Advances in Biochemistry and Biotechnology (ISSN: 2574-7258)

Article / research article

"DNA Barcoding and Evolutionary Lineage of Some Economically Important Scarabaeid Beetles in South India"

K Srinivasa Murthy*, Syeda Lubna Banu, A Ranjitha, Sharad Pattar

ICAR-National Bureau of Agricultural Insect Resources, Bangalore, Karnataka, India

*Corresponding author: K Srinivasa Murthy, ICAR-National Bureau of Agricultural Insect Resources, P,B.No 2491, H.A. Farm post, Bellary Road, Karnataka, India 560024. Email: ksm239@rediffmail.com

Received Date: 29 November, 2017; Accepted Date: 26 December, 2017; Published Date: 04 January, 2018

1.      Abstract

Proper identification of the scarabaeid beetle’s species and knowledge of their bio ecology is essential for developing environmentally compatible integrated pest management strategies. Lack of taxonomic understanding has been a major impediment to the study and management of scarabaeid beetles. Identification of scarabaeid species is a challenging task due to variable morphological differences among species and delineation among the immature forms, the grubs and adults. DNA barcoding facilitates prompt identification of the pest utilizing fragmentary body parts. Barcodes were generated to identify the scarabaeid beetles from various geographical locations in South India, based on the mitochondrial Cytochrome c oxidase I (COI) gene (648-656 bp size). Genomic DNA of 23 scarabeaid beetles obtained from different geographical locations were characterized and a total of 19 barcodes were generated with Barcode Identity Number (BIN) in Bold Systems V3 database. Phylogeny for evolutionary relationship and divergence was assessed using MEGA (Molecular Evolutionary Genetic Analysis) program and Neighbor -Joining (NJ) methods, the overall mean distance was 0.215% and the pairwise genetic distance ranged from 0.000 to 0.384 The phylogeny revealed formation of distinct species-specific clusters. Nucleotide composition, genetic variations and sequences similarities were worked out. The composition of the mitochondrial sequence of the COI gene in the present study was expectedly AT biased. Molecular sequence information from NCBI revealed relatedness in all the collected scarabaeids, accurately as revealed by their morphological characters. The implications of the information generated for species delineation of scarabaeid beetles and increased accuracy in their management is presented

2.      Keywords: Barcoding; COI; DNA; Phylogeny; Scarabaeid Beetles

1.      Introduction

Scarabaeidae is the second largest family within the order Coleoptera, and is cosmopolitan in distribution [1]. Scarab beetles are the most diverse and widely distributed insects which belong to the largest order Coleoptera under superfamily Scarabaeoidea, the family Scarabaeidae is composed of about 91% of all scarabaeoids and represented by 30,000 species worldwide [2,3]. Maximum numbers occur in the tropical areas of the world, particularly in the African and Oriental regions. 

Persusal of the data on the collection of scarabaeid beetles from the different geographical locations and crops and their identification had revealed the diversity of beetles in the country. An array of phytophagous beetles (Table 1) belonging to the subfamilies (dynastinae, melolonthinae and rutelinae) were recorded. Such a diversity was earlier reported from various locations of the country. [33], reported the diversity and relative abundance of pleurostrict scarabaeidae in the Achanakmar-Amkarkantak biosphere reserve in Chattisgarh state. About 22 species belonging to 11 genera and 6 subfamilies were reported from the region, while in Madhya Pradesh, 47 species were reported [6,34,35]. The genus Anomala predominated over among all the scarabeids in both the states. In Maharshtra, the occurrence of different species of Holotrichia was widespread on sugarcane, sorghum, groundnut and soybean crops in South konkan and Vidharba regions [34] and in Pune [36,37]. Holotrichia serrata was predominant among all the species recorded. In Himachal Pradesh, Anomala sp. followed by Brahmina sp. were dominant in Chamba, Kanra, Kullu and Shimla areas [38,39]. Congenial habitat, natural vegetation, food availability and appropriate soil type contribute to the diversity of scarabaeids and the species richness [40,41] In addition, climatological factors rainfall, humidity, temperature and wind velocity play a decisive role on the emergence, movement, distribution and bioecology of scarabaeids [3]. Knowledge on species diversity, abundance, richness and dominance through surveys would be helpful in planning strategies for conservation of natural enemies, habitat management, design and develop pest management strategies.

1.1.  Nucleotide Analysis

Nucleotide analysis of the sequences was carried out in order to find out the MCL (Maximum Composite Liklyhood) estimate of the pattern of nucleotide substitution, AT%, GC% and the AT content at first, second and third codon position. The MCL pattern showed the probability of substitution (r) from one base (row) to another base (column) [42]. The sum of r values was made equal to 100. Rates of different transitional are shown in substitutions which were 18.79, 21.13, 9.83 and 8.97 and the transversionsal substitutions are given in (Table 2). The nucleotide frequencies are 27.99% (A), 40.02% (T/U), 18.63% (C), and 13.36% (G). The transition/ transversion rate ratios are k 1 = 3.253 (purines) and k 2 = 2.558 (pyrimidines). The overall transition/ transversion bias is R = 1.288, where R = [A*G* k 1 + T*C* k 2]/[(A+G)*(T+C)].

The analysis also revealed that the percentage of AT was comparatively more i.e., 33.25% ranging between 29.8-34.9% than that of GC which is 16.8% with a minimum of 15.1% and maximum of 19.4% (Table 3), indicating that the sequences were AT biased. This difference was attributed to the AT percentage at different codon position. The AT content at first codon position ranged between 46-48% with average of 44%, and the AT percentage at second and third codon position is nearly invariant 27% and 28% respectively (Table 3). Therefore, higher genetic distance was observed at first codon position comparatively to the second and third codon position. The higher values at first codon position indicated that detailed studies on first codon position may reveal possible evolutionary information among the closely related species.

The utility of DNA data in taxonomy and species diagnosis in the scarabaeid beetles was reported by [43,44] based on the sequence variation in DNA based groups which was highly structured. The population of scarabaeids from various locations were characterized using Cytochrome C oxidase subunit I (COI) gene, which has been recognized as an effective marker not only for species identification and also for phylogenetic relationship [15,41,45]. In the present studies, the isolated genomic DNA of scarab beetles from various locations was characterized through COI gene fragment (648-656 bp size). The amplified gene was sequenced and the Blast done with NCBI database to decipher the identity of the scarabaeids from various locations and crops (Table-1). Molecular sequence information from NCBI revealed relatedness in all the collected scarabaeids, accurately as revealed by their morphological characters. Our observations, corroborate with the reports of [46-49,45,25]. [47], suggested that where sequence information is available in Genbank for morphologically defined species, which can be matched with some DNA based clusters, close relationship can be identified readily in sequence variation in field collected field samples and these clusters are likely to correspond to previously described unknown species [21,50]. reported that the sequence information based on mitochondrial markers can be utilized for species delineation of adults and grubs of scarabaeids inferring larval taxonomy. Our studies indicate the relevance of DNA sequencing to match different forms of scarabs and address the issues of having to depend exclusively on morphological features and avoid misdiagnosis.

1.2.  Phylogenetic Analysis

Analysis involved 22 nucleotide sequences which involved the 1st+2nd+3rd+Noncoding codon positions. A total of 133 positions for comparison were obtained in the final dataset using Kimura 2-parameter method, where all the positions containing gaps and missing data were eliminated. The overall mean distance was 0.215% and the pairwise genetic distance ranged from 0.000 to 0.384 (Table 4). The lowest genetic variation was observed between Hybosorus illigeri (Theni-GB-3b) and Oryctes rhinocerous (Theni-GB-2[b]) i.e., 0.008, whereas the highest genetic variation was 0.384 observed between Onthophagus auritus (Mudhi-Sc-2) with Calcinemis obesa (Theni-GB-2[a]) and Oryctes rhinocerous (Theni-GB-2[b]) (Table 4).

A phylogenetic tree of the species using Neighbour-Joining method [31] was drawn on the basis of multiple sequence alignment of COI gene. e tree elucidates of 22 concatenated samples of the COI genes from 12 different areas in Karnataka, Tamilnadu and Gujarat states. The phylogeny clearly indicated the phylogenetic generalized least squares (PGLS) and revealed formation of distinct species-specific clusters based on the geographical location of the scarabaeid population obtained. All the Onthophagus nuchicornis collected from parts of Karnataka were placed in the top cluster with the bootstrap value and branch length range of 19-100 and 0.00-0.10 respectively (Table 5).

The Anomala ruficapilla (sub family - Rutelinae) was clustered with Exomala pallidipennis (Rutelinae) and diversified as a polyphyletic clade in the tree, the interesting observation among the polyphyletics was that one cluster was from Karnataka (cluster-2) whereas the other one was from Tamilnadu (cluster-4). The geo-specie specific cluster 2 and 4 was mediated by Adoretus flavus obtained from Gujarat which clustered with that of Copris tripartitus (Scarabaeidae) from Karnataka. Apart from the above said clusters (1-4), three samples of garden chafer Phyllopertha horticola were placed Cluster-5 which were all from Karnataka region.

Our findings, concur with the observations made by [16,51], on the significance of phylogeny based on DNA data. Weak phylogenetic foundation is related to issues like rare information about scarabaied taxa at the family level. The classification of the world dynastinae is fairly well established, while in melolonthinae, rutelinae and cetoniinae that are poorly known taxonomically, new genera cannot be reliably identified. In the present study the species specificity might be due variations in the geographical location, the cropping pattern and the prevailing environmental condition.

Scarabaeidae species are often soil dwelling, their population densities and the associated damage risk probably depend on the site characteristics that influence soil temperature, soil moisture and humus content or the impact of management strategies imposed on the crop. The environmental conditions that are determining grub populations and the damage risk are fairly understood.

Nevertheless, a larger study of many populations and different genes may help reconstruction of the phylogeny and understand the evolutionary relationship. Further, phylogenetically, closely related species are likely to have a comparable physiology [15] which would facilitate precision management of the pest with insecticides. The present studies do not lead to exclusive inference, since a large number of scarabaeids are yet to be analyzed.

COI barcoding has the added advantage of not being limited by polymorphism, sexual form(asexual/sexual) and life stages of the target species [42]. Overall rate of nucleotide substitution, ratio of two specific instantaneous rates of substitution rate at which transitions and transversions occur and the rate variation among sites play a significant role and are necessary for accurate reconstruction of phylogeny [12,36]. The present study was based on the molecular identification of scarabeid beetles attacking crops and comprised of 23 nucleotide sequences were identified by COI.

Results indicated that the COI-based pest identification was extremely effective for the beetles based on the COI marker profile. Most of the phylogenetic information has been derived from mitochondrial DNA variations and recently DNA sequence data have been employed successfully to elucidate the relationships of many groups of insect species at generic level. The composition of the mitochondrial sequence of the COI gene in the present study was expectedly AT biased and this was generally observed in several previous studies [37]. In general, the frequency of transitional substitutions is known to be higher than transversion substitutions in the genome [27], According to 10X rule the percentage of nucleotide divergence between the intraspecies should be less than 3% and that of interspecies should be more than 3%. Hence the sequences analyzed in the present study exhibited high inter species variability on the basis of nucleotide sequences. Therefore, the intra specific divergence was higher enough to discriminate between the individuals.

Molecular identification was done for several pests worldwide, in Orius (Hemiptera: Anthocoridae) [19] and potato flea beetles (Coleoptera: Chrysomelidae) [18], Japanese beetles [52] and other scarabaeids [53]. Moreover, discrimination of aphids of 32 species collected in various host plants in South India was also reported [49]. This investigation of COI barcoding could potentially be applied in agricultural and horticultural researches to rapid identification of pests. The phylogenetic signal is a direct function of the length of the branch (in units of the expected number of substitutions per site), which sheds light the evolutionary relationship [45].

DNA barcoding using COI genes could be an effective method for screening insect pests and to shed light on their genetic variations in addition the integration of traditional taxonomy especially for the scarabaeids where the species delineation and larval taxonomy is a challenging task Our findings contribute to a better understanding of the identification of pests by COI genes and aid in formulating better management strategies.

2.      Conclusion

The diversity of phytophagous scarabaeid beetles from various geographical locations of India occurring in crops were morphologically identified and characterised using molecular tools, Molecular sequence information from NCBI revealed relatedness in all the collected scarabaeids, accurately as revealed by their morphological characters. Phylogenetic tree revealed the genetic relatedness among the beetles and understand the evolutionary relationship. The relevance of DNA sequencing to match different forms of beetles and address limitations in morphological identification is indicated. Knowledge on species diversity, abundance, richness and dominance through surveys would be helpful in planning strategies for conservation of natural enemies, habitat management, design and develop pest management strategies.

3.      Acknowledgements

The authors are thankful to the Director, ICAR-NBAIR, for providing the facilities. Identification of the scarabaeids by Dr ARV Kumar Department of Entomology, GKVK, Bangalore and Dr. K.Sreedevi , Division of Entomology, Indian Agriculture Research Institute, New Delhi is duly acknowledged. We also thank Dr. Veena kumari, Dr. A.N.Shylesha Principal Scientists and Dr. Jagdish Patil, Scientist of ICAR –NBAIR for the collections.


 

A

T

C

G

A

-

8.26

3.84

8.97

T

5.78

-

9.83

2.76

C

5.78

21.13

-

2.76

G

18.79

8.26

3.84

-


Table 2: MCL estimate of the pattern of nucleotide substitution of COI sequences of scarabaeid beetles.

 

Identified species

 

AT%

GC%

First

Second

Third

Protaetia cuprea (DAST-SC-16)

29.8

20.2

36

24

29

Phyllopertha horticola (Anekal-SC-4)

34.9

15.1

47

29

29

Exomala pallidipennis (Valam-SC-1)

32.3

17.7

43

26

28

Holotrichia serrata (DAST-SC-14)

29.9

20.1

38

24

28

Anomala ruficapilla (DAST-SC-19)

33.8

16.2

45

28

28

Onthophagus nuchicornis (Chick-SC-1)

34.5

15.5

47

29

28

 Adoretus flavus (Guj-SC-4)

34

16

47

27

29

Phyllopertha horticola (Doddashiv-SC-1)

34.9

15.1

47

29

29

Phyllopertha horticola (Doddashiv-SC-2)

34.5

15.5

45

28

29

Exomala pallidipennis (Yella-SC-1)

34.7

15.3

48

29

28

Onthophagus auritus (Mudhi-Sc-2)

33.8

16.2

46

27

28

Onthophagus coenobita (Nand-SC-1)

33.1

16.9

43

28

28

Onthophagus coenobita (Nand-SC-2)

32.9

17.1

43

28

28

Copris tripartitus (Nand-SC-3)

34.3

15.7

47

27

29

Onthophagus coenobita (Nand-SC-5)

32.9

17.1

43

27

28

Onthophagus nuchicornis (Chikka-SC-7)

34.3

15.7

47

28

28

Onthophagus nuchicornis (Chin-SC-1)

34.8

15.2

48

29

28

Onthophagus nuchicornis (Rajan-SC-1)

34.5

15.5

47

29

28

Anomala ruficapilla (Then-GB-1[a])

33.9

16.1

46

27

29

Calicnemis obese (Then-GB-2[a])

30.7

19.3

38

26

28

Oryctes rhinoceros (Then-GB-2[b])

31.4

18.6

39

26

29

Hybosorus illigeri (Theni GB-3b)

30.6

19.4

38

26

28

MEAN

33.2

16.8

44

27

28


Table 3: AT%, GC% and AT% at first, second and third codon position of scarabaeid beetles.


1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

2

0.25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

0.258

0.158

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

0.149

0.218

0.247

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5

0.207

0.197

0.159

0.217

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6

0.28

0.168

0.178

0.291

0.158

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7

0.216

0.197

0.187

0.217

0.131

0.159

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8

0.25

0.015

0.177

0.208

0.206

0.178

0.207

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9

0.25

0.015

0.177

0.208

0.206

0.178

0.207

0

 

 

 

 

 

 

 

 

 

 

 

 

 

10

0.258

0.206

0.178

0.247

0.15

0.158

0.15

0.206

0.206

 

 

 

 

 

 

 

 

 

 

 

 

11

0.313

0.197

0.208

0.296

0.237

0.159

0.197

0.206

0.206

0.227

 

 

 

 

 

 

 

 

 

 

 

12

0.315

0.291

0.281

0.318

0.216

0.168

0.237

0.302

0.302

0.269

0.178

 

 

 

 

 

 

 

 

 

 

13

0.315

0.291

0.281

0.318

0.216

0.168

0.237

0.302

0.302

0.269

0.178

0

 

 

 

 

 

 

 

 

 

14

0.247

0.197

0.258

0.228

0.206

0.177

0.123

0.206

0.206

0.207

0.227

0.268

0.268

 

 

 

 

 

 

 

 

15

0.315

0.291

0.281

0.318

0.216

0.168

0.237

0.302

0.302

0.269

0.178

0

0

0.268

 

 

 

 

 

 

 

16

0.28

0.168

0.178

0.291

0.158

0

0.159

0.178

0.178

0.158

0.159

0.168

0.168

0.177

0.168

 

 

 

 

 

 

17

0.28

0.168

0.178

0.291

0.158

0

0.159

0.178

0.178

0.158

0.159

0.168

0.168

0.177

0.168

0

 

 

 

 

 

18

0.28

0.168

0.178

0.291

0.158

0

0.159

0.178

0.178

0.158

0.159

0.168

0.168

0.177

0.168

0

0

 

 

 

 

19

0.247

0.168

0.237

0.259

0.187

0.227

0.159

0.187

0.187

0.197

0.177

0.248

0.248

0.217

0.248

0.227

0.227

0.227

 

 

 

20

0.281

0.269

0.259

0.269

0.237

0.292

0.25

0.258

0.258

0.384

0.315

0.259

0.259

0.269

0.259

0.292

0.292

0.292

0.279

 

 

21

0.281

0.269

0.259

0.269

0.237

0.292

0.25

0.258

0.258

0.384

0.315

0.259

0.259

0.269

0.259

0.292

0.292

0.292

0.279

0

 

22

0.27

0.258

0.248

0.258

0.227

0.28

0.239

0.247

0.247

0.372

0.303

0.27

0.27

0.258

0.27

0.28

0.28

0.28

0.268

0.008

0.008


Table 4: Pairwise genetic distance for partial COI sequences of scarabaeid beetles.

 

Sl. No.

 

Organism name

1

Protaetia cuprea (DAST-SC-16)

2

Phyllopertha horticola (Aneka-SC-4)

3

Exomala pallidipennis (Valam-SC-1)

4

Holotrichia serrata (DAST-SC-14)

5

Anomala ruficapilla (DAST-SC-19)

6

Onthophagus nuchicornis (Chick-SC-1)

7

Adoretus flavus (Guj-SC-4)

8

Phyllopertha horticola (Doddashiv-SC-1)

9

Phyllopertha horticola (Doddashiv-SC-2)

10

Exomala pallidipennis (Yella-SC-1)

11

Onthophagus auritus (Mudhi-Sc-2)

12

Onthophagus coenobita (Nand-SC-1)

13

Onthophagus coenobita (Nand-SC-2)

14

Copris tripartitus (Nand-SC-3)

15

Onthophagus coenobita (Nand-SC-5)

16

Onthophagus nuchicornis (Chikka-SC-7)

17

Onthophagus nuchicornis (Chin-SC-1)

18

Onthophagus nuchicornis (Rajan-SC-1)

19

Anomala ruficapilla (Then-GB-1[a])

20

Calicnemis obesa (Then-GB-2[a])

21

Oryctes rhinoceros (Then-GB-2[b])

22

Hybosorus illigeri (Theni-GB-3b)


Table 5: Chronological order of the scarabaeid beetles as indicated in the similarity matrix.

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Citation: Murthy KS, Banu SL, Ranjitha A, Pattar S (2017) DNA Barcoding and Evolutionary Lineage of Some Economically Important Scarabaeid Beetles in South India. Adv Biochem Biotechnol 2: 151. DOI: 10.29011/2574-7258.000051

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