Genetic diversity of mango leafhopper, Amritodus atkinsoni (Hemiptera: Cicadellidae) based on mtCOI gene sequences from India

Abstract

The mango leafhopper, Amritodus atkinsoni is a serious and an endemic insect pest of mango in India. We analyzed mtCOI gene sequences from six Indian populations of A. atkinsoni for genetic diversity and population structure. The analysis of mtCOI sequence revealed 14 unique haplotypes and a low level of nucleotide diversity. mtCOI gene sequence analysis also revealed that A. atkinsoni specimens were clearly differentiated from other closely related species with a high level of accuracy.

Keywords:

• Genetic structure
• haplotypes
• phylogeography

Introduction

Mango leafhopper Amritodus atkinsoni (Lethierry) is one of the most economically important insect pests of mango (Mangifera indica L.) distributed mainly in South Asian and South East Asian countries (Waterhouse 1993). Amritodus atkinsoni is considered as the native of India and Pakistan (Pena et al. 1998) and causes 20–100% yield loss (Choudhary et al. 2012). Inspite the status of the pest, information on the population genetics of A. atkinsoni is not available from any region of the world.

Mitochondrial COI gene has been extensively used in population studies and identification of insects have unique features i.e. non recombinant, high copy numbers, simple maternal inheritance, high rate of evolution and robust evolutionary markers for determining intra- and inter-specific relationships of various invertebrate taxa including many insect species (Lunt et al. 1996; Armstrong and Ball 2005; Choudhary et al. 2018).

Therefore, in this study, we have employed the mitochondrial COI gene to infer the genetic diversity of A. atkinsoni collected from different regions of India. The dataset of mtCOI gene developed for the species has the potential for rapid and accurate identification.

Materials and methods

Adult A. atkinsoni were collected from six locations within India (Table 1 and Figure 1(A)). The collected adults were identified on the basis of morphological descriptions given by Viraktamath and Mohan (2004). Voucher specimens were preserved with voucher ID: A1, A2, B1, B2, B3, B4 P3, P5, P8 and P6 in the Entomology Laboratory, ICAR RCER, Research Centre, Ranchi, India.

Figure 1. (A) Sites of Amritodus atkinsoni collections for the present study. BH: Bihar, JH: Jharkhand, OD: Odisha, TL: Telangana, GU: Gujarat and RJ: Rajasthan. (B) Median joining network of mtCOI haplotypes of Amritodus atkinsoni. Each circle represents haplotype, and size of the circle is proportional to the haplotype frequency. Colours indicate the proportion of individuals of different populations present in a haplotype. (C) Neighbor-joining tree of 102 mtCOI gene sequences of different species representing family Cicadellidae. Bootstrap values based on 1000 replications are shown near nodes.

Table 1. Details of sampling locations and genetic diversity indices of Amritodus atkinsoni.

Location, State	ID code	Latitude (N)	Longitude (E)	Elevation m (amsl)	Collection year	n	H	Hd	K ± SD	π ±SD
Ranchi, Jharkhand	JH	23°35′	85°33′	651	2012	18	5	0.48366	0.69935 ± 0.55347	0.00131 ± 0.00115
SK Nagar, Gujarat	GU	24°12′	72°12′	209	2012	3	3	1.0	1.33333 ± 1.09834	0.00248 ± 0.002551
Sangareddy, Telangana	TL	17°62′	78°09′	524	2013	20	6	0.57368	0.76316 ± 0. 58387	0.00142 ± 0.001214
Patna, Bihar	BH	25°61′	85°14′	58	2013	18	6	0.49020	0.77778 ± 0.59406	0.00145 ± 0.001237
Bhubaneswar, Odisha	OD	20^o27′	85^o84′	45	2013	17	3	0.22794	0.23529 ± 0.28567	0.00044 ± 0.000595
Jaipur, Rajasthan	RJ	26°92′	75°82′	431	2013	9	1	0.0	0.0 ± 0.0	0.0 ± 0.0
All						85	14	0.88291	0.641 ± 0.039	0.00164 ± 0.00019

n: Number of samples; H: Number of unique haplotypes; Hd: Haplotypes diversity; K: Average number of nucleotide differences; π: Nucleotide diversity; SD: Standard deviation.

Total genomic DNA was extracted from individual specimens with methods described in Prabhakar et al. (2009). The mtCOI gene was amplified using UEA7 (F) and UEA10 (R) developed by Lunt et al. (1996) with PCR conditions mentioned in Prabhakar et al. (2012). These were then freeze-dried and custom sequenced using same upstream and downstream primers (Xcelris Labs Limited, India).

GenBank Accession Numbers: KC513467- KC513474 and KY084430-KY084442.

Nucleotide sequence analysis

The 85 mtCOI gene sequences of A. atkinsoni were aligned using ClustalW implemented in MEGA 6.0. (Tamura et al. 2013). Descriptive statistics of sequences were calculated with DnaSP (Librado and Rozas 2009). A median-joining network was constructed using NETWORK 4.6 (Bandelt et al. 1999). Phylogenetic reconstruction with 102 sequences representing the family Cicadellidae were analysed with the neighbor-joining method in MEGA 6.0. The “best close match” method in the programme TaxonDNA (Meier et al. 2006) was performed to test the frequency of successful identification.

Results and discussion

mtCOI gene sequences of 537 bp were obtained and aligned from 85 individuals of A. atkinsoni from six populations in India. The number of haplotypes per population (H) ranged from 1 to 6, haplotype diversity (Hd) from 0 to 1.000, average number of nucleotide differences within populations (K) from 0 to 0.23529 ± 0.28567 and nucleotide diversity (π) ranged from 0 to 0.00248 ± 0.002551 (Table 1). A total of 14 haplotypes were identified. The most common haplotype was H2 and H5 comprised of 43 and 28 individuals of A. atkinsoni, respectively. Haplotype 2 (H2) was shared by five populations [JH (Jharkhand), GU (Gujarat), TL (Telangana), BH (Bihar) and OD (Odisha)] except RJ (Rajasthan) (Figure 1(B)). The analysis of mtCOI sequences demonstrated low values of nucleotide diversity and relatively high value of haplotype diversity with no geographic pattern. Most of the populations shared one or both of the most frequent haplotypes (H2 and H5). When values of haplotype (Hd > 0.5) and nucleotide (π > 0.5%) diversity are high, the population appears to be stable with a relatively long evolutionary history (Rosetti and Remis 2012). None of the population presently examined meet these conditions, suggesting recent and unstable evolutionary history of the species. Low values of π and high values of Hd suggest the existence of small populations that have suffered recent population growth. Except R, this situation is observed for all the populations studied, where haplotype diversity is relatively high and nucleotide diversity relatively low. When both π and Hd values are low, the population has probably undergone a reduction in population size or a recent colonization event (Rosetti and Remis 2012). RJ is characterized by low values in both indices, reflecting recent population colonization.

The mtCOI gene analysis in the present study reveals A. atkinsoni to exhibit a high degree of diversity when compared with other hopper species. In Scaphoideus titanus Ball, COII gene sequences with 22 European and 8 Asian populations showed Hd and (π) value of 0.583 ± 0.072 & 0.203 ± 0.042 and 0.0046 ± 0.0027 & 0.0013 ± 0.0001 for nucleotide diversity, respectively, and showed moderate values for haplotype and nucleotide diversity (Papura et al. 2012). Genetic analysis of mtCOI gene sequences among 11 populations of rice brown planthopper, Nilaparvata lugens (Stål) and 5 populations of whitebacked planthopper, Sogatella furcifera (Horvath) from southeast Asia, showed low values of haplotype diversity 0.000–0.299 and 0.188–0.382 for N. lugens and S. furcifera, respectively (Mun et al. 1999), that were substantially lower than those observed in present study.

One hundred and two sequences from the family Cicadellidae were analysed for the accurate identification of A. atkinsoni. Intraspecific genetic divergence of 85 mtCOI gene sequences of A. atkinsoni ranged between 0 and 0.9% with an average of 0.50%. The best close match methods identified the 95% intraspecific genetic divergence threshold value 0.44% of A. atkinsoni identified all A. atkinsoni specimens correctly. Our results showed large gaps between intra and interspecific genetic distances for cicadellid species are consistent with previous reports on mtCOI gene based identification ofof insect species (Choudhary et al. 2018). DNA species barcode gaps can be detected by recording the overlap between the highest intraspecific and the lowest interspecific genetic distances (Meier et al. 2008). Phylogenetic analysis using the neighbor-joining method (Figure 1(C)) gave a strong bootstrap support (99%) for the monophyly of A. atkinsoni. The mtCOI gene sequences developed in the present study and its availability in GenBank could also be useful in identification of the species in the future (Virgilio et al. 2010; Jinbo et al. 2011). Incorrect species identification using the mtCOI is largely down to insufficient geographic sampling of the taxa (Jinbo et al. 2011). In this context and in relation to A. atkinsoni, our present results significantly increase the number of mtCOI gene sequences available, which will encourage the molecular identification of this and related important mango leafhopper species.

Acknowledgements

Authors are grateful to Mr. Deepak Tanti and Mr. Pradeep Lakra for collection of mango leafhoppers.

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

This work was supported by the Indian Council of Agricultural Research (ICAR), New Delhi, for providing financial assistance for this study under the National Innovations in Climate Resilient Agriculture (ICAR-RCER/RC R/E.F./2011/29).