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Threatenedtaxa.Org | 26 March 2014 | 6(3): 5544–5552 5545 Morphological and Molecular Identification of Grasshoppers Nazir Et Al

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Threatenedtaxa.Org | 26 March 2014 | 6(3): 5544–5552 5545 Morphological and Molecular Identification of Grasshoppers Nazir Et Al :ŽƵƌŶĂůŽĨdŚƌĞĂƚĞŶĞĚdĂdžĂͮǁǁǁ͘ƚŚƌĞĂƚĞŶĞĚƚĂdžĂ͘ŽƌŐͮϮϲDĂƌĐŚϮϬϭϰͮϲ;ϯͿ͗ϱϱϰϰʹϱϱϱϮ DÊÙ֫ʽʦ®½ÄÃʽç½Ù®Ä㮥®ã®ÊÄÊ¥Ù®® ÊÃÃçÄ®ã®ÊÄ ¦ÙÝÝ«ÊÖÖÙÝ;Ù®®͗KÙã«ÊÖãÙͿ¥ÙÊÃWÊÊÄ«®ò®Ý®ÊÄ͕ /^^E þ:ÃÃç<ݫîÙ͕W»®ÝãÄ KŶůŝŶĞϬϵϳϰʹϳϵϬϳ WƌŝŶƚϬϵϳϰʹϳϴϵϯ EĂŝůĂEĂnjŝƌϭ͕<ŚĂůŝĚDĞŚŵŽŽĚϮ͕DƵŚĂŵŵĂĚƐŚĨĂƋϯΘ:ƵŶĂŝĚZĂŚŝŵϰ KWE^^ 1,2,4 Department of Entomology, University of Poonch, Rawalakot, Azad Jammu Kashmir 12350, Pakistan 3 ŝŽĚŝǀĞƌƐŝƚLJ/ŶƐƟƚƵƚĞŽĨKŶƚĂƌŝŽ͕hŶŝǀĞƌƐŝƚLJŽĨ'ƵĞůƉŚ͕KŶƚĂƌŝŽ͕ĂŶĂĚĂ͕Eϭ'Ϯtϭ 1 [email protected] (corresponding author),2 [email protected],3 [email protected], 4 [email protected] ďƐƚƌĂĐƚ͗dŚĞƉƌĞƐĞŶƚƐƚƵĚLJǁĂƐĐŽŶĚƵĐƚĞĚƚŽƌĞƐŽůǀĞĐŽŶŇŝĐƚƐŝŶƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨŐƌĂƐƐŚŽƉƉĞƌƐƉĞĐŝĞƐŽĨƚŚĞĨĂŵŝůLJĐƌŝĚŝĚĂĞ;KƌƚŚŽƉƚĞƌĂͿ ŽŶƚŚĞďĂƐŝƐŽĨŵŽƌƉŚŽůŽŐLJĂŶĚEďĂƌĐŽĚŝŶŐ͘'ƌĂƐƐŚŽƉƉĞƌƐƌĞƉƌĞƐĞŶƟŶŐϮϲƐƉĞĐŝĞƐŽĨƚŚĞĨĂŵŝůLJĐƌŝĚŝĚĂĞǁĞƌĞĐŽůůĞĐƚĞĚĨƌŽŵĚŝīĞƌĞŶƚ ŚĂďŝƚĂƚƐĂŶĚŚŽƐƚƉůĂŶƚƐĨƌŽŵWŽŽŶĐŚĚŝǀŝƐŝŽŶŽĨnjĂĚ:ĂŵŵƵ<ĂƐŚŵŝƌ͕WĂŬŝƐƚĂŶ͘^ƉĞĐŝŵĞŶƐǁĞƌĞŝĚĞŶƟĮĞĚƚĂdžŽŶŽŵŝĐĂůůLJĂŶĚEƐĞƋƵĞŶĐĞĚ ĨŽƌƚŚĞĐLJƚŽĐŚƌŽŵĞĐŽdžŝĚĂƐĞ;K/ͿďĂƌĐŽĚĞƌĞŐŝŽŶ͘ĂƌĐŽĚĞƐŽĨϭϵŵŽƌƉŚŽůŽŐŝĐĂůƐƉĞĐŝĞƐǁĞƌĞƐƵĐĐĞƐƐĨƵůůLJŽďƚĂŝŶĞĚĂŶĚƚŚĞƐĞƋƵĞŶĐĞĚĂƚĂ ǁĂƐƵƐĞĚƚŽƐĞƉĂƌĂƚĞƐƉĞĐŝĞƐďLJEĞŝŐŚďŽƌͲ:ŽŝŶŝŶŐĐůƵƐƚĞƌĂŶĂůLJƐŝƐ͘ĂƌĐŽĚĞĚĂƚĂƐƵĐĐĞƐƐĨƵůůLJĚŝƐĐƌŝŵŝŶĂƚĞĚϭϴƐƉĞĐŝĞƐ͕ǁŚŝůĞƚǁŽ͗Patanga japonica;ŽůŝǀĂƌ͕ϭϴϵϴͿĂŶĚP. succincta;:ŽŚĂŶŶƐŽŶ͕ϭϳϲϯͿĐŽƵůĚŶŽƚďĞĚŝƐƟŶŐƵŝƐŚĞĚƐŝŶĐĞƚŚĞLJƐŚĂƌĞĚƚŚĞďĂƌĐŽĚĞƐĞƋƵĞŶĐĞĂŶĚĐůƵƐƚĞƌĞĚ ƚŽŐĞƚŚĞƌŽŶƚŚĞEĞŝŐŚďŽƌͲ:ŽŝŶŝŶŐ;E:ͿƚƌĞĞ͘DŽƌƉŚŽůŽŐŝĐĂůůLJ͕ƐƉĞĐŝŵĞŶƐŽĨShirakiacris shirakii;ŽůşǀĂƌ͕ϭϵϭϰͿǁĞƌĞŝĚĞŶƟĮĞĚĂƐŽŶĞƐƉĞĐŝĞƐ͕ ďƵƚďĂƌĐŽĚĞĚĂƚĂƌĞǀĞĂůĞĚƚŚĂƚŝŶĂĚĚŝƟŽŶƚŽShirakiacris shirakii;ŽůşǀĂƌ͕ϭϵϭϰͿƚǁŽŽƚŚĞƌƐƉĞĐŝĞƐŽĨƚŚĞŐĞŶƵƐShirakiacris are present in ƚŚĞƌĞŐŝŽŶ͘^ŝŵŝůĂƌůLJ͕ŽŶƚŚĞďĂƐŝƐŽĨŵŽƌƉŚŽůŽŐŝĐĂůĐŚĂƌĂĐƚĞƌƐƚǁŽƐƉĞĐŝĞƐǁĞƌĞŝŶĚĞŶƟĮĞĚŝŶƐƵďĨĂŵŝůLJĂƚĂŶƚŽƉŝŶĂĞ͕Catantops erubescens ;tĂůŬĞƌ͕ϭϴϳϬͿand Xenocatantops brachycerus;tŝůůĞŵƐĞ͕ϭϵϯϮͿ͕ďƵƚďĂƌĐŽĚĞĚĂƚĂƐƵŐŐĞƐƚƚŚĞƉƌĞƐĞŶĐĞŽĨĂŶĂĚĚŝƟŽŶĂůCatantops species ŝŶƚŚĞƌĞŐŝŽŶ͘dŚĞƐĞĮŶĚŝŶŐƐƐŚŽǁƚŚĞƵƐĞĨƵůŶĞƐƐŽĨďĂƌĐŽĚĞĚĂƚĂŝŶĚŝƐĐƌŝŵŝŶĂƟŶŐŐƌĂƐƐŚŽƉƉĞƌƐƉĞĐŝĞƐĂŶĚŝŶĚŝĐĂƚĞƚŚĂƚƐƵĐŚĚĂƚĂĐĂŶďĞ ƌĞůŝĂďůLJƵƐĞĚĨŽƌĚĞǀĞůŽƉŝŶŐƌĞĨĞƌĞŶĐĞůŝďƌĂƌŝĞƐĨŽƌƐƉĞĐŝĞƐŝĚĞŶƟĮĐĂƟŽŶǀŝĂƐĞƋƵĞŶĐĞŵĂƚĐŚĞƐ͘ <ĞLJǁŽƌĚƐ͗ĐƌŝĚŝĚĂĞ͕K/͕EďĂƌĐŽĚŝŶŐ͕<ĂƐŚŵŝƌ͕ŵŽƌƉŚŽůŽŐŝĐĂůŝĚĞŶƟĮĐĂƟŽŶ͘ K/͗ŚƩƉ͗ͬͬĚdž͘ĚŽŝ͘ŽƌŐͬϭϬ͘ϭϭϲϬϵͬ:Ždd͘ŽϯϱϬϳ͘ϱϱϰϰͲϱϮͮŽŽĂŶŬ͗ƵƌŶ͗ůƐŝĚ͗njŽŽďĂŶŬ͘ŽƌŐ͗ƉƵď͗ϯϮϭϱϲͲϱϳ&ͲϰϮϮͲϵϲϱϱͲϰϲϳϰϰϬϬ ĚŝƚŽƌ͗R.K. Avasthi, Rohtak University, Haryana, India. ĂƚĞŽĨƉƵďůŝĐĂƟŽŶ͗ϮϲDĂƌĐŚϮϬϭϰ;ŽŶůŝŶĞΘƉƌŝŶƚͿ DĂŶƵƐĐƌŝƉƚĚĞƚĂŝůƐ͗DƐηŽϯϱϬϳͮZĞĐĞŝǀĞĚϮϵ:ĂŶƵĂƌLJϮϬϭϯͮ&ŝŶĂůƌĞĐĞŝǀĞĚϭϱDĂƌĐŚϮϬϭϰͮ&ŝŶĂůůLJĂĐĐĞƉƚĞĚϭϴDĂƌĐŚϮϬϭϰ ŝƚĂƟŽŶ͗EĂnjŝƌ͕E͕͘<͘DĞŚŵŽŽĚ͕D͘ƐŚĨĂƋΘ:͘ZĂŚŝŵ(2014). DŽƌƉŚŽůŽŐŝĐĂůĂŶĚŵŽůĞĐƵůĂƌŝĚĞŶƟĮĐĂƟŽŶŽĨĂĐƌŝĚŝĚŐƌĂƐƐŚŽƉƉĞƌƐ;ĐƌŝĚŝĚĂĞ͗KƌƚŚŽƉƚĞƌĂͿĨƌŽŵ Poonch division, Azad Jammu Kashmir, Pakistan. Journal of Threatened Taxaϲ;ϯͿ͗ϱϱϰϰʹϱϱϱϮ; ŚƩƉ͗ͬͬĚdž͘ĚŽŝ͘ŽƌŐͬϭϬ͘ϭϭϲϬϵͬ:Ždd͘ŽϯϱϬϳ͘ϱϱϰϰͲϱϮ ŽƉLJƌŝŐŚƚ͗ ©EĂnjŝƌĞƚĂů͘ϮϬϭϰ͘ƌĞĂƟǀĞŽŵŵŽŶƐƩƌŝďƵƟŽŶϯ͘ϬhŶƉŽƌƚĞĚ>ŝĐĞŶƐĞ͘:ŽddĂůůŽǁƐƵŶƌĞƐƚƌŝĐƚĞĚƵƐĞŽĨƚŚŝƐĂƌƟĐůĞŝŶĂŶLJŵĞĚŝƵŵ͕ƌĞƉƌŽĚƵĐƟŽŶĂŶĚ ĚŝƐƚƌŝďƵƟŽŶďLJƉƌŽǀŝĚŝŶŐĂĚĞƋƵĂƚĞĐƌĞĚŝƚƚŽƚŚĞĂƵƚŚŽƌƐĂŶĚƚŚĞƐŽƵƌĐĞŽĨƉƵďůŝĐĂƟŽŶ͘ &ƵŶĚŝŶŐ͗^ĞƋƵĞŶĐĞĂŶĂůLJƐŝƐǁĂƐŵĂĚĞƉŽƐƐŝďůĞďLJĂŐƌĂŶƚĨƌŽŵ'ĞŶŽŵĞĂŶĂĚĂĂŶĚƚŚĞKŶƚĂƌŝŽ'ĞŶŽŵŝĐƐ/ŶƐƟƚƵƚĞŝŶƐƵƉƉŽƌƚŽĨƚŚĞ/ŶƚĞƌŶĂƟŽŶĂůĂƌĐŽĚĞŽĨ>ŝĨĞ WƌŽũĞĐƚ͘&ŝŶĂŶĐŝĂůƐƵƉƉŽƌƚǁĂƐĂůƐŽƉƌŽǀŝĚĞĚďLJ,ŝŐŚĞƌĚƵĐĂƟŽŶŽŵŵŝƐƐŝŽŶWĂŬŝƐƚĂŶďLJŐƌĂŶƚ,EŽ͘ϮϬͲϭϰϬϯͬZΘͬϬϵ͘ ŽŵƉĞƟŶŐ/ŶƚĞƌĞƐƚ͗dŚĞĂƵƚŚŽƌƐĚĞĐůĂƌĞŶŽĐŽŵƉĞƟŶŐŝŶƚĞƌĞƐƚƐ͘ ƵƚŚŽƌŽŶƚƌŝďƵƟŽŶĂŶĚĞƚĂŝůƐ͗E®½Eþ®ÙͲdŚĞƉƌŝŶĐŝƉůĞĂƵƚŚŽƌ͕ŝƚǁĂƐŚĞƌD^Đ;,ŽŶƐ͘ͿƌĞƐĞĂƌĐŚǁŽƌŬ͘EŽǁƐŚĞŝƐǁŽƌŬŝŶŐĂƐĂůĞĐƚƵƌĞƌ͘<«½®D«ÃÊÊ ͲĐŚĂŝƌŵĂŶĂŶĚƐƵƉĞƌǀŝƐŽƌĚƵƌŝŶŐƚŚĞƐƚƵĚLJ͘,ĞŝƐĂŶŽƌƚŚŽƉƚĞƌŝƐƚĂŶĚĐƵƌƌĞŶƚůLJǁŽƌŬŝŶŐŽŶƐŽŵĞŐĞŶĞƌĂŽĨĐƌŝĚŝĚĂĞ͘Dç«ÃÃÝ«¥ØͲĐŽͲƐƵƉĞƌǀŝƐŽƌ͕ŚĞ ǁĂƐǁŽƌŬŝŶŐĂƐĨŽƌĞŝŐŶƉƌŽĨĞƐƐŽƌŝŶE/'&ĂŝƐůĂďĂĚ͕WĂŬŝƐƚĂŶ͘,ŝƐƌĞƐĞĂƌĐŚŝŶƚĞƌĞƐƚƐĂƌĞŵŽůĞĐƵůĂƌďŝŽůŽŐLJĂŶĚEďĂƌĐŽĚŝŶŐŽĨĂƌƚŚƌŽƉŽĚƐ͘,ĞĐŽŶƚƌŝďƵƚĞĚŝŶ planning, carrying out the study and analyzing the sequence data. :çÄ®Z«®ÃͲ assisted during the research in all aspects. ĐŬŶŽǁůĞĚŐĞŵĞŶƚƐ͗&ŝƌƐƚĂƵƚŚŽƌŝƐŐƌĞĂƚůLJƚŚĂŶŬĨƵůƚŽ/ŶƐĞĐƚDŽůĞĐƵůĂƌ>ĂďE/';EĂƟŽŶĂů/ŶƐƟƚƵƚĞŽĨŝŽƚĞĐŚŶŽůŽŐLJĂŶĚ'ĞŶĞƟĐŶŐŝŶĞĞƌŝŶŐͿ&ĂŝƐůĂďĂĚĨŽƌ ƉƌŽǀŝĚŝŶŐŵĞƌĞƐĞĂƌĐŚĨĂĐŝůŝƟĞƐĂůůƚŚŽƐĞƉĞŽƉůĞ;^ůĞĞŵŬŚƚĞƌ͕DĂƌLJƵŵDĂƐŽŽĚ͕ZŽŵĂŶĂ/ŌŚŬĂƌͿƚŚĞƌĞĂƐƐŝƐƚĞĚŵĞĚƵƌŝŶŐŵLJǁŽƌŬ͘/ǁŽƵůĚůŝŬĞƚŽŽīĞƌŐƌĞĂƚ ƐĞŶƐĞŽĨŐƌĂƟƚƵĚĞƚŽǁĂƌĚƐŝŽĚŝǀĞƌƐŝƚLJ/ŶƐƟƚƵƚĞŽĨKŶƚĂƌŝŽ͕hŶŝǀĞƌƐŝƚLJŽĨ'ƵĞůƉŚ͕ĂŶĂĚĂĨŽƌƉƌŽǀŝĚŝŶŐŵĞĨĂĐŝůŝƟĞƐĨŽƌEďĂƌĐŽĚŝŶŐ͘DLJƐŝŶĐĞƌĞƚŚĂŶŬƐWƌŽĨ͘ ƌ͘D͘ZĂĮƋƵĞ<ŚĂŶĨŽƌƐƵƉƉŽƌƚ͕WƌŽĨƌ͘D͘ZĂŚŝŵ<ŚĂŶ͕DŝƐƐŶƐĂdĂŵŬĞĞŶ͕Dƌ͘ďĚƵů'ŚĂīĂƌ͕^ŚƵŵŝůĂƌŝĨ͕DƵŶĂnjnjĂ<ŚƵƌƐŚŝĚĨŽƌƚŚĞŝƌĂƐƐŝƐƚĂŶĐĞĚƵƌŝŶŐŵLJ ƌĞƐĞĂƌĐŚǁŽƌŬ͘/ĂŵŐƌĞĂƚůLJŝŶĚĞďƚĞĚƚŽDƌ:ƵŶĂŝĚZĂŚŝŵĨŽƌĂůůƐŽƌƚŽĨĂƐƐŝƐƚĂŶĐĞĚƵƌŝŶŐŵLJƌĞƐĞĂƌĐŚǁŽƌŬĂŶĚŵŽƌĂůƐƵƉƉŽƌƚ͘Support from Dr. Paul Hebert, ^ĐŝĞŶƟĮĐŝƌĞĐƚŽƌŽĨŝK>ŝƐĂĐŬŶŽǁůĞĚŐĞĚ͘ ϱϱϰϰ Morphological and molecular identification of grasshoppers Nazir et al. INTRODUCTION (Colgan 1991; Chapco & Litzenberger 2003; Rowell & Flook 2004). Several researchers have used DNA data Grasshoppers are the most prevalent pests in all in phylogenetic analysis to identify grasshopper species sorts of vegetation in pastures and grasslands. Family (Chapco & Litzenberger 2002; Mukha et al. 2001; Song Acrididae encompasses the short-horned grasshoppers & Wenzel (2007) Ketmaier et al. 2010). Use of DNA data and locusts, phytophagous insects that are widely has also been used in combination with morphological distributed throughout the world and considered ruinous data to establish species relationships (Brust 2008). in the arid zone (Watts et al. 1982). Taxonomists generally Keeping in view the economic importance of use morphological identification for studies used to plan grasshoppers and their damage to crops in Azad control strategies, but this method of identification has Kashmir, a need for correct identification of this group several limitations (Scotland et al. 2003). Cryptic species of pests has emerged. Azad Jammu & Kashmir lies (sibling species) may be incorrectly identified due to between 73–750N and of 33–360E and comprises an phenotypic malleability. Morphologically enigmatic taxa area of 5,134m2 (13,297km2) (Fig. 1). Poonch division are common in many groups neglected by this approach of Azad Jammu Kashmir comprises an area of 2,792km2. (Jarman & Elliott 2000). Morphological keys are often Its topography is mainly hilly, climatic conditions and limited to particular life stages, limiting the effectiveness floristic composition significantly varies from place to of identification. Finally, a high level of proficiency is place. Administratively, this division consists of four required to use the keys to avoid misdiagnoses. This has districts, Bagh, Poonch, Sudhnoti, and Haveli. A survey led to the use of molecular data to resolve cryptic species was conducted to identify grasshopper species of family (Xiao et al. 2010). In micro genomic identification, Acrididae from Poonch division. Major contributions system differences among DNA sequences are used to to the Acrididae fauna of Kashmir have been provided identify the different organisms (Wilson 1995). In fact by some entomologists like Kirby (1914), Fletcher these sequences are genetic barcodes enclosed in each (1919), Mahmood (1995), Mahmood & Yousaf (1999), cell. The barcode region, a 658-bp nucleotide fragment Mahmood & Yousaf (2000); Mahmood et al. (2002); of mitochondrial COI has been accepted by scientists for identification of animal species (Hebert et al. 2003). N The use of short standardized gene regions as internal W E species tag to recognize the species is an accurate, S reliable, and rapid method. Due to copious benefits in identification, DNA barcoding is getting considerable concentration in the field of science (Hebert et al. 2004). The basic scientific advantage of DNA barcoding is fast and digital species identification at any life stage or piece of an organism, and the simplification of species explorations (Janzen et al. 2005). The selected DNA sequence precisely separates the species on the basis of interspecific and intraspecific variations (Matz & Nielsen 2005). Barcoding has helped in resolving cryptic species complexes (Burns et al. 2007; Deng et al. 2012) and performing ecological studies on various animal phyla (Valentini et al. 2009). The generated data is also being used to construct barcode reference libraries for identification of unknowns by matching sequences with the known species (Guralnick & Hill 2009; Janzen et al. 2009). A combination of molecular and morphological data can produce reliable data sets to be used in barcode libraries (Emery et al. 2009). Use of PCR as a tool to amplify and sequence genes and then exploit the nucleotide data for phylogenetic analysis and develop evolutionary relationships among grasshopper species Figure 1. Map of Azad Kashmir illustrated Poonch division with has previously been practiced by a number of researchers highlighted collection localities Journal of Threatened Taxa | www.threatenedtaxa.org | 26 March 2014 | 6(3): 5544–5552 5545 Morphological and molecular identification of grasshoppers Nazir et al. Table 1. Characteristics of collection localities of study area cyanide and stretched out on the stretching board with District Locality Altitude(ft) Latitude Longitude the help of standard entomological pins (No. 16–40). Rawalakot 5393 33.80N 73.80E The specimens were dried, examined with the use of a Leica MZ6 microscope and identified using keys (Bie- Hajeera 3167 33.60N 73.30E Bienko & Mischenko (1951), Drish (1961), Ritchie (1982), Jandali 6785 33.30N 73.30E Poonch and Mason (1973), Suhail (1994), Mahmood (1995). The 0 0 Banjosa 6212 33.0 N 73.9 E terminology of Kirby (1914) and Bie-Benko & Mischenko 0 0 Tolipeer 8800 33.0 N 73.9 E (1951) were used in this identification process. The Abbaspur 4261 33.60N 73.00E specimens of each identified species were confirmed Mang 4842 33.20N 73.90E from (Eades et al. 2011). Plundari 4000 33.20N 73.90E Sudhnoti Baloch 5304 33.80N 73.00E Sequencing/ DNA barcoding Trarkhel 5600 33.90N 73.70E Morphologically identified grasshopper specimens Khautta 5189
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