and cytosine (Table 1). Sequence composition of GC base pairs among individuals of five studied families also varied non-significantly. No clear variation within different species of same family or among families was found.
When different species of family Araneidae were compared for nucleotide frequency distribution significant difference was observed in G% distribution only (P =0.04). However, the variations in other nucleotides among different species were statistically non-significant (P> 0.05; Table 2). Although, there was variation in nucleotide frequency distribution among species of family Lycosidae, Salticidae, Clubionidae and Oxyopidae but statistically non-significant differences were found (Table 2).
Cytochrome c oxidase subunit I (CO1) gene of mitochondrial DNA (mtDNA) was used to separate the insect species but in this study no significant variation was found in nucleotide frequency distribution within members of same species or even within different species of same family. Previously this aspect has not been discussed in literature by researchers. The samples were compared collected from agricultural fields of central Punjab, Pakistan. Although spiders were collected from different habitats but the difference in the climatic conditions and microhabitats were almost the same. This might be the possible difference of non-significant difference in nucleotide frequency distribution in members of same species or different species of same family. There is need to collect samples from different geographical ranges to establish the role of nucleotide frequency distribution of nucleotide in species delimitation.
It is concluded that the variation in nucleotide frequency distribution in CO1 gene may not be useful for identification of spiders in the study area.
Acknowledgement
Authors thank to the Office of Research, Innovation and Commercialization (ORIC), Government College University, Lahore for providing research grant for this study under the project grant No. ORIC/101. We also thank to the Centre for Biodiversity Genomics, University of Guelph, Canada.
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