DNA BASED IDENTIFICATION AND GENETIC DIVERSITY STUDIES OF SPECIES OF PAKISTAN

BY

SALEEM AKHTAR

A dissertation submitted to The University of Agriculture, Peshawar in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (PLANT GENOMICS & BIOTECHNOLOGY)

DEPARTMENT OF PLANT GENOMICS & BIOTECHNOLOGY PARC INSTITUTE OF ADVANCED STUDIES IN AGRICULTURE, THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN APRIL, 2018 DNA BASED IDENTIFICATION AND GENETIC DIVERSITY STUDIES OF ANTLION SPECIES OF PAKISTAN By

SALEEM AKHTAR

Approved by:

Foreigner Examiner 1

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Dr André Prost, M.D, PhD Secretary International Association of Neuropterology, Rue de l'église F 39320 Loisia- France Foreigner Examiner 2

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Prof. Dr. Prof. Roberto A. Pantaleoni Università degli Studi di Sassari Dipartimento di Protezione delle Piante Sezione di Entomologia, via Enrico De Nicola – 07100 SASSARI SS Italia

TABLE OF CONTENTS

ACKNOWLEDGEMENT i LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT viii I. INTRODUCTION 1 II. REVIEW OF LITERATURE 6 III. MATERIAL AND METHODS 11 IV. RESULTS 22 V. DISCUSSION 142 VI. SUMMARY 154 VII. RECOMMENDATION FOR FUTURE RESEARCH 155 SPECIES CHECKLIST MYRMELEONTIDAE OF 156 PAKISTAN LITERATURE CITED 158

ACKNOWLEDGEMENT

First and foremost, I would like to express my deepest gratitude to Allah Almighty.

I would like to thank my committee chair and advisor, Dr. Yusuf Zafar (T.I), for the opportunity to pursue a degree under his guidance and for all the hours of his invaluable advice and training. I would also like to thank my other committee members, Dr. Muhammad Ashfaq, Dr. Shoukat Ali, Dr. Syed Ahmed Zia, Dr. Ghulam Muhammad Ali and Dr. Farhatulla for their assistance during my research and in the completion of this thesis. Thanks also to the faculty, staff, and fellow students in the PARC Institute of Advance Studies in Agriculture, National Agricultural Research Centre (NARC), Islamabad for their excellent teaching, assistance and help in making my time here as a Ph.D. student enjoyable. I would like to specifically thank the members of the NIGAB and PEP laboratories for taking the time to help me during my research. I express my gratitude to the Higher Education Commission Islamabad Pakistan for financial support under 5000 Indigenous Scholarship and International Research Support Initiative Program (six month research training at Texas A & M University USA). International research scientists who helped me a lot to work in this hard area of study Dr. Levente Abraham Rippl-Rónai Museum, Natural History Department, Kaposvár, Hungary, Dr. Victor A. Krivokhatsky Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia, Dr. Lionel A. Stange Center for Systematic Entomology, Gainesville, Florida, USA, Dr. Andre prost, International Association of Neuropterology, Rue de l'église Loisia- France and Dr. Joshua Jones Department of Biology, Utah Valley University,USA and Dr. Roberto A. Pantaleoni of Università degli Studi di Sassari, Italy for their help in this manuscript preparation, taxonomic identification and species verification. I am also thankful to people from TEXAS A & M University, Professor Dr. John Oswald for six month training and hosting in his lab. Also for his consistent support and round the clock help during my stay over there. Also to other professors at A & M, Dr. James B. Woolley, Dr. Albert Mulenga, Dr. J. Spencer Johnston, Dr. Mariana Mateos who added into my professional skill enhancement. Also to the student fellows, Tracy James, Lauren Ward, Lindsey porter, Meaghan Pimsler, Suhas Vyavhare, Charity Grace Owings, Ordom Brian Huot. Benjamin Diehl, Renato Jose

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Pires Machado, Muhammad Umer, Sabheeullah, Sardar Bacha, Samiullah Jabarkhel, Mr. Abid Ali, for making my stay at Texas A & M University, USA enjoyable. I also thank to the director NIBGE and fellows Dr. Muhammad Asif Qadri, Dr. Arif Muhammad Khan, Dr. Akhtar Rasool, Dr. Muhammad Tayyab Nassem, Dr. Inam Ullah, Dr. Rafu Ahmed Khan, Dr. Khadim Hussain Khosa, Dr. Ahmed Zaheer, Dr. Amir Raza, Dr. Roman Iftikhar, Maryam Masood, Huma Mumtaz, Qamar Abbas, Qamar Islam, Muhammad Zia, Silahudin and Shahswar,. There is list of people who helped me in this venture in lot of different ways the top most was moral support. My friends, relatives and my class mates from past and present Irfan Shabbir Khan, Sami Ullah Khan, Syed Sada Hussain, Muhammad Sajid, Taqi Jawad, Muhammad Ali, Mansoor Baloch, Rana Muhammad Aadil, Muhammad Irfan, Javed Iqbal, Hidayat Ullah Kakar, Abdul Rehman Azeemi, Zahid Mehmood, Sikandar Bhagat, Abdul khaliq, Akash Zafar, Munawar Abbas, Muhammad Waseem Baloch, Dr. Abdul Jabbar, Abdul Qadir Baloch, Dr. Zaheer Abbas, Usman sial, Dr. Zulfiqar Ali, Amir Zia, Sabahat Noor, Nazia Rehman, Asia Sakeena, Sumera Aslam, Almas, Dr. Sara Sadiq, Almas, Khansa Jameel and My late friend Malik Nasir Abbas. At last I would thank to my inspiration towards this achievement my high school teacher Ijaz Hussain Bhutta, my father Allah Wasayia Khan my cousin Ameer Muhammad Khan. In end I would like to thank my Mother, my wife, brothers and sisters for continuous endurance and prayers.

(Saleem Akhtar)

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Table No. List of tables Page No. Table No. .1 Localities visited and their geographical information 11 Table No. 2. Barcode Index Numbers (BINs) and maximum 129 intraspecific distances for Antlion species of Pakistan and other countries.

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Figure No. LIST OF FIGURES Page No. Fig.1. Physical map of Pakistan 16 Fig.2. Head and its morphology 22 Fig.3. Head and thorax morphology 23 Fig.4. Wing morphology and its parts 24 Fig.5. Leg morphology and its parts 25 Fig.6. Abdomen and Male genitalia 25 Fig. 7. Indopalpares pardus habitus, head and male genitalia 27 Fig. 8. Palparellus astutus habitus, face and female 30 terminalia Fig. 8. Palparellus astutus habitus, face and female 31 terminalia Fig.10. Palpares rajasthanicus habitus and male terminalia 34 Fig.11. Palpares trichogaster habitus, thorax and male 36 terminalia Fig.12. Parapalpares solidus habitus and male terminalia 39 Fig.13. Stenares clarus habitus and male terminalia 41 Fig.14. Stenares improbus habitus 43 Fig.15. Acanthaclisis neozelandica habitus and male 45 terminalia Fig.16. Acanthaclisis obscura habitus, thorax and male 48 terminalia Fig.17. indica habitus, thorax and male 50 terminalia Fig.18. Centroclisis thari n.sp habitus and male terminalia 52 Fig.19. Echthromyrmex orientalis habitus, face and pronotum 54 Fig.20. asirensis habitus, thorax and other 56 characters Fig.21. Neuroleon egenus habitus, wings and other parts 59 Fig.22. Neuroleon (Ganussa) leptaleus habitus, wings and 61 other parts Fig.23. Neuroleon (Ganussa) tenellus habitus, wings and 63 other parts

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Fig.24. Indoleon vartianorum habitus, wings and other parts 66 habitus Fig.25. Gepus invisus habitus, wings and other parts 68 Fig.26. Iranoleon vartianae habitus, wings and other parts 70 Fig.27. Myrmecaelurus laetus habitus, thorax and male 71 genitalia Fig.28. Myrmecaelurus longiprocessus habitus, thorax and 75 male genitalia Fig.29. Myrmecaelurus paghmanus habitus, wings, thorax 77 and male genitalia Fig.30. Myrmecaelurus (Nohoveus) vanharteni habitus, wings, 79 thorax and male genitalia Fig. 31. Solter felderi habitus and other body parts 81 Fig. 32. Solter hardei habitus and other body parts 84 Fig. 33. Cueta lineosa habitus and body parts 86 Fig. 34. Cueta minervae habitus and body parts 89 Fig. 35. Cueta plexiformia habitus and body parts 91 Fig. 36. Cueta striata habitus 93 Fig. 37. Cueta thaliae habitus and body parts 95 Fig. 38. Epacanthaclisis alaica habitus and body parts 97 Fig. 39. Psudoformicaleo gracilis Habitus and body parts 99 Fig. 40. Delfimeus intricatus habitus and body parts 102 Fig. 41. cyrus habitus and body parts 105 Fig. 42. Distoleon nefandus habitus and body parts 107 Fig. 43. Distoleon obducens habitus and body parts 110 Fig. 44. Distoleon tesselatus habitus and body parts 112 Fig. 45. Ganguilus pulchellus habitus and body parts 114 Fig. 46. asamiances habitus and body parts 116 Fig. 47. Myrmeleon bore habitus and body parts 118 Fig. 48. Myrmeleon hyalinus habitus and body parts 120 Fig. 49. Myrmeleon inconspicuus habitus and body parts 122 Fig. 50. Myrmeleon noacki habitus and body parts 125 Fig. 51. Myrmeleon trivialis habitus and body parts 127

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Fig. 52. Nighbour joing cluster for species in subfamily 132 Palprinae Fig. 53. Nighbour joing cluster for species of sub-family 133 Fig. 54. Neighbour joining cluster for species of genus 133 Neuroleon Fig. 55. Neighbour joining cluster for species of genus 134 Myrmelecerus and other close taxa Fig. 56. Neighbour joing cluster for species of genus Cueta 135 Fig. 57. Neighbour joing cluster for species of genus Distoleon 136 Fig. 58. Neighbour joing cluster for species of Genera 137 reprsented by single species Fig. 59. Neighbour joing cluster for species of genus 138 Myrmeleon Fig. 60. Maximum intraspecific desistance versus Nearest 139 Neighbor distances Fig. 61. Mean intraspecific distance versus Nearest Neighbor 139 distances Fig. 62. Number of individuals per species verses maximum 140 Intra-specific distance Fig. 63. Frequency histogram of distance to nearest neighbor. 140 Fig. 64. Histogram of Pairwise distance 141 Fig. 65. Ranked Pairwise distances 142

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DNA BASED IDENTIFICATION AND GENETIC DIVERSITY STUDIES OF ANTLION SPECIES OF PAKISTAN Saleem Akhtar and Yusuf Zafar Department of Plant Genomics and Biotechnology PARC Institute of Advanced Studies in Agriculture The University of Agriculture Peshawar, Pakistan ABSTRACT , a group of popular predatory , comes under the family Myrmeleontidae and the order . Both adults and larvae are predatory in nature. The proper identification of an organism is a key factor in understanding its biology. Due to their unique biology and mysterious living antlions generally remain under-explored. A large number of antlion species have been described from around world; still there is a lot of confusion in their . In Pakistan, antlion fauna is not fully explored prior to this study only 28 species have been reported so far from the country. This study steps forward in exploration of Pakistan’s rich antlion faunal diversity. Still lot of it needs to be explored. This study improved the species number of Pakistan to 44. Which blong to 2 subfamilies 8 tribes and 20 genera. Subfamily of Pakistan is represented with five genera and eight species. Species Palparellus astutus and Palpares rajasthanicus first time reportd from Pakistan. While Palpares trichogaster Navas, 1913 reinstate as valid species also P. cephalotes by Iqbal and Yousuf (1997) identified as junior synonym for the species. Genus Stenares Hagen, 1866 two species recorded Stenares hyaena clarus Navas, 1932 and Stenares improbus (Walker, 1853). Species S. improbus first time recorded from Pakistan. Author was not able to collect any specimen of genus Tomatares from Pakistan. Subfamily is represented by seven tribes, eighteen genera and thirty five species. Tribe Acanthaclisini Navas, 1911 constitutes two genera Acanthaclisis and Centroclisis. Acanthaclisis obscura Hölzel, 1972 first time reported form Pakistan. Genus Centroclisis is represented by two species with one new species refered as Centroclisis thari.Tribe Dendroleontini Banks, 1912 is represented by one species Epacanthaclisis alaica first time recorded from Pakistan. In Tribe Banks, 1911 represented by first time reported species Pseudoformicaleo gracilis.

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Tribe Neuroleontini Navas, 1912 with seven genera; genus Delfimeus Navas, 1912 monotypic Delfimeus intricatus. Genus Distoleon Banks, 1910 with four species Distoleon nefandus, Distoleon cyrus, Distoleon obducens and Distoleon tesselatus all four first reported from Pakistan Genus Ganguilus Navas, 1912 one species Ganguilus pulchellus (Banks, 1911). Genus Indoleon Banks (1913) presentdd with one species Indoleon vartianorum (Hölzel, 1972). Genus Neuroleon Navas, 1909 four species Neuroleon tenellus, Neuroleon (Ganussa) leptaleus, Neuroleon egenus and Neuroleon asiricus all first time reported. Myrmecaelurini Esben-Petersen, 1918 having six genera: Genus Gepus Navas, 1912 with one species Gepus invisus Navas, 1912 first time recorded from Pakistan. Genus Iranoleon, Hölzel, 1968 monotypic Iranoleon vartianae Hölzel, 1968. Genus Myrmecaelurus Costa, 1855 having four species. Myrmecaelurus laetus, Myrmecaelurus paghmanus and Myrmecaelurus (Nohoveus) vanharteni first time reported from Pakistan. Genus Solter with two species and Solter Solter felderi first time reported from Pakistan. Tribe Nesoleontini Markl, 1954 with three genera: Genus Cueta having five specie. While four species Cueta striata, Cueta thaliae, Cueta minervae and Cueta plexiformia first time recorded from Pakistan. Tribe only genus Myrmeleon with six species. Five species Myrmeleon trivialis,Myrmeleon bore, Myrmeleon hyalinus, Myrmeleon noacki and Myrmeleon inconspicuous are first time reported from Pakistan. Species were identified morphologically and 703 specimens were selected for DNA barcoding. Maximum 10 specimens from each species were selected. The Barcode Index Number (BIN) analysis using BOLD Systems Version 3.0 assigned 40 BINs to total 359 Antlion DNA sequences recorded for DNA barcode region. To find maximum distance distribution in species Barcode Gape Analysis and Automated Barcode Gap Analysis was performed using BOLD and online resource respectively. Results showed that maximum intera specific distance was 0 to 2.69%. While comparison with other countries was not possible as there was no data available for same species from other countries. This may be due to non processing of specimens from other countries or species endemism. The analysis showed that no species have been already sequenced and added to BOLD. So it has been added first time at least for local fauna. Value of maximum interspecific distance was less than nearresrt neighbor distance. Distance analysis of the barcode sequences showed an interspecific viii divergence of at least 2%. Nearest neighbor joining (NNJ) tree also revealed that close taxa were grouped together more precisely than distant species. An online DNA based reference library was created, for species collected in the present study, on the Barcode of Life Data Systems (http://www.boldsystems.org). Results revealed that antlions exist throughout Pakistan in all geographical and environmental conditions and were not restricted to dry hot and arid areas. They are much abundant in undisturbed and non polluted areas. Collection and survey records revealed that in intensive crop zones due to habitat loss and higher chemical pollution antlion population has almost been eliminated.

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I. INTRODUCTION Antlions, a popular predatory group, belong to family Myrmeleontidae and the order Neuroptera. Family Myrmeleontidae got its name from two Greek words [myrmex (= ant) and Leon (= lion)] Myrmeleontidae is further divided into four subfamilies i.e. Myrmeleontinae, Palparinae, Araripeneurinae and Palaeoleontinae. Among them, Araripeneurinae and Palaeoleontinae are fossile species (Stange, 2004). Antlions may be misidentified with dragonflies by amateurs, yet they are quite different from odonates in their antennal size, flight pattern and many other morphological features. Antlions are distributed worldwide. There is a misunderstanding that they are restricted to arid zones only. However, antlions can live in very hot deserts to very cold regions with seasonal snowy mountains. They have acclimatized to extreme environmental conditions (Abrahám, 2003). Antlions are holometabolous; complete life cycle in four metabolic stages i.e. egg, larva, pupa and adult. Larval stage has three instars and it is the longest stage of their life cycle. In optimum conditions, depending on the availability of prey, antlion life cycle may range from one to several years. Most species spend their winter as larvae. However, adults generally emerge in summer. Antlions lay eggs singly in less disturbed areas and safe from rain. Eggs can be found under vegetation (bushes, trees, grass), in caves, under rocks, tree holes and/or even sheltered under buildings. These are coated with a secretion from glands that promotes adhesion to sand or soil particles (New, 1999). Antlion’s larvae received much attention due to its prominent pit making habit as compared to their adult stages. In a few genera, upon hatching, larvae make pits in loose soil or ground to use them as ambush bunkers for trapping their prey. Many antlion species larvae do not make pits (New, 1999; Matsura et al., 2005; Satar et al., 2006). Antlions larvae are commonly found in dry, shaded places and loose substrates such as sand (Day and Zalucki, 2000) and are known from arid, semiarid subtropical and tropical regions of African, Australian, Asian, and the American continents. Larvae generally prefer dwelling in open woodlands, scrub grasslands, and dry sandy areas. Species such as members of the tribe Dendroleontini inhabit tree holes where they can hide themselves and can get their prey (Stange, 2002).

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Larvae do not have mouth parts like other insects groups. They possess large jaws carrying strong spines (setae like) to hold prey. They hold their prey firmly and inject liquid juice from their mid gut into the prey body through siphon like structure. This liquid carries toxin and digestive enzymes. Liquid starts digesting their prey from inside. Juicy contents of their prey are sucked by the larvae and carcass is thrown out of the pit (Topoff, 1977; Griffiths, 1980; Gepp and Hölzel, 1989). Larvae mostly feed on ants but not exclusively rely on them. They also consume aphids, caterpillars, beetles, moths, flies and even other antlions as their food. Adult antlions are nocturnal predatory insects. They are weak and fragile and can be seen attached to other sources like Rocks, walls, tree trunks, branches and leaves of vegetation during day time. However, very few species exhibit behavioral difference by their crepuscular and diurnal activity. Adults commonly feed on insects like Aphids, caterpillar, flies and moths (Stange, 1980). The mating occurs by attachment of male terminal end with female sitting on branch or some source. Antlion adults have unique gut micro-flora that can be used for many beneficial aspects. Fifteen species of microbes under group Eugregarinorida from genus Gregarina are recorded from the guts of adult antlions (Devetak and Klokočovnik, 2011). While larvae also possess very diverse gut micro flora with 222 strains of reported bacteria (Dunn and Stabb, 2005). Some bacteria have been reported for having insecticidal activity that can be a potential source of bio pesticide or bio control (Nishiwaki et al., 2007). Antlions have been a subject of considerable taxonomic research. There are more than 2000 described species of antlions worldwide with around 1600 valid species. From interior Asia (i.e. Arabia, Iran, Afghanistan, West- Pakistan, Turkey and the adjacent parts of Kazakhstan more than 250 species are known (Hölzel, 1972). There are about 125 species reported from India. The major work was done by Walker (1853), Needham (1909), Dover (1921), MacLachlan (1868), Navás (1912, 1914, 1932), Markl (1954), Banks (1911, 1913a) and Ghosh (1984). China has recorded fauna of 150 Myrmeloenid species reported by Wan and Wang (2003, 2004), Bao and Wang (2004, 2006a, 2006b), Zhan et. al. (2011) and Ao et. al. (2009, 2010). About 110 species are known from Iran. Most of these are recorded and described by Hölzel (1968), Krivokhatsky (2002b), Aspöck (1980), Mirmoayedi (1998, 2002, 2003, 2007, 2006, 2013a, 2013b, 2013c) and Navás (1912). The species toll for Afghanistan goes above 60, with major work contributed by Hölzel (1968, 1972), Navás (1912).

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To date taxonomic studies performed in family Myrmeleontinae are based on morphological characters. The main characters considered for morphological identification include wing venation, body colour, wings markings pattern, number and type of setae on various body parts, shape of different parts of male and female genitalial morphology. Most developed identification keys and literature is based on male morphological characters. The larval stage also has specific species based differences and can be identified on the basis of shape of jaws, their setae (on jaws, body and posterior digging setae). There are still improvements required for establishing comprehensive knowledge about antlions morphological taxonomy (Stange, 1970b; Mansell, 1992; Insom and Carfì, 1988) To make taxonomic studies easy, reliable tools are required to overcome ambiguities of morphological identification. Scientists have used different characters as support e.g. behavior (breeding season, flight pattern, habitat exploitation, feeding) and physiological features (sounds, biochemical composition, and growth rate). Most features have their limitations in order to reach some firm inference. Then taxonomy moved toward next phase with some reliable sources for species identification. Hence use of molecular methods was started as a reliable support to morphological studies. Molecular methods used for insect taxonomy include comparative analysis of some chemical contents viz. Cuticular Hydrocarbons (Dapporto, 2007; Hay-Roe et al., 2007), Allozymes (Foley et al., 2007; Grill et al., 2007). Also some genomic content comparison techniques including i.e. Karyotypes (Martin et al., 2006; Kandul et al. 2007), Randomly Amplified Polymorphism (Naegele et al., 2006; Beebe et al., 2007; Li et al., 2007) Restriction Fragment Length Polymorphic DNA (Kumar et al., 2001; Vandewoestijne and Baguette, 2002; Al-Barrak et al., 2004) , Amplified Fragment Length Polymorphism (Gompert et al., 2006; Scheffer and Hawthorne, 2007; Mock et al., 2007), Microsatellites (Meglecz et al., 2007; Zakharov and Hellmann, 2008), Direct Nucleotide Sequencing (Elias et al., 2007; Linnen and Farrell, 2007; Nazari et al., 2007), Single Nucleotide Polymorphisms (Morin and McCarthy, 2007; Niehuis et al., 2007; Wondji et al., 2007), Expressed Sequence Tag (Papanicolaou et al., 2008; Wahlberg and Wheat, 2008) and Microarray (Turner et al., 2005; Frey and Pfunder 2006) All techniques have their limitations; wing colour spots change, venation disfiguring during wing spread and fallen setae may lead to ambiguous taxonomy. Hebert et al., (2003) introduced a concept for species discrimination named DNA

3 barcoding. DNA barcode provided much firm results for species discrimination than any other method applied. DNA barcode region constitute 658bp region of COI- 5ʹ gene. DNA barcode differentiates species at a very small difference of 2% and has generally been accepted by the scientific community for species discrimination. Later a mega project started for DNA barcoding of Eukaryotes and bacteria. The project became fully functional during 2004 by administrative Consortium for the Barcode of Life (CBOL). The CBOL have established a collection of reference database of DNA barcodes reface libraries of world organisms for molecular identification of species. The DNA barcode sequence data is being maintained at the Barcode of Life Data (BOLD) (Ratnasingham and Hebert, 2007), an online source freely available for developing a barcode reference library. The data can be used for comparison and identification of unknown species with species present in reference library (Virgilio et al., 2010) The BOLD organizes the barcode data in Barcode Index Numbers (BINs) (Ratnasingham and Hebert, 2013). BINs contain all information regarding Taxonomy, collection, sequence and indexing. BOLD supports data analysis and helps eliminating confusions like synonymy and also helps to organize information regarding species taxonomy. The BIN analysis revealed species grouping in most cases similar to numerical taxonomy (Zahiri et al., 2013). BIN analysis plays an important role in species level taxonomic registration (Hausmann et al., 2013), new species discovery (Landry and Hebert, 2013; Mutanan et al., 2013; Ashfaq et al., 2014a), species complex analysis, species turnover over the time and local biodiversity assessment (Trontelj and Fiser, 2009; Bickford et al., 2007; Young et al., 2012; Carr et al., 2011). Stange (2004) listed possible existence of 38 antlion species in Pakistan based work of The major work involved Hölzel (1972), Gosh (1984) and Iqbal and Yousuf (1990; 1991; 1991b). Which include seven species in sub family Palparinae = 7 species. While in sub family Myrmeleontinae Tribe Nemoleontini= 12 species , Tribe Myrmecaelurini = 10 species, Tribe Nesoleontini = 2 species, Tribe Myrmeleontini = 3 species and Tribe Acanthaclisini = 4 species in time stretch from 1967 to 1997). The insect fauna of Pakistan shows transitional state. Records have shown that its Oriental species are continuous with those of Indian Punjab and Rajisthan. Palearctic species are continuous with those of Iranian Baluchistan, eastern Afghanistan, Tajikistan (Tadzhikistan) and Turkmenistan, northwestern and eastern China (Zia et al.,

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2011). Ethiopian influence runs along the southern coastal areas of Sindh and east Makran in Baluchistan (Ashfaq et al., 2013). Pakistan’s diverse ecological zones promote a broad spectrum of antlion fauna. Already known rich fauna from neighboring countries highlights great potential in diversity of fauna yet to be explored. Available knowledge about species description and number of antlion species is very limited and unreliable. Taxonomic keys established based on local fauna are also poor. Most of the previously described species have been synonymized. In lieu of this, countrywide survey was conducted to explore more species from Pakistan. An updated record for extant species of country was made. Objectives of this study were as follow.  To collect antlion specimens adults and larvae  To rear larvae get adults for morphological and molecular studies.  Morphological characterization by using microscopy  Molecular/DNA based characterization using COI gene (barcode region) and phylogenetic analysis.

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II. REVIEW OF LITERATURE The order Neuroptera was first described by Linnaeus (1758) in his book Systema Natura. In his book, Odonata were included among Neuroptera. Later Fabricius (1775) included Neuroptera in a large group Synistata which extended to Hymenoptera, Isoptera, and Crustacea. This classification omitted Odonata from Neuroptera as documented by Linnaeus. Latreille (1825) published another systematic catalog which conformed to Linnaean grouping. Brauer (1855) took first step towards considering Neuroptera as a distinct group called “Family Megaloptera”,. Withycombe (1925) first introduced divisions of Neuroptera into five families including Myrmeleontidae. Later there were many changes but the name for the family remained valid for antlion. Hölzel (1967) described four species of genus Solter including one new record from Pakistan i.e. Solter vartianae (Hölzel) from the south west of Pakistan at an elevation of 900m about 150 Kilometer from Quetta. He again in 1968 in his publication on antlion of Iran included two species from Pakistan. He provided description of tribes Myrmecaelurini, Gepini (Myrmecaelurini), Nesoleontini, Myrmeleontini, as Formicaleonini (Neuroleontina) and Creoleonini (Neuroleontina) of Iran. He reported Maracanda iranica from 150 Km west of Quetta. Which later Krivokhatsky (1996) synonymized as Subgulina iranica valid to date. Second species reported was Pignatellus laetus later synonymized as Delfimeus laetus by Aspöck et al., (2001). He also reported another species Iranoleon vartianae from south west of Quetta, Pakistan. Hölzel (1972) published a manuscript on Neuroptera of East Asia. In his paper he described few species from Pakistan including Acanthaclisis occitanica from Swat at an altitude of 2100m 17 kilometer north of Kalam at Gabral-Tal. Acanthaclisis neozelandica from Toba Afghan district near Quetta, Pakistan. Myrmeleon paghmanus from Swat, Gabral-Tal, Kalam and from Madyan. Neuroleon erato from Quetta west, Distoleon cyrus from Madyan, Distoleon luteomaculatus from Kalam Swat, Indophanes vartianorum and Mesonemurus harterti. Stange (2004) removed Mesonemurus harterti from fauna of Pakistan. Acanthaclisis neozelandica was assigned new combination to Cosina neozelandica by Navas (1911) while Indophanes vartianorum synonymized as Indoleon vartianorum. Ghosh (1984) accounted forty five species from India constituting three sub families splitting into nine tribes and generic count of twenty one His records indicated seven new species including five from Orissa and two from Assam. He documented 6 eight species as new record from the region. These were Stenares hyaena, Indopalpares pardus, Tomatares pardalis, Myrmeleon assamensis and Myrmeleon clothilde. While he reported Cueta facile, Centroclisis indica and Centroclisis eustalacta under wrong tribe and Syngenes horridus a wrong combination. He indicated about the presence of two genera Palpares and Neuroleon in Pakistan. Iqbal and Yousuf (1990) published a manuscript on Myrmeleontidae. They reported genus Palpares from Punjab province and reported four species viz. Parapalpares solidus from Rawalpindi and Indopalpares pardus from Faisalabad. Two species from Ranjan Pur; Palpares astarte and Palpares cephalotes. In their listing out of four species two were first record from Pakistan and one was first record from indo Pak sub-continent. Iqbal and Yousuf (1991a) recorded genus Nohoveus from Pakistan for the first time. They described two species including N. virgrilatus and N. implexus. Among these Nohoveus virgulatus was reported as new to science. Iqbal and Yousuf (1991b) reported two genera; Stenares from Mianwali and Tomatares from Pakptan Sharif. They presented each genus with a single species each. Genus Stenares with species Stenares hyaena and genus Tomatares with the species Tomatares pardalis. Iqbal and Yousuf (1992) re-described two species of genus Myrmeleon; Myrmeleon assamensis Ghosh, 1984 and Myrmeleon clothilde Banks, 1913. Iqbal and Yousuf (1997) made a comprehensive study on the antlion fauna of Punjab provincewith a total of twenty six species from Punjab. They reported four new species Macronemurus notofasciatus, Neuroleon punjabensis, Myrmecaelurus longiprocessus and Myrmecaelurus ghoshi. Hölzel (2002) studied the collection from Yemen Arab republic. He reported 65 species from all seven families of Neuroptera. He reported thirty five new records of family Myrmeleontidae along with three new species Myrmecaelurus fallax, Myrmeleon yemenicus and Nicarinus secundus. Krivokhatsky (2002a) studied male genitalia of members in family Myrmeleontidae and discussed their morphology and physiology as well. Krivokhatsky (2002b) described two new species of antlions Myrmeleon valentine and Myrmecaelurus solaris and stated that species differ from previously known close species by organ pilula axillaris present in one and absent in other. He suggested that the absence of the pilula axillaris should be considered as evidence of convergence. 7

Hence subgenus Morter by Navas (1915) should be considered polyphyletic rather than monophyletic group. Mirmoayedi (2003) reared and described larva of Cueta luteola Hölzel, 1972 for the first time from west Iran Hassanabad Sananadaj area. The larva was reared through imago and description of both larvae and imago were also reported. Bao and Wang (2004) reviewed five species from the tribe Acanthaclisini of China. They reported five species with Acanthaclisis pallida and Stiphroneura inclusa as new records. They also proposed Alloclisis hainanica a synonym for Centroclisis negligens. Stange (2004) published a systematic catalog describing classification of the world antlions. He mentioned almost every species known up to the year 2000. In the catalogue, he described 1509 extant species in 191 genera. He attributed most taxonomic work to early workers including Walker (1853), McLachlan (1867), Nathan Banks (1892-1943), Longino Navas (1903-1940), Peter Esben-Petersen (1906-1939), Lionel A. Stange (1963 to 2000), Herbert Hölzel (1964 to 2000), Mervyn Mansell (1974 to 2000), Robert B. Miller (1985 to 2000), Timothy New (1985 to 2000), Chi-kum Yang (1986 to 2000), and Victor Krivokhatsky (1990 to 2000). Wan et al., (2004) studied genus Dendroleon of China and described its five species along with identification key. They reported three new species i.e. Dendroleon callipterum, Dendroleon perlistigma and Dendroleon lii. They also brought seven species viz. Dendroleon angulineurus, Dendroleon decorillus, Dendroleon decorosus and Dendroleon tripunctatus under genus Gatzara. Bao and Wang (2006a) added two new species to the Myrmeleontidae fauna of China. They described Myrmeleon circulis and Myrmeleon trigonois as new species. Along with the description of new species they also provided identification key to the previously known species of genus Myrmeleon from China. Bao and Wang (2006b) reviewed genus Euroleon from China. They provided identification keys and discussed distribution of seven species viz. Euroleon coreanus, Euroleon polyspilus, Euroleon parvus, Euroleon coreanus, Euroleon polyspilus and Euroleon parvus. Species Euroleon parvus and E. polyspilus were reported as first record from china. They proposed Euroleon sanxianus as a new synonym for Euroleon coreanus. Mirmoayedi (2006) studied the antlion species from different regions of Iran. He reported seven species viz Acanthaclisi occitanica, remanei, Cueta 8 lineosa, Cueta luteola, Mymecealurus trigrammus, Myrmeleon hyalinus and Palpares libelloides as new records. Abraham (2007) described new species Macronemurus farsensis from Iranian locality Chesmedy Chenar, Fars, Iran. Mirmoayedi (2007) reported three new species of antlions from Iran i.e. Cueta clara, Gymnocnemia variegate and Neuroleon (Ganussa) lukhtanovi Saji and Whittington (2008) made a survey for adult of Myrmeleontidae captured twenty seven species viz. Centroclisis cervina, Centroclisis speciosa, Iranoleon arabicus, Lopezus fedtschenkoi, Myrmecaelurus laetus, Myrmecaelurus lobatus, Myrmecaelurus luridus, Myrmecaelurus parvulus, Myrmecaelurus peterseni, Myrmecaelurus philbyi, Ganguilus pallescens, Creoleon pullus, Distoleon asiricus, Gepus cunctatus, Gepus invisus, Geyria arabica, Geyria lepidula, Geyria pallida, Geyria saharica, Neuroleon asirensis, Quinemurus cinereus, Solter hardei, Solter parvulus, Cueta lineosa, Cueta striata, Palpares cephalotes and Palpares dispar. Out of these 24 species were new records for Abu Dhabi. Aspöck (2010) reviewed the past thirty years of research progress in Neuropterology, and emphasized the importance of molecular techniques in Neuropterology. Ábrahám (2012) described a new species i.e. Nohoveus vanhartenia from Oman. He provided detailed descriptions. He also reported Nohoveus palparis and N. implexus from Pakistan. Pantaleoni and Badano (2012) reported Myrmeleon punicanus as new species from Sicily and Pantelleria. They provided species description and also performed DNA based studies. They established Cytochrome Oxisade one (COI) based data for molecular identification. They established molecular data profile for six species viz. Myrmeleon inconspicuous, Myrrneleon formicarius, Myrmeleon gerlindae, Myrmeleon caliginosus, Myrmeleon punicanus and Zhan and Wang (2012) studied wing outlines of Myrmeleon bore, Myrmeleon immanis, Myrmeleon fuscus, Euroleon coreanus, and Euroleon flavicorpus. They performed multiple variate analysis for numerical data from wing out line shapes and variations. According to them statistical data arranges all five species in two groups. The species from genus Myrmeleon were grouped separately than the Euroleon species group. They also prepared UPGMA phylogenetic cladogram on the basis of hind wing

9 shape it resulted in the same clustering order. So they inferred that the numerical analysis system of wing shape may resolve taxonomic complexities. Hazarmany et al. (2013) collected neuropteran insects from Kurdistan, Iran using light traps. They collected individuals from Chrysopidae, Myrmeleontidae, Ascalaphidae and Nemopteridae family. They identified their collection into sixteen different species distributed in four families. They listed six species of Myrmeleontidae family. Mirmoayedi et al. (2013) studied molecular genetic diversity in Myrmeleontidae family. They used Random Amplified Polymorphic DNA (RAPD). They used seventeen primers to get amplification. They performed parsimony analysis and Nei’s index and used thirty six specimens belonging to twelve different species. They also constructed phylogenetic tree using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and found that used primers differentiated all of these in proper twelve clusters. Although the grouping of species may swap to resolve complexities. Mohammad and Mirmoayedi (2013) collected and analyzed antlions from Iran and Kermansha during 2009-2010. They identified twelve species of antlions from the regions. They reported three new records Cueta thaliae, Distoleon curdicus and Distoleon nuristanus from Iran and one new record species (Myrmeleon lineari) from Kirman shah.

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III. MATERIAL AND METHODS Morphological identification of antlion specimens was carried out at the National Insect Museum Integrated Program (NIMIP), NARC during 2008 through 2014 and Texas A & M University, USA (6 months during 2011-2012). DNA based studies were carried out at National Institute for Genomics and Advanced Biotechnology (NIGAB) (during 2009-2014), the National Agricultural Research Centre (Islamabad), Insect Molecular Biology Laboratory (IMBL); National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad (during 2010-2014), and Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, Canada. All the specimens analyzed in this study were collected from different geographic regions of Pakistan during 2008 - 2014. 3.1 Localities Visited Collection expeditions were carried out from February to October of the collection years. The main areas covered for collections were Azad Jammu & Kashmir (AJK) Pakistan Part, Balochistan, Khyber Pakhtunkhwa (KPK), Northern Areas (Gilgit and Baltistan), Sindh and Punjab. A total 120 sites were visited for sample collection. Geographic information of collection sites was obtained by hand held GPS (Garmin model Etrax 150) and photographs of locations were taken by Olympus Coolpix µ9000 and Cannon Power Shot SX 150IS. Some frequently visited localities and their information are given below Table No. 3.1 Localities visited and their geographical information Sr. District/Area Location Latitude Longitude Elevation No. Azad Jammu and Kashmir 1 Bagh Mujahid Abad 33.920303 73.723807 1068 2 Harighel 33.937061 73.708135 957 3 Chattar1 34.019275 73.782586 1252 4 Birpani 34.046681 73.759633 1674 5 Muzafarabad Kohala Pull 34.096092 73.495033 603 6 Pir Chanasi 34.382236 73.526927 1775 7 Dhani 34.425143 73.691611 1007 8 Sharda 74.137571 74.137571 1826 9 Kel seri 74.303365 74.303365 1999

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10 Garhri Dupatta, 73.593683 73.593683 907 Mujohi 12 Poonch Khai Gala 33.847721 73.823377 1710 13 Banjosa 33.810449 73.815770 1793 14 Hajeera 33.767085 73.895738 953 15 AbbassPur 33.814437 73.968117 1157 16 Titrinot 33.742703 73.978331 862 17 Sudhnuti Palandir 33.723179 73.676576 1310 Baluchistan 18 Barkhan Rakhi Munh 29.967111 70.082300 926 19 Quetta ARS, Sariab 30.115929 66.980665 1723 20 Ziarat 30.396115 67.739566 2546 21 Loralai Killi chapli 30.388895 68.668408 1387 22 Killa Saifulla Killa Saifulla 68.908869 68.908869 1489 23 Zob Zob 31.411901 69.394206 1322 24 Turbat Jaussak Road 26.017350 63.061749 141 25 Barkhan Barkhan 69.513425 69.513425 1144 Islamabad Capital Area 26 Islamabad PMNH 33.686959 73.075634 528 27 Rawal Dam 33.697775 73.140958 636 28 Simli Dam 33.709926 73.333948 656 29 Shahdra 33.772694 73.173801 675 Khyeber Pukhtunkhwa 30 Abbottabad Changla gali 33.999272 73.382732 2519 31 Ayubia 34.037136 73.390587 2403 32 Garhi Habib 34.379480 73.388099 882 Ullah 33 Thandiani 34.230908 73.352142 2635 34 Balakot Balakot 34.530286 73.347598 991 35 Shogran 34.640679 73.460960 2340 36 Chitral Rhamboor 35.742670 71.709502 1740 38 Bhaumborat 35.696377 71.683624 1986

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39 Dargai Karakai 71.880941 71.880941 492 40 Batkhela 34.628169 72.004612 675 41 Kaghan SRS, Kaghan 34.770417 73.524614 2054 42 Mingora Banjot 34.776986 72.467164 1162 43 Azizabad 72.574402 72.574402 2010 44 Shakardara 34.867762 72.407565 1103 45 Madyan 35.136073 72.534995 1426 46 Swat Mingora 34.791543 72.391253 947 47 Kalam 35.511684 72.609191 2085 48 Kalam 72.592022 72.592022 1965 Northren areas Gilgit Baltistan 49 Chilas Babusir 74.049678 74.049678 2971 50 BabusirGah 35.249494 74.084651 2401 51 Besham kachi- 35.009885 72.887856 632 chaki 52 Pattan 35.108138 73.002075 765 53 Dasu 35.269543 73.217317 954 54 Geen Nala 35.403919 74.212472 1034 55 Gilgit Jaglot 35.676235 74.617580 1302 56 Kargah Nallah 35.909904 74.265098 1728 Punjab 57 Bhakkar, Darya Khahwar Kalaan 31.734167 71.108030 178 58 Khan Panj Girain 31.922220 71.156423 183 59 Basti Wagan 31.786599 71.073103 165 60 Bhawalpur Solang 29.323254 71.738694 122 61 Lal Sohanra Park 29.366553 71.965685 129 62 Chakwal Tobah 32.58016 72.81631 199 63 Kalar Kuhar 32.7692 72.69507 672 64 Ratuchah 32.704656° 73.005606° 739 65 BARI 32.926142 72.722516 452 66 Qadir Mill 32.906165 72.896635 509 67 Chiniot Chenab Nagar 31.756461 72.938555 185

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68 Dera Ghazi Khan Choti Bala 29.808107 70.333541 170 69 Sakhi Sarwar 29.951983 70.373967 225 70 Rakhi Gaj 29.956873 70.035071 1081 (Gorge) 71 Dina Jindi Reserve 33.061847 73.473957 385 72 Faisalabad Samundri Chak 31.125938 72.994197 175 387 GB 73 PARAS 31.393296 73.015706 178

Gujar Khan Dhoke Imam 33.236747 73.345216 449 74 Bakhash 75 Rawalpindi Koral 33.595926 73.146756 461 76 Salgran 33.819938 73.277270 805 77 Jehlum Gura Ahmed 32.919347 73.615529 243 78 Mangla Dam 33.127657 73.666163 320 79 Malot 32.945069 73.618198 240 80 Diliwar 32.707904 73.515957 250 81 Misri More 32.697366 73.506514 233 82 Jhang Basti Rahseed 31.142028 72.139820 147 pur 83 Bhawalpur KhanPur, Chak 28.657833 70.766934 89 – 1/p 84 Khushab Uchali 32.527178 72.034315 881 85 Jauharabad 32.293440 72.302212 188 86 Khewra 32.646859 73.007066 300 87 Layyah Dhori Ada 30.832005 71.209396 144 88 Chaubara 30.912725 71.476816 146 89 Mailisi Chahwaiwala 29.765788 72.186485 130 90 Jhok Lai 29.721779 72.242615 131 91 Mandi Bhaudin Jalalpur Sharif 32.65755 73.42243 206 92 Darapur 32.721189 73.539518 218 93 Mianwali Khairabad 32.913077 71.614402 237 94 Chashma 32.431054 71.388220 192

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95 Mianwali 32.570479 71.508736 196 96 Murree Aliot 73.476306 73.476306 1574 97 PRS, Murree 33.902660 73.382904 2016 98 Muzafar Garh Saraikiastan 30.073164 71.120151 120 99 Pind Dadan Khan Pind Dadan Khan 32.59309 73.02221 205 100 Rahim Yar Khan Chak- 55/p 28.298642 70.444381 82 101 Rajan Pur Kot Mithan 28.976523 70.361786 91 102 Sadiqabad Chak -160/P 28.16018 70.19576 86 103 Near Tibi 28.284078 70.125921 78 Waghawar 104 Sargodha Risala No. 5 32.118715 72.675859 191 105 Shah Pur 32.296799 72.405846 175 106 Shakar Garh Wadapai 32.328633 75.298956 282 Masroor 107 Shakargarh Chak Amru 32.373276 75.172875 288 108 Taunsa Mangrotha 30.691265 70.625270 173 109 Mangrotha 30.691265 70.625270 173 Sindh 110 Naukot Naukot 69.466777 69.466777 15 111 Umer Kot Umer Kot 25.375898 69.818881 23 112 Islamkot Bhalwah 24.614379 70.649670 24 113 Nagar Parker 24.348304 70.736604 149 114 MirPur Khas Khipro 25.779917 69.484921 12 115 Mithi Mithi 24.728641 69.819958 45 116 Islamkot 24.698438 70.265879 71 117 Islamkot, Ada Ke 70.459930 70.459930 50 Thar 118 Sanghar Makhi Belo 26.169940 68.969829 32 119 Umer Kot Umer Kot 69.820467 69.820467 29 120 Tano Bula Khan Pokhin Jheel 67.924793 67.924793 100

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Fig. 3.1. Physical map of Pakistan 3.2 Insect collections The Following steps were carried out for antlion collection. 3.2.1 Light trap Adult antlions were collected by light trap installed preferably during dark nights. The light trap was set using white sheet 3×3 meter supported by metal rods. Three types of mercury vapor light bulb were used. Bulb range included 160 Watts, 170 watts mercury vapor (General Electronics USA), 200, 100 watt Incandescent tungsten bulb (Philips Pakistan) and 18 Watt tube black light white (GE, USA) and 40 watt tube white light (Philips Pakistan). All light sources run by ballast except tungsten. Power source used was petrol operated 850 watt generator and vehicle battery attachable 1200 watt; 12 volts DC to 220-240 volts AC converter. Light trap collection was started after sun set till 23:55 hours. Collection was also made from lights at various public places streets, cafes, hotels and petrol pump stations.

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3.2.2 Sweep netting Hand net with 15 inches diameter and 72 inches handle was used for collection of adult antlions. Net sweeping aided by disturbing flora was done during day and also with help of hand held light source during the night. Net sweeping was made over the grasses to extract antlions hidden in cover. While at night grasses and shrubs were shaken to make antlions fly in front of search light for hand net collection. Antlion specimens were collected and killed by using killing jars containing Ethyl Acetate or potassium cyanide (Srivastava, 1996). The dead specimens were either field pinned or after killing the specimens were paper wrapped singly. Some specimen were kept in 98% Ethanol for long term preservation to conduct DNA studies. paper triangular envelops were used for transporting specimens to laboratory. Data were recorded on envelops. Field notes were also taken regarding ecology of antlion. 3.2.3 Larval collection Larvae were collected by excavating them from their pits using steel spoon. The pit building material was scoped and filtered to reveal larvae. Larvae were handled by using feather light forceps (Bio-Equip, USA) to avoid physical damage and stress on them. Then larvae were transferred to a glass vial with screen lit to transfer to the laboratory. The larvae those died while rearing were kept in 98% ethanol to preserve them from spoilage and DNA analysis. 3.2.4 Larval rearing The larvae were reared in the laboratory at a temperature 27+3 °C. The larvae were reared in glass vials of size 2.54cm × 6.3cm size capped with a rubber loose cap. Antlion larvae fed with their natural prey ants, aphids and sometime termites collected from field. Ants collection was done by using aspirator (locally made manual and motorized). Single ant was offered to larvae every day once. Only third instar larvae were fed twice a day. Once they stopped feeding for three days offering prey was stopped. They were left for one week and then pupae were extracted transferred to plastic cups of size 5.0 cm (bottom) × 7.62cm (top) × 7.62cm (deep). A fine stick wrapped in tissue paper was placed in jar to make adults grip easy and let them expend their wings. The pupae were placed in cage to avoid any escape on emergence. The pupae were kept for imago emergence at 27 +3 Celsius.

3.2.5 Pinning and preservation

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The lab reared adults and field collected adults were processed in laboratory for curation. Specimens desiccated or hardened during transport (from field to laboratory) were softened by placing specimens in a glass container with moist sand in the bottom. The specimens were kept till they were soft enough to work with. Antlions were pinned by using different size entomological pins ranging 00, 0, 1, and 2 according to the body size of the specimens. The specimens were spread on setting board and dried for three days in summer. While during winter season one week drying was done. All the collections were labeled following standard entomological procedures. Label information contained locality, GPS coordinates, date of collection, collector name and collection method. The specimens were stored in custom made boxes with dimensions 33.02cm × 38.1cm× 7.62cm. The boxes were treated with insect repellents and stored in custom made shelves with supply of Phenolphthalein pellets to save the specimens from predatory insects. 3.3 Morphological characterization Morphological characters of antlions were studied by using stereo microscopes models Olympus stereo Model 314465, Olympus Zoom Stereomicroscope SZX7, (Japan), Labomed CZM6 Trinocular Microscope (USA) and an eyepiece mountable microscope camera (China). The measurements were recorded by using ocular micrometer fitted in microscope eyepiece. While for measuring wing and body length; digital vernier calipers (INSIZE series 1112-150, Insize, FL, USA) were employed. 3.3.1 Dissection & genitalia preparation Some insect body parts, especially terminalia, desiccate and shrink and require special treatment for analyzing their morphological features. The specimens were handled in a dissecting tray and abdominal terminal part i.e. (6th or 7th segment) was removed by using Micro Scissors (eye surgical scissors, Sialkot Pakistan) and feather light forceps (BioEquip, USA). For maceration, terminalia were kept in 10 percent Potassium hydroxide (KOH) solution for 3 to 12 hours depending upon condition of the specimen. Tissues were extracted from macerating solution and then flushed by using Ethyl Alcohol (EtOH) or diluted Acetic acid to remove any debris or macerated stuff. The terminalia were stained by using Chlorazol Black E (1937-37-7) for 1 to 2 minutes. After colouring once again tissue were rinsed with 70% EtoH to remove extra colour. Caution was taken to avoid extra staining which may hinder in observation of complex exoskeletal structures. The structures were suspended in glycerin by gently teasing with special needle (micro pin mounted on fine bamboo stick) and observed 18 under microscope. Structures were saved in Polyethylene Genitalia Vials #1133C (BioQquip, Inc, USA). 3.3.2 Terminology used The terminology used was based on details presented by Stange (1970). Most of his terminology includes details of Stitz (1931) and Sundermeier (1940). While some terminology modifications from Mark1 (1954) were adopted. The primary terminology related to wings by Comstock (1918), Adams (1957, 1958), Tillyard (1915), Esben- Petersen (1917) and Banks (1928). While regarding genitalia Tillyard (1915), Tjeder (1954), Acker (1960) and Oswald (1993). Other than Stange (1970) some general terms by Nichols (1989) have been used. The recent details by Krivokhatsky (2002) attributed as append to Tjeder (1954, 1956), Acker (1960), Aspöck et al. (1980) and Shvanvitsh (1949) have been consulted. The detail listing of characters is given below. 3.4 Molecular identification Molecular identifications were carried out by sequencing of DNA barcode region of mitochondrial gene Cytochrome Oxidase I (COI-5ʹ);. The DNA barcode has been established for sequence based identification of and is currently the method of choice to identify and resolve species complexes. 3.4.1 DNA barcoding 3.4.1.1 DNA extractions and PCR amplifications A 96-well micro plate was filled with 30 µl of 95% ethanol in each well. A claw from single leg was removed from each specimen with a sterile forcep and then transferred to 96-well micro plate well. DNA extraction, PCR amplification, and sequencing were performed at the Canadian Centre for DNA Barcoding (CCDB) following standard protocols (Ivanova et al., 2006, 2007; Ivanova & Grainger 2007a, 2007c). DNA extractions were performed by following the protocols developed for invertebrate barcoding (Ivanova et al., 2006). Amplification of the COI-5’ barcode region was performed with primer pair LCO1490/ HC02198 or LepF1/LepR1 (Table 1) (Hebert et al., 2004b) using the following PCR conditions: 94°C (1 min); 5 cycles of 94°C (30 s), 45°C (40 s), 72°C (1 min); 35 cycles of 94°C (30 s), 51°C (40 s), 72°C (1 min); and final extension of 72°C (10 min). PCR was carried out in 12.5 µL reactions containing standard PCR ingredients and 2 µL of DNA template. PCR products were analyzed on 2% agarose E-gel® 96 system (Invitrogen Inc.). Amplicons were sequenced bidirectional using BigDye Terminator Cycle Sequencing Kit (v3.1) on an ABI 3730XL DNA Analyzer. The forward and the reverse sequences were assembled and aligned 19 using CodonCode Aligner (CodonCode Corporation, USA). Sequences were also inspected and translated in MEGA V5 (Tamura et al., 2011) to verify that they were free of stop codons and gaps. Assembled sequences were uploaded to the project "MAALN" (Antlions of Pakistan) on the Barcode of Life Data System (BOLD) (Ratnasingham and Hebert, 2007) for curation and analysis. 3.4.1.2 Sample preparation for DNA barcoding or automated DNA sequencing After sample collection DNA barcoding was done in four steps. 3.4.1.3 Data sheet preparation Data sheet contains basic information about the specimen. Information regarding data on field identification, taxonomic identification, identifier, voucher type, collectors, collection date, province, region, locality, latitude, longitude and elevation were entered on a spreadsheet. 3.4.1.4 Imaging The images of insect specimen's Images were taken for dorsal, lateral and ventral all three positions or whatever can provide more information. Images were taken in an ascending order corresponding to label attached. Imaging was done at maximum resolution (4320×3240) and good light condition using cool daylight Philips Bulbs. 3.4.1.5 Tissue sampling A small tissue a single claw from leg or 2 - 3 mm from larger insect was removed using aseptic fine forceps (BioQuip Inc., USA) and kept in 30μl absolute Ethanol pre added to wells of 96 well plates in the corresponding well. Maximum five specimens of each species were used for molecular studies. 3.4.1.6 DNA extraction DNA extractions were performed following standard protocols (http://ccdb.ca/docs/CCDB_DNA_Extraction.pdf) at the Canadian Centre for DNA Barcoding (CCDB) at the Biodiversity Institute of Ontario, University of Guelph, and at the NIBGE, Faisalabad. 3.4.2 Manual protocols Manual protocols for the above procedures are given in Annexure -1

3.4.2.7 Sequencing Isolated plasmids and PCR products were submitted for sequencing through Sanger Di-Deoxy Nucleotide chain termination method (Sanger et al., 1977) using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on an Applied 20

Biosystems 3730XL DNA Analyzer. Sequences aligned and edited using CodonCode Aligner (CodonCode Corporation, USA). The sequences were tested and verified for any stop codons and contamination. The sequences were also blasted for their homology with antlion or order Neuroptera. If sequences had no homology with neuropteran or had out group homology like plants and animals e.g. human; were considered contaminations and discarded. The correct sequences were uploaded to Barcode of Life Data Systems (BOLD) for further analysis. 3.4.2.8 Data analysis Data analysis for Species discrimination using DNA and Genetic diversity and phylogenetic analysis was performed using computer software MEGA-6 and BOLD online data analysis tool following Ashfaq et. al. (2013) and Ashfaq et. al. (2014). 3.5 Species discrimination using DNA barcodes Identification Request" function was used to "Blast" compare sequence from each species barcode to sequences of known species in GenBank. Most insect species show 2% sequence divergence at COI (Hebert et al., 2003) which was used for species delimitation for antlions. All the sequences were assigned to BINS (Ratnasingham & Hebert, 2013).

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IV. RESULTS 4.1 Morphological characters of Antlions (Myrmeleontidae: Neuroptera) Many scientists have contributed to elaborate morphological characters in Neuroptera. In Neuroptera, sometime inter family defined characters and specific characters are utilized. Here some morphologically important characters are briefly discussed. 4.1.1 Coloration Members of family Myrmeleontidae or Myrmeleonids (antlions) expresses an array of coloures to camouflage themselves in their habitat. These colourations have been used in their taxonomy by many scientists. The important colouration include vertex marking, face colour and markings, pronotum, thorax, and abdominal markings. Colours of wing patterns, different parts of leg, colour of the setae and hair are also used as distinguishing characters. The markings are sometimes unique in some species or groups e.g. round eye like shape in Dimarini and Pesudimerlla, vertex and pronotum shape in Acanthaclisini, wing markings in Acanthaclisini and Palparinae. Also abdominal streaks in Palparinae. The extent of utility may vary in different species but this coloration also helps in discrimination of species from very close allies (Ghosh, 2004). 4.1.2 Head Vertex (Ver); upper dome shape of head; (top dorsally in middle of the eyes; starts beyond antennae and ends before occiput). Vertex may be raised, medium or indent in different species. Vertex divides into three rows, anterior, middle and posterior. Markings of vertex important in Fig.2. Head and its morphology species distinction in various groups. Genae (Ge): front part of head area between eyes and clypeus and mouth parts. Frons (Fr); 22 sclerite between epicranium and clypeus. Clypeus (Clp), Labrum (lbr), Mandibles (md), Maxillae (mx), Maxillary Palpi (mxp) divided to Palpomeres (plm); a basal palpomere stalk like cylindrical part and apical palpomere little dilated part. Terminal palpomere present in various form e.g. spindle shape or club shaped. The palpomere part also has a sensory spot round, oval and slit like varying in shape or length in various species. Eyes (E); very prominent large eyes present on head in a lateral orientation, ocular rim (ocr) marginal line around eye at inner side ocular rim setae (ors) their presence, their size and number important in species discrimination. Antennae (ant) encircled by cone shape antennal sclerite (asc) relatively short clavate in nature. They have easily distinguishable three parts, scape (scp), pedicle (ped) and flagellum (fl). Relative Inter ocular distance and inter antennal distances used in generic discrimination in antlions. Occiput (ocp); posterior part of epicranium between vertex and neck. Used as endings reference point for lines rising from vertex. 4.1.3 Thorax Thorax divided into three distinct parts prothorax, mesothorax and metathorax. Last two further divided into three parts scutum, scutellum and postnotum. Colour patterns (body colour, streaks and marks), groves, and presence of setae, hairs act as important generic and species determination characters. 4.1.4 Wings Antlions have two pairs of membranous wings. Forewings slightly larger in most species. Wings mostly glassy transparent that may have markings on them. Wings may carry setae on veins and marginal hairs. Fig.3. Head and thorax morphology The wing venations, shapes and marking play a great role in species distinctions. Within wing venation Costa (C), anterior margin of wing (from base to the point of junction) outer most vein of the wing. Subcosta (Sc); vein next to costa. Costal area; area between costa and subcosta.

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The venation and arrangement of cells can be used as distinguishing generic character. Pterostigma (Pt) a thickened, opaque spot on the costal margin with branching vein of a wing. Radius (R): A long vein parallel to the sub costa and fuses with costa these also fuse with subcostal apically. Hypostemagtic cell (hcl) an elongate cell present just below the pterostigma at junction of costa and radius; radial sector (Rs) vein originating from radius joins that branch of median (Ma). The further division of this composite vein (Rs+Ma) give raise to Fig.4. Wing morphology and its parts a network of veins known as radial sector. The media (Ma) further bifurcates; bifurcation point called medial fork. Median give rise to M1 anterior (MaA) and M2 posterior (MP), area before Ma and Rs junction known as presectorial area. Number of presectorial veins distinct in different genera. Cubitus (Cu) vein next to media. Further divided into two veins i.e. anterior Cu1 (CuA) and posterior Cu2 (Cup). Banksian line (bln) when branches of wing bend in such a way that they form a straight line. Anterior Banksian line; line formed by bending of veins of radial sector. While if formed by cuA and Mp then called as posterior Banksian line. Anal veins (A) last veins in wing i.e. three inner most 1A, 2A and 3A. Rarely extended to middle of wing and generally restricted to base. Anal veins have distinctive arrangement and formation in different genera. Small veins joining main branches of veins called cross veins and cross veins connecting branches of radial sector before wing margin are called gradates. 4.1.5 Legs Legs in antlions prominent and sometime think stout or slender. They have all division clearly separated into coxa, trochanter, femur, tibia and tarsus.

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They sometime may have tibial spurs and sensory hairs on femora. They carry five tarsal segments. Presence of sensory hairs, presence and shape of tibial spurs along with tarsal claws very important in species Fig.5. Leg morphology and its parts discriminations. 4.1.6 Abdomen Antlions have 10 segmented abdomen with first segment generally fused to metanotum. While segment 2-8 well developed with clear distinction between tergite and sternite sclerites. Last two segments (9 -10) modified or fused to form genital structures. Antlions bear 8 pairs of spiracles generally present on 2nd to 8th segment. Male copulatory organs present between anal opening and 9th sternite. Gonarcus (gon); main copulatory organ in males having a hood like structure called mediuncus. Paramere (pa); attached by a muscle that allow paramer to move to opening from gonarcus. The 10th tergite modified into a single plate Fig.6. Abdomen and Male genitalia like structure (without any process) called ectoproct (ect). In case of female the 9th sternite modified into a pregenetal plate. Females have two pairs of gonapophysis; lateral gonapophysis and posterior gonapophysis. Generally interior gonapophysis very small and invisible. While lateral gonapophysis and posterior gonapophysis much prominent. Females have large setae on their gonapophysis. Generally setae on ectoproct believed to be digging setae i.e. they help them to dig in soil for oviposition.

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Shape, size and position of both sex genitalial parts very important in species discrimination. Presence of different types of setae and hairy structures also important in some species complex analysis. 4.2 Subfamily Palparinae 4.3 Tribe Palparini 4.3.1 Genus Indopalpares Insom and Carfi, 1988 This mono species genus is endemic from Pakistan and India 4.3.1.1 Indopalpares pardus (Rambur, 1842) Indopalpares pardus (Rambur) : Insom & Carfi, 1988; Palpares pardus Rambur, 1842 ; MacLachlan, 1868 ; Ghosh, 1984 ; Iqbal and Yousuf, 1990; Ghosh, 2000; Myrmeleon nepalensis Klug after Hagen, 1866; Myrmeleon expertus Walker, 1853;Banks, 1913; Palpares pardus partitus Banks, 1911;Palpares pardus stellata Navas, 1912;Palpares pardus asanai Kuwayama, 1933 Diagnosis Rows thick black on setae at scape, vertex grove forming cleft. Upper half clypeus with four rows of long black setae and labium with three rows setae. Abdominal setae hair like; frequent at base become thick and sparse as proceed to end.presence of long setae on inner side of ectoproct distinguishing character. Description Measurements (mm) Based on 2 males; forewing length: 51-52 mm; hind wing length: 48-49 mm and body length (from vertex to end of 7th segment tergite): 46 mm. Head Face, clypeus and labrum all creamy yellow. Labrum with thin shining pale setae. Epicranial and internatennal mark creamy yellow. Vertex brown dived by deep suture throughout and with small setae on interior margin. Labial palpi yellow at the base and apical segment gets brown towards the apex. Palmicula very small and oval. Antennal sclerite bright yellow coloured. Scape yellowish brown encircled by long black setal brush. Flagellum light colour, thin at base thick and dark at towards apex. Interantennal mark fine brown line with brown speculation. Thorax Pronotum, broad, yellow with one median brown line expending posteriorly. A very fine brown line on lateral sides and indent in middle. Mesothorax brown with two yellow strips on sides. Metathorax brown with two yellow strips on sides and two 26 creamy yellow spots on metascutum discs. Meso and metathorax covered with creamy yellow fine hair like setae.

(a)

(b) (d) (e)

(c)

Fig. 7. Indopalpares pardus habitus, head and male genitalia (a) habitus (b) vertex (c) Front view of face (d) male genitalia (e) inner side of male gentalia Wings Wings broad, convex posteriorly and have brown quadrate marks in costal area. Very small brown dots along posterior margin of wing start form base in forewing and in hind wing beyond recurrent vein. Forewing 6 cross veins before redial sector, colour of costa, Radius and cubitus upto fork area brown, all rest veins creamy white with brown at speculation spots scattered in wing. Wing margins covered with fringe of small setae, veins and veinlets also have fine setae. Pilula axillaris light to dark shining golden brown. Hind wing with more prominent markings; first spot two cells beyond recurrent vein starting from Mp1 to third branch of recurrent vein, second spot staring form radius to middle of wing near third branch of recurrent vein, third spot just before hypostigmatic cell about three cell wider towards base ( do not reach to posterior margin

27 of the wing ). Forth spot at apex beyond pterostigma with two eyes like clear spots in between brown area. All speculation in both wings light brown. Legs Coxae dark brown, femora brownish yellow with large black setae, tibiae brownish yellow with large black setae, tarsi starting from yellow to dark brownish black, tibial spurs slightly curved and length equal to first three segments of tarsi. Abdomen Light yellowish brown at base and dark at end. The tergits with transverse bright yellow ring like band at endings, sternite with small spot in middle. Abdomen covered with medium fine light yellow and dark brown setae. Male ectoproct yellow colour and postventral length lobe equal to 6th tergite. Ectoproct have one thick seta on inner side of either lobe right at genital opening. While a brush of thick setae on inner side of postventral lobe and very fine setae on outer side. Material examined 2 ♂ BARI, Chakwal, 2-ix-2009, light trap, Coll. Saleem Akhtar Distribution Species have been recorded from Pakistan (Iqbal and Yousuf, 1990) and India (Ghosh, 1984) Habitat Species occurs in Acacia trees dominated ecology; scrub wood lands with longer grass species and undulating hill zone. 4.3.2 Genus Palparellus Navas, 1912 Only one species recorded from Pakistan 4.3.2.1 Palparellus astutus Walker, 1853 Myrmeleon astutus Walker, 1853; Palpares astutus (Walker): Hagen, 1860; Tomatares astutus (Walker): Hagen ,1866; Filparellus astutus (Walker): Navas,1912 ; Palpares (Palparellus) astutus (Walker): Banks, 1913; Palparellus dubiosus Navas, 1925; Peringuey, 1910 Diagnosis Wings highly marked with blackish brown and mustard yellow background. Clypeus bicolored upper part black and lower part yellow. Labial palpai dark blackish brown and sensory opening round.

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Description Measurements Based on 1 ♀, forewing: 42 mm; hind wing: 40 mm; length of body: 36 mm. Head Face black, internatennal mark, epicranial mark and vertex all black. The clypeus upper half blackish and lower light yellow. Labrum blackish brown, clypeus and labrum with small dark brown setae. Labial palpi brownish black, distal segment dilated in middle, the palmicula with round sensory spot. Antennal sclerite yellow, scape dark blackish brown with black setae and flagellum brownish black. Ocular rime setae present very small white colour. Thorax Pronotum dark brown: with three rows of setae one in middle and two on each margin. Meso and meta thorax dark blackish brown covered with creamy white setae and light brown setae on discs of metascutum. Wings Wings hyaline mustred green colour with prominent markings, Fore wing radial sector starts six veins later. Costal area with markings froming bubble like round areas. Four grey brown markings: first mark starts at radial sector start and follows hind fork of cubal vein toward posterior margin of wing. Second mark three cells next also reaches to hind margin in scattered manner. Third marking before hypostegmatal cell infuses to hind margin in splashes. Fourth epical kidney shape convex outward. Hind wing three major spots: first spot at division of upper arm of RS six to seven cell wide moves down to posterior margin. Mark splits in three fingers before reaching margin. Second four cells beyond first and before hypostegatal cell. Similar to first starts from radius reaches to hind margin of wing. Third at apex with a clear spot in middle. Radius brown on both wings. Legs Coxae, femora, tibia and tarsi all blackish brown. Setae present little larger on femora and tibia, also mixed with small white setae. Tibial spurs less curved with length equal to two basal tarsi.

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(a)

(b) (c)

Fig. 8. Palparellus astutus habitus, face and female terminalia (a) habitus (b) face (c)female terminalia Abdomen Light brown with very small hair like setae. In case of female its smaller while in males makes 2 mm beyond the wings. Male 30erminalia postventral lobe longer than 8th tergite: yellowish in appearance with setae on it. Material examined 1♀ Sindh, Makhi Below, 21-viii-2012, light Trap, Coll. Saleem Akhtar Distribution Species distribution ranges between India (Mansell, 1996) and first time recorded from Pakistan Habitat The species occurs in scrub thorny sandy desert zone with some olea species trees

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4.3.3 Genus Palpares Rambur, 1842 4.3.3.1 Palpares astarte Banks, 1913 Palpares astarte Banks: Iqbal and Yousuf, 1997 Diagnosis Large wings with yellow colour and have brown markings. Thorax bright yellow has blackish brown streaks. Male post ventricular lobe long robust. Description Measurements (mm) On the basis of ♂ forewing length: 61mm; hind wing length: 58 mm; body length: 75 mm

(a)

(c)

(b)

Fig. 9. Palpares astarte habitus and male terminalia (a) habitus (b) male terminalia (c) male terminalia spines

Head Face, clypeus and labrum all pale colour. Labrum with four rows of setae and clypeus has one row of setae. Antennal sclerite turned into wart like structure, scape

31 brown with black setae pedicel brown and flagellomere dark brown with clavate epically. Vertex raised and suture deep. Labial palpi yellow palmicula about ¼ of the distal segment sensory opening slit like. Thorax Pronoturn yellow with one wide median brown strip and brown on lateral sides. Pronotum with brown thick setae and weak white setae. Mesothorax and metathorax bright yellow; with medial wide brown band and very fine brown streaks on sides. Meso and meta thorax covered with tufts of white hair like setae. The disks of meta scutum light golden brown setae, meso and meta thorax have tufts of creamy yellow and white colored setae. Wing Wings pale yellow with prominent brown markings. Fore wing have five markings. First one runs along the arm of cubitus staring form base to drop of lower cubital arm. Second three cells beyond radial sector; starts at radius and reaches three cells down in cubital area. Third three cells away from second mark broad at base and narrows toward posterior margin of wing. Mark ends moving two cells inside cubital area. Three small spots at apex of wing. Hind margin lined with brown colure band. Hind wing has three presectorial veins before radius. Five markings firs at cubital fork small dot like. Three major markings; dropping from radius but not reaching posterior margin of wing. First mark three cells after RS six cells wide. Second mark four cells wide three cells after first. Third mark at hypostigmatic cell all drop down but end few gradates before hind margin. Fourth mark at apex with one circular spot in center. Pilula axilaris present brown in colour. Legs Coxae, femora, tibia all brown and tarsi dark brown. Tibial spurs present and equal to the first three segments of tarsi. Tibial spurs gently curved. Abdomen Abdomen pale brown; with fine white hairs on first four segments and dark brown to black on later segments. In male ectoproct postventral lobe larger than 8th tergite. Material examined 1 ♂ Phar Pur, Dera Ismail khan, 9-ix-2009, light trap, Coll. Saleem Akhtar.

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Distribution Species distribution ranges between India (Banks, 1913) and Pakistan (Iqbal and Yousuf , 1990; 1997) Habitat Species occurs in dry hill zones with high temperature and much rocky and Scrub forest areas 4.3.3.2 Palpares rajasthanicus Gosh, 1991 Diagnosis Male ectoproct have three strong seate on inner sides near paramere. Male ectoproc slightly curved. Epicranial marks makes “V” shape. Abdomen brown with white setae encircled by brown base. Abdomen has brown streakes beyond 5th segment some time turns completely dark brown. Description Measurements (mm) Based on ♀ Forewing: 49-41 mm; hind wing: 45-38 mm; body length: 35-33 mm. Based on ♂ forewing length: 44mm; hind wing length: 40mm; Body length: 40.23mm. Head Face, clypeus and labrum all light yellow. Labrum has fine pale setae. Vertex raised and divided by slight suture. Vertex has median brown line and with off-white setae on anterior row. Labial palpi very long, last 1/3 of distal segment dilated. Palmicula slit like starts form base to apex of dilated portion and crosses to more than half length on other side. Thorax Pronotum pale brown with one median dark brown strip in middle and two narrow on sides. Meso and meta thorax yellowish with three lines; one thick central and two on sides. Pro and meso thorax covered with white setae. The discs of metascutum pale brown. Thorax covered with fine creamy white hairs. Wings Fore wing with splashes of brown colour. Costal first four cells free remaining area biareolate. Radius brown and Rs stars twelve veins later. Speculation along upper arm of cubitus prominent and a small patch in median area followed by similar mark. Posterior margin lined with two rows of brown spots; one at of veins end gradates and second row their landing on posterior margin. Hind wing have three markings; first at 33 fork of cubitus reaching to recurrent vein. Second at split point of upper arm of RS remains suspended in radial and cubital area. Third spot below hypostigmatic cell narrow slender hanged in radial area horizontally.

(a)

(b) (c)

Fig.10. Palpares rajasthanicus habitus and male terminalia (a) habitus (b) male terminalia (c) male terminalia spines Legs Coxae, femora, tibia, and tarsi all dark brown. Tibial spurs present meta tibial spur smaller equal to two basal segments of tarsi. While others littler longer equal to first three basal segments. Tibial spurs slightly curved. Tibial spurs smaller equal to two basal segments of tarsi. Dark brown setae present on legs. Abdomen Pale brown in colour from base to 5 segments. Beyond 5th segment have brown bands. Tergites have middle yellow line and sternites completely blackish brown. Abdomen with small fine white setae brown. Setae start thickening and darkening after 5th segments. At seventh segments setae become thick almost equal to female digging setae. Female gonapophysis small with very thick stout straight digging setae.

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Material examined 5♀ and 1 ♂ Lal Sonhanra National Park, Bhawalpure, 8-v-2012, light trap, Coll. Saleem Akhtar and Qamar Abbas. Distribution Species distribution ranges between India (Gosh, 1991) and first time record from Pakistan Habitat The species is distributed through sandy desert with thorny forest dominated by acacia trees. 4.3.3.3 Palpares trichogaster Navas, 1913 Diagnosis Very large species. Male abdomen with thick stout setae. Male ectoproc have two seate on inner side near paramere. Description Measurements Based on ♀ forewing: 55-54 mm; hind wing: 51-50 mm; body length: 49-48 mm. Based on ♂ forewing length: 53 mm; hind wing length: 50 mm; Body length: 63 mm Head Face pale yellow. Inter antennal distance less than the width of scape. Vertex inflated. Vertex with brown longitudinal stripe starts from middle extending to occiput. Labial palpus brown and apical 1/2 of the palpus clavate with sensory slit reaching to apex. Antennal scape light brown; encircled with tufts of small white setae and 2 to 3 large around 2 mm hard black frontal setae . Pedicel light brown. Flagellomeres black. Thorax Pronoturn yellow with one wide median brown strip and a brown strip on lateral sides. Prominent frontal and posterior furrows. Pronotum have large white setae along ridges. Furrows centrally lined with setae. Mesothorax yellow with medially brown, along groves brown, discs yellow and with median brown strips.

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(a)

(d)

(b) (c)

Fig.11. Palpares trichogaster habitus, thorax and male terminalia (a) Habitus (b) thorax (c) male terminalia (d) male terminalia spines

Meso thorax medially brown and has tufts of white hair like setae. Meta thorax yellow with light brown spot in center and covered with tufts of white hair like setae. Wings Narrow at base and convex centrally. Forewing costa and subcosta brown. Costal area simple. Most wing venation yellow with brown spots. Stigma yellow. Five presectorial veins only three free and rest two split to make biareolate cells. Cu brown little beyond fork. Three prominent markings. First at second branch of RS and second on third branch of Rs about two cells wide were forming a streak downward to the middle of wing. Third mark near apex oblique in shape with two cells row having distinct white broad gradates. Hind margin delineated with light brown colour. 1A reaching margin, 2A with two branches, 3A also reaches hind margin. All connected to each other with strong uniform vein. Hind wing costa and sub costa yellowish brown. Radius also yellow in colour three presectorial veins. Five major marking; first at cubital fork split into two parts lower one looks triangular. Second starts at second branching of Rs and third at second branching of RS upper arm. Narrow streak like expending posteriorly but not reaching to wing margin. Fourth and fifth marking divided in to two spots. In fourth marking

36 upper spot right on the hypostigmal cell and second part just below it forming little oval shape. Fifth spot at apex; upper part scattered while lower in solid shape. A1 fused with A2 for short distance but separates later to from three branches reaching margin of the wing. A2 and A3 also reach hind margin of the wing. A2 and A3 have dark black setae. Legs Coxae brown with white small setae. Femora dark brown have large black setae and smaller white setae. Tibiae dark brown have setae very small white and few larger black. Tibial spurs present slightly curved have length equal to first three tarsal segments. Tarsi dark brown, last tarsal segment very long. Tarsal spurs slightly curved. Abdomen Light brown; tergites light brown both in male and female. Females have white hair like setae on first segment. Second segment have small white setae and have strong black setae along ventral surface. Fourth to sixth segment covered by small strong stout black setae. At apex of each segment setae turn in a ring like formation. Size and strength of setae reduces as proceeds from 7-9th segment. Female gonapophysis greatly reduced strong digging setae present. In males abdominal setae long and strong throughout abdominal length. Males have finger like structures with prominent two strong setae at base. Ectoproct post ventral lobe dilated. Setae on inner surface small thick and outer setae long weak. Material examined 10♂ and 12 ♀, Sindh, Thar, Hatongo, 14- vii-2012, light trap, Coll. Saleem Akhtar Distribution Species records have been made only from Pakistan previously Navas (1913) reported from Karachi. Habitat Species wide spread in sandy desert area scrub lands dominated by Acacia and cactus species

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4.3.4 Genus Parapalpares Insom and Carfi, 1988 4.3.4.1 Parapalpares solidus (Gerstaecker, 1894) Palpares solidus Gerstaecker, 1894; Parapalpares solidus (Gerstaecker): Insom & Carfi, 1989; Palpares klapaleki Navas, 1912; Palpares zugmayeri Navas, 1912; Palpares validus Navas, 1927 Diagnosis Wings very large and broad. Wings veins have thick stout setae and setae on 3A large. Thorax brown and have white hairy setae. In male abdominal setae on end of second segment arranged comb like. Black thin setae present on sternites absent in tergites. Ectoproct with long postventral lobe. Description Measurements (mm) Based on 2 ♂; Forewing length: 57 mm; Hind wing length: 53mm and Body length: 59mm. Based on 2 ♀; Forewing length: 60 mm; Hind wing length: 64mm and Body length: abdomen missing. Head Face pale yellow. Interantennal distance less than diameter of scape. Vertex highly inflated. Epicranial mark area indent. Vertex has brown spots. Occiput has a suture passing through. Vertex has very few small white setae. Labial palpi very large dark brown. Palmicula less than1/3 of distal segment of palpus. Plaimcula clavate with sensory slit reaches the apex of the club. Antenna1 scape light brown with small dark brown and black setae. Pedicel light brown. Flagellomeres black. Thorax Pronotum brown colour, short, broad, indented in middle. Meso and metathorax brown densely covered with white fine setae. Meta scutum has light yellowish brown tint on discs. Wings Wings narrow and smaller in males. Wings convex in middle and veins have thick small setae. Setae on 3A in fore wing and hind wing much larger. Fore wing with brown spots scattered all over the wing especially at veinlets. Only two larger spots. First spot present 3 cells beyond RS; three cells wide reach to middle of cubital fork. Second in middle of first spot and apex 2-3 cells wide and 5 cells log suspended in medial area. Hind wing with 5 major markings and clear background. First small marking at start of cubital fork to the recurrent vein. Second 5-6 cell beyond origin of radial sector. Third

38 and fourth mark start with gap of 3 cell to preceding mark. All start from radius and end in cubital area none of marks reaches to anal margin. Fifth spot covers the apex of wing. All spots colour blackish brown.

(a)

(b)

(c)

Fig.12. Parapalpares solidus habitus and male terminalia (a) Habitus (b) male terminalia (c) male terminalia spines Legs Coaxe brown with tufts of white hairs. Femora, tibia dark brown to black colour have long black and small white setaep. Tibial spurs present slightly arched almost equal to three basal segments of tarsi. Tarsi dark brown to black in color. Abdomen Abdomen brown in color with fine hair like whiter setae at base upto 3rd segment. While rest black, smaller and little thick. Setae make a comb like formation at end of third segment. In male postventral lobe longer encircled with medium size setae on outer side and very small on inner side. Material examined 1 ♂ Chitral, Rahmboor (near Afghan border) 18-vi-2011, light trap, Coll. Saleem Akhtar. 1♀ Quetta, Ziarat, 05-vi-2010, light trap, Coll. Saleem Akhtar

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Distribution Species well distributed through Afghanistan and Iran (Kuwayama, 1966b, Hölzel, 1972), Pakistan (Iqbal & Yousuf, 1990; 1997) and Turkey. Species previous records are Pasni, Mekran, and Balochistan area (Hölzel, 1972). Recent collections were made from two new localities i.e. Bhamborat Chitral and Ziarat Quetta. Habitat The species occurs in high mountain areas with alpine vegetation clear water springs and snow covered winters. 4.3.5 Genus Stenares Hagen, 1866 4.3.5.1 Stenares clarus n.sp Diagnosis Costal area biareolate and wings apex oblique. Vertex much raised, face without any black lines. Six black spots around median streak at middle row of vertex make a palm like mark. Male ectoproct postventral lobe length more than three time length of 8th tergite. Description Measurements Based on 1♂ forewing length: 57mm; hind wing length: 52 mm; Body length: 38mm Head Vertex much raised, face creamy white, clypeus creamy white and labrum creamy white. Face without any black lines. Epicranial mark creamy white. Six black spots around median streak at middle row of vertex make a palm like mark. Interantenal mark creamy white. Antennal sclerite dark brown. Scape dark brown with black setae and falgellomer dark brown to black. Labial palpai longer brown. Palmicula less than 1/3rd of distal segment. Sensory slit starts on base and crosses over before end. Clypeus and labrum has small pale brown setae. Thorax Pronotum with one median black strip and two lateral. Two round dots on both sides of pronotum and long brown setae on margins. Meso and meta thorax have median black strip and two on sides. Meso and meta thorax covered with fine white setae. Discs of scutums brown. Pilula axilaris light golden brown.

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(a)

(b)

(c)

Fig.13. Stenares clarus habitus and male terminalia (a) Habitus (b) male terminalia (c) paramere Wings Wings oblique at apex. Costal area biareolate except first three cells. Pterostigma white and small. Fore wing veins and veinlet covered with brown spots. Spots make a line like appearance on radius and upper arm of cubitus. An oblique spot on apex starting from middle point moves back like line upto lower arm of first division of cubitus. Hind margin lined with brown spots on the veinlet landings. Hind wing shorter than forewing, oblique posteriorly. Radius brown and cubitus upto medial fork brown. Two small brown spots 2 to 3 cells wide present along upper arm of cubitus. Two brown marks delimited to radial area 5 to 6 cells wide present on both sides of pterostigma. Hind margin forms a line like spread of brown spots starting from middle to apex. Legs Legs well build stout. Coxae, femora and tibia all brownish black. Legs coved with blackish brown setae. Tibial spurs present and length almost equal to first two segments of tarsi. Tibial spurs slightly arched.

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Abdomen Thick and smaller than wing span. Tergites yellowish brown in middle while dark blackish brown on edges. Sternites dark brown to black. First two segment covered with fine white hair like setae while rest with small brown setae. Postventral lobe very long about 4 times of 8th tergite. Posterventral lobe have fine setae on outer side and thick stout setae brush on inner side. Material examined 1♂ Rehna Sadat, Chakwal, 5-vi-2010, light Trap, Coll. Saleem Akhtar. Distribution Species has wide distribution through Central Africa (Prost, 1995), India (Ghosh, 1984) and Pakistan (Iqbal & Yousuf, 1991; 1997) Habitat The species occurs in low to high mountain areas with different tree species. 4.3.5.2 Stenares improbus (Walker), 1853 Myrmeleon improbus Walker, 1853; Stenares improbus (Walker): MacLachlan, 1868 Diagnosis Costal area biareolate.Vertex raised with small patches of dark black marks. Forewing splashed with brown spots especially at veinlet. Hind wing has oblique distinctive markings in epical region. Description Measurements Based on ♀ forewing length: 65 mm; hind wing length: 60 mm and body length: abdomen damaged. Head Labrum, clypeus and face all yellow. Antennal sclerite brown, scape brown with setae and flagellomere form base to apex calvating. Vertex raised with small black spots on anterior row of vertex. Labial palpai black colour; with last ¼ as palmicula has sensory opening slit like which crosses apex and goes to half on other side. Thorax Pronotum twice wider than length with one prominent large median black streak. One black round dot on both sides of median stripe. Meso and Meta thorax black in colour. Meso and meta thorax have one median strip and thin lines on sides. Meso

42 and meta thorax covered with brown and white setae. Discs of meta scutum with brown setae brush.

(a)

Fig.14. Stenares improbus habitus (a) Habitus Wings Fore wing larger than hind wing. Costal region biareolate. Pterostigma small whitish. Ten cross veins before RS. Wing colour brown because of brown tint on every cross vein. Veins normally white in colour except costa covered with brown spots. Near apex of the forewing forms a dark oblique brown spot made by cross veins. Hind wing smaller than fore wing. Mostly veins brown tinted, with few dark brown spots. First spot floating below costa and fork of median. Mark 3 cells wide to 4 cells long reducing pattern to posterior margin. Second spot in middle of wing at eighth cross vein of lower branch of median. Spot 3 cells wide and reaches to anal margin of the wing. Next small spot right after it at anal margin. Spot next to that larger but restricted in branches of recurrent vein. Another spot at RS spreading in both direction form base to apex and attaches to anal margin. Before stigma there is a clear dark brown spot. Apex of the wing have two spots facing each other in lips like formation. Legs Robust, coxae dark brown, covered with white and black setae. Fermora dark brown to black in colour and covered with large thick setae. Tibiae black in colure covered with stout thick black setae. Tibial spurs present and larger than first tarsal segment i.e. almost equal to first two segments. Tibial spurs much thick and curved. Tarsi black in colour. Abdomen Damaged only 3 segments were there. First segment dark brown in colour with dense cover of hair like white setae. Second segment little lighter in colour but with few setae. Third segment with light yellow spots on tergite and very small setae. First

43 segment larger, second segment smallest and third segment largest of all three available segments. Material examined 1♀, Darkot, Islamabad, 19-vi-2010, Coll. Ahmed Zia, 1♀ Dadar, Manshera, (Jhon O Dell, United Kingdom personal collection) Distribution Species distribution ranges between India (Navas, 1929). First time reported from Pakistan. Habitat Species occurs in high mountain areas with much green forest area and high rainfall area. 4.4 Subfamily Myrmeleontinae 4.5 Tribe Acanthaclisini Navas, 1911 4.5.1 Genus Acanthaclisis Rambur, 1842 4.5.1.1 Acanthaclisis neozelandica (Navas), 1912 Cosina neozelandica Navas, 1912 Diagnosis Large body heavily covered white and black hairs and setae. Tibial spurs curved at 90° angle reaching to third tarsal segment. Male ecto proct longer and have two lobes Description Measurements Based on ♂ forewing length: 53 mm; hind wing length: 47 mm; body length: 45 mm. Head Vertex little raised with thick black hairs like setae. Vertex divided by suture in middle and also split in a lobe formation by another grove on either side. Epicarinal mark brown covered with white setae. Interantenall mark yellow in colour covered with white hairs and antennal sclerite yellow. Scape brownish yellow with white hairs, flagellomer with segments black at base and ending in light cream colour with small white setae. Clypeus yellow have black hairs on upper boarder area and sides also lined with black setae. Labrum yellow with few small yellow setae. Labial palpi black and distal segment last three quarters swollen. Sensory slit starts from base and reaches to apex.

44

(a)

(b) (c)

(d)

Fig.15. Acanthaclisis neozelandica habitus and male terminalia (a) Habitus (b) Male terminalia (c), (d) Paramer different views Thorax Pronotum broader than longer. Base brownish yellow with one black median streak and two on lateral side. A broken line on either side of median line. Pronotum covered with mix of black and white setae. Meso and meta thorax blackish in colour with inner faces of discs yellow. Discs of meta scutum with golden glow brown velvet spot. Meso and Meta thorax also coved with black and white hair like setae. Wings Forewing costal area biareolate. Radial sector starts after 7 cross veins. Cup folds back by taking two turns one sharp 90 degree while second slight curved and joins cubitus. Wing venation black and white. Radius and cubitus prominent due to black colour. Vein 2A takes sharp turn and fuses with 3A. Peterostigma not much distinct with white and black colour. Interior and posterior bankisian lines present. Hind wing shorter than forewing oblique posteriorly. Radial sector starts after 5 cross veins Veins brown and black with white patches also. Radius and cubitus dark colour makes them prominent. Hind margin with long white hairs on posterior margin in basal area upto drop of 2A on wing margin. Vein 3A joints with lower arm of cubitus and joint vein reaches margin of wing. Vein 2A reaches to wing margin in a slight drop slope. Vein

45

1A very reduced and restricted in distance equal to first cross vein of costal area. Pilula axilaris yellowish brown. Legs Pro coxae white, meso and meta coxae brownish black. All coxae covered with white tufts of setae. Femora and tibia black with some yellow colouration. Tibia black with yellowish white spots on outer side. Tibia spurs strongly arched about 90° and reach upto 3rd tarsal segments. Tarsal segments black, much reduced and have white setae. Tarsal claws present and highly arched. Abdomen Abdomen long black and covered with white setae hair. Hair pencils present on 6th segment. Post ventral lobe has an extra lobe in the middle and post ventral lobe has long black setae on tubercles on it. Material Examined 1♂, 1♀ Chitral, Ziarat, 2-vi-2011, light trap, Coll. Saleem Akhtar Distribution Species well distributed through India; Tadzhikistan (Krivokhatsky, 1998); Tibet (Yang, 1987), Afghanistan and Pakistan (Hölzel, 1972). Habitat The species occurs in high altitude alpine forest areas dominated by juniper and alpine forests. 4.5.1.2 Acanthaclisis obscura Hölzel, 1972 Diagnosis Costal area biareolate and apex of wings acute. Pronotum broader than longer with 5 yellow strips. Distinctive yellow coloured discs of thoracic regions. Males have tuft of hairs (hair pencils) at end of 6th segment. Elongated postventral lobe extended with one extra lobe raised in middle. Description Measurements (mm) Based on ♂ forewing length: 40 mm; hind wing length: 36 mm; body length: 31 mm; Based on ♀ forewing length: 41 mm; hind wing length: 35 mm and body length: 31 mm. Head Face, clypeus and labrum all yellow. Antennal sclerite yellow and have white setae. Interantennal mark yellow. Scape yellow, falgellomer yellow anteriorly and 46 blackish appearance on ventral side. Labial palpi yellow in colour with palimicula brown. Epicarinal mark brown with white setae. Vertex raised, mostly black with markings on front right above epicarinal mark. A brown spot along all width of vertex. On interior vertex a yellow spot on both sides. Median grove clearly observed. Thorax Pronotum wider than its length. Pronotum with five yellow spot; one in center of median black strip. Two “C” shape yellow spots on both sides limited to frontal furrow region only. Two lateral yellow lines interrupted by a black spot. Pronotum with small white setae in center while margins covered with long white and black setae. Meso thorax majorly black with distinctive yellow discs. Meta thorax dark with discs of meta scutum light orange yellow. Long dense white hairs tufts present on thorax. Wings Wings hyaline with black and white colour. Costal area in forewing biareolate. Apex of wings acute. Forewing 8-9 presectorial veins before RS, only 2 free other attached forming biareolate region. RS with 9 branches in forewing. Anterior and posterior bankisian lines present. Hind wing narrower than forewing and more acute. Five 5 cross vein before RS and RS has 9 branches. Cup begins well distal and rejoins Cu. A3 fused with lower fork of cubitus before reaching to the wing margin. Legs Coxae yellowish brown, femora blackish brown and tibia blaksih with white circles. Sensory hairs 3 in pro, 2 in meso and 1 in case of meta femur. Tibial spurs present with flanges sharply bend at 90° and reaching upto apex of 3rd tarsomere. Tarsal claw strongly curved. Abdomen Abdomen with white hair like setae at base become short and stout as proceed toward apex. Apex of tergits have yellow circular bands. Males have tuft of hairs at end of sixth segment. Postventral lobe elongated with extra lobe raised in middle. Distinctive tubercle present before secondary lobe in apical area. In females posterior gonapophysis long cylinder like and larger than interior gonapophysis. Ectoproct well developed and have thin black hairs. Material examined 08 ♂ and 10♀ Lal Sonhara Narional Park, Bhawalpur, 15-v-2013, light trap, Coll. Saleem Akhtar.

47

(a)

(c) (d)

(b)

Fig.16. Acanthaclisis obscura habitus, thorax and male terminalia (a) Habitus (b) thorax (c) Male terminalia (d) Paramer Distribution Species have been reported from Afghanistan (Hölzel, 1972) and recently collected from Pakistan. Species first time reported from Pakistan. Habitat The species have been collected sandy lands with sparse scrub vegetation and acacia as dominant species. 4.5.2 Genus Centroclisis Navas, 1909 4.5.2.1 Centroclisis indica Banks, 1911 Diagnosis Antennal sclerite yellow and scape brown with white setae on it. Ocular rim setae present. Internal antennal mark brown.Hind wing has 7-8 cross veins before RS while fore wing has 10.Males have eversible sacs on 6th segment of abdomen. Tergits yellow with brown boarders. Description Measurements (mm) Based on ♂ forewing length: 39 mm; hind wing length: 23 mm and body length: 32mm. Based on ♀ forewing length: 43 mm; hind wing length: 38 mm and body length: 39 mm.

48

Head Face yellow with white setae. Clypeus yellow with white setae and few black setae on upper line and lateral sides. Clypeus yellow with row of small brown setae. Labial palpai light yellow and with brown tint. Apical ½ of distal segment dilated and palmicula sensory slit spread all along dilated area and little raised. Antennal sclerite yellow scape brown with white setae on it. Antennae pale brown on one side and brownish black on other side. Long white ocular rim setae present. Internal antennal mark brown vertex divided into rows by groves. Groves have long thick black hair like setae. Thorax Pronotum wider than its length. Pronotum covered with white setae along border area and black setae between borders. Pronotum has one median blackish brown strip and lateral side’s also brownish black. In between median and lateral line a discontinuous light colour line present from front to end of pronotum. Meso and meta thorax have yellow marking on discs. Meta scutum discs bright orange yellow. Wings Wing longer with acute apex. Wing venation with black and white colour. Costal area simple, peterostigma small white in colour. Fore wing has costal area simple, 10 cross veins before RS and Rs have 12 branches. Cup rejoins cubitus at very short distance. Apex of median and cubitus have small black spots. Posterior bankisian line present. Hind wing narrow and acute at apex. Radial sector starts after 7 to 8 cross veins. Second to fifth cross vein have special elbow shape formation towards apex and do not attach to radius. RS with 12 branches posterior bankisian lines present. Pilula axilaris blackish in colour. Legs Coxae yellowish brown, femora brown and tibiae blackish with yellowish spots on them. Sensory hairs; 2 Pro, 2 Meso and 1 meta femoral sense hair present. Tibial spurs curved at about 90° have flanges and reaching upto apex of third tarsal segment. Tarsai black tarsal claws greatly arched.

49

(b)

(a)

(e) (c) (d)

Fig.17. Centroclisis indica habitus, thorax and male terminalia (a) Habitus (b) thorax (c) eversible sacs (d) Male terminalia (e) Paramer Abdomen Sternites dark blackish brown in female while little lighter in males. Tergites have sclerite centers much yellow colour and edges brown prominent in males. Males have eversible sacs. Extended postventral lobe with setae on it and pregenetal plate triangular pointed. In females posterior gonapophysis slenderical and larger than lateral gonapophysis. Lateral gonapophysis has digging setae. Ectoproct well-built has fine black and few white hairs. Material examined 6♂ and 15♀ Lal Sonhara Narional Park, 18-ix-2014, light trap, Coll. Saleem Akhtar. Distribution Species distribution ranges between India (Ghosh, 1984) and Pakistan (Iqbal and Yousuf, 1997) Habitat The species has been collected from sandy lands with sparse scrub vegetation and acacia as dominant species.

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4.5.2.2 Centroclisis thari n.sp Diagnosis Epicarinal mark brown. Antennal sclerite yellow on lower and brown on upper side. Labial palpi pale yellow with black mark on apex of basal segment. Males have eversible sacs and post ventral lobe. Ocular rim setae present. Description Measurements (mm) Based on ♂ forewing length: 30 mm; hind wing length: 27 mm and body length: 29 mm. Based on ♀ forewing length: 34 mm; hind wing length: 29 mm and body length: 30 mm. Head Face, clypeus and labrum all yellow. Inter antennal mark yellow. Face and clypeus covered with white setae. Labrum has yellowish light brown setae. Labial palpi pale yellow with black mark on apex of basal segment. Labial palpai distal segment ¾ dilated to form Palmicula. Sensory slit elongate and little raised. Antennal sclerite yellow on lower and brown on upper side. Falagellomers yellowish brown dorsally and dark blackish on ventral side. Epicarinal mark brown. Vertex raised, yellowish white mark on anterior row of vertex. Anterior row of vertex shining and dived in to two lobes. While a grove with black hairs behind it. Posterior row into four parts two small inner and one outer all lobes black and shining. Ocular rim setae long and white. Thorax Pronotum wider than its length. Width extends posteriorly. One median broad black stipe. One oblique black line along posterior furrow and a lateral line on both sides of median line. Pronotum covered with white and black setae. Meso thorax with one line on pre scutum narrowing posteriorly. Two lines on meso scutum and meso scutellum. Meta scutum dark blackish with discs orange brown. Thorax covered with fine white hairs like setae. Wings Forewing broader and hind wing narrow. Wings acute at apex and costal area simple. Pterostigma small black. Fore wing 6-7 presectoral veins. Cup runs back to fuse with Cu at very short distance. Lower arm of cubital fork reaches the wing margin. Posterior bankisian line present. Rs with 9-10 branches. Hind wings 6 cross veins before RS and RS with 10 branches. Vein 3A fuses with lower arm of cubital fork before reaching hind margin of wing. Posterior banksian line present. 51

(a)

(c) (b)

Fig.18. Centroclisis thari n.sp habitus and male terminalia (a) Habitus (b) Male terminalia (c) Paramere Legs Coxae yellowish brown, femora blackish brown and tibia blackish brown with white marking on posterior side. Legs covered with white and few black setae. Femoral sense hairs present viz. 2 pro, 2 meson and one meta femoral sense hair present. Tibial spurs present; with flange curved at 90° angle and reaching apex of 3rd tarsal segment. Tarsal claws present and strongly curved. Abdomen With large hair like setae on basal segment, later covered with small black and white setae. In females sternites dark brown to black. While tergites have some yellow marking much prominent streaks in males. Female posterior gonapophysis elongate cylindrical, anterior very small posterior gonapophysis small and have setae, ectoproct well developed and with hair like setae. Sixth and seventh tergits have silvery appearance. Male have well developed postventral lobe with slight angle. Have setae hair like, pregenetal plate triangular and pointed with dark colour on edges and yellow in middle.

52

Material examined 1♂ Lal Sonhara Narional Park, 18-ix-2014, light trap, Coll. Saleem Akhtar. 1♂, 2♀ Naukot, Thar Sindh, 18-vii-2010, Coll. Saleem Akhtar, 5♀ Hatongo, Khipro, Thar, Sindh, light trap, 5-vii-2010, Coll. Saleem Akhtar. Distribution Species widely distributed through Thar Desert. Collection records have shown existence in all areas starting from Bhawalpur to end areas of Nagger Parker. Habitat The specie inhabits in hot climate regions. Specially recorded from Thar and Cholistan region of Pakistan. The figures have been presented in previous species from Lal Sohanra Park and Thar. 4.6 TRIBE DIMARINI NAVAS, 1914 4.6.1 Genus Echthromyrmex Genus Echthromyrmex represented by only one species and firtst report from Pakistan. 4.6.1.1 Echthromyrmex orientalis MacLachlan, 1873 Diagnosis Pilula Axilaris present, No femoral sense hairs, fore wings tinted with brown colour patches Description Measurements Based on one ♂ forewing: 39 mm; hind wing: 39 mm; length of body: abdomen damaged Head Face Brown, internatennal mark and epicranial mark blackish brown. Vertex yellow with irregular brown spots and have small setae. The clypeus light yellow with few black and most setae yellow. Labrum yellow with yellow setae. Labial palpi brownish black, distal segment dilated at apical part, the palmicula with slit like sensory spot. Antennal sclerite brwon, scape brown with small black setae and flagellum brownish. Thorax Pronotum brown wider than length. Pronotum have four light yellow spots two in center fainted and two on the side much prominent. Meso and meta thorax dark brown.. Tarsal claws slightly curved.

53

(a)

(b) (c) (d)

Fig.19. Echthromyrmex orientalis habitus, face and pronotum (a) Habitus (b) face (c) pronotum (d) pilula axilaris Wings Wings clear with brown splashing and spots. Forewing ostal area simple and four cross veins before radial sector. Radial sector divided into seven branches. Forewing mostly clear with brown splashing the veins colour also brown at splash point. MP2 originates after the first cross vein merges with CuA. Hypostigmatic cell present. Hind wing costal area simple and two cross veins before radial sector. Radial sector divided into eight branches. Pilula axilaris present. A large brown spot present in the apical area majorly present in radial area extending down to medial area forming two buble like clear spots with posterior margin Legs Coxae, femora, tibia and tarsi all yellowish. Thick small present little larger on femora and tibia. While all tibia have two brown spot line one in middle and second on the point of tibial spur base. Tibial spurs strong less curved with length more than one tarsal segment but less than two segments in fore and meso leg. While equal to first tarsal segment

54

Abdomen Damaged so no observation were recorded Material examined 1♂ Ptan, Kohistan, Khyber Pakhtunkhwa 17-vi-2013, light Trap, Coll. Saleem Akhtar Distribution Species distributed in India (Gosh, 1984) and Pakistan. Species is first time reported from Pakistan Habitat Species occurs in the area with Acacia and other tree species near riverine area. The majorly occupied with rocky and sandy soils. 4.7 Tribe NEUROLEONTINA NAVAS, 1912 4.7.1 Genus Neuroleon Navas, 1909 4.7.1.1 Neuroleon asirensis Hölzel, 1983 Diagnosis Small size species yellow colour most body. Fore femur have apically swollen white setae. Tibial spurs absent. Two rows of spots on vertex. The brown lines run in paralle from pronotum to throughout thorax. Description Measurements (mm) Based on ♂ length of forewing: 19.5 mm; length of hind wing: 20 mm and length of body: 23 mm. Head Face yellow, clypeus yellow and labrum yellow. Labial palpi distal segment not much swollen and sensory spot round. Interantennal mark, epicranial mark and scape black. Vertex little raised, base colour yellow, anterior row have five spots and posterior row have 6 spots. Thorax Pronotum yellow with two median stripes separated apart by yellow base. Four small brown spots two on anterior and tow on posterior margin near lateral brown lines. Pronotum have scattered yellow setae and also have 3-5 strong whitish yellow setae. Meso and Meta thorax have one median and two lateral wide brown bands. Median band divided by very fine yellow line. Lateral sides have fine yellow setae and with brown and yellow spots.

55

(a)

(b)

(c)

(e) (f) (d)

(g) (h)

Fig.20. Neuroleon asirensis habitus, thorax and other characters (a) habitus (b) forewing (c) hindwing (d) labial palpi (e) thorax (f) face (g) male terminalia (h) paramere different views Wings Wings elongate and narrow with mostly black venation. Some veins with white spots. Wings pointed at apex and both wings almost equal in size. Fore wing have 7

56 cross veins before radial sector and radial sector divided into 9 cross veins. Hind wing have single cross vein before RS and RS have 9 branches. Cross veins and forks black. Legs Coxae yellow and fore coxa have comb of white setae. Femora yellow only fore femur with blackish colour and swollen. Femoral sense hair present less than half length of femur. Tibia predominantly yellow with some blackish ring in meta and meso tibia. Fore tibia little more black in colour. Apical tarsus longest most followed by basal and rest are small. Tarsal claws smaller than apical tarsus and gently curved. Abdomen Tergites have brown colour and sternites yellow in colour. Abdomen have small yellow setae thin in basal and thick in apical segments. Male terminalia ectoproct well developed and pregenital plate much reduced. Material examined 1♂ Matta Swat, 29-viii-2012, light trap, Coll. Saleem Akhtar, Distribution Species well distributed in northern parts of the country Habitat Species inhabits in rainfed area with mountains receiving snow fall during winter season. Area soils sandy and sometime loam in nature. Most vegetation alpine while plain areas host some grasses species.

4.7.1.2 Neuroleon egenus Navás, 1914 Myrmeleon sticticus Navas, 1903; Myrrneleon sticticus Blanchard, 1845; Nelees cyprius Navas, 1940; Neuroleon egenus stirpis Steffan, 1975 Diagnosis Species have spots on the abdomen only on 2nd 3rd and 5th tergits but 4th tergite dark brown to balack.Pterostigma black at strat and white on apex. Description Measurements (mm) Based on ♂ length of forewing: 29.5 mm; length of hind wing: 27.5 mm and length of body: 34 mm. Based on ♀ length of forewing: 32.5 mm; length of hind wing: 30.5 mm and length of body: 30 mm. Head Yellow with upper part black, clypeus yellow with erect black setae and labrum yellow with setae on lower part. Labial palpai basal segment yellow and distal segment blackish and sensory spot round. Epicarinal mark and inter antennal mark blackish 57 brown. Antennal sclerite yellow and scape brown with some yellow setae. Flagellomer segments have base colour brown and apex yellow. Vertex raised with blackish brown colour dominating on yellow base. Anterior and posterior row of vertex blackish brown. Middle row yellow in colour and have black setae. Thorax Pronotum broader than its length and have base colour brown. Pronotum have one median fine yellow stripe from anterior to posterior. Two oval yellow spots before lateral margin near posterior margin and reach upto middle. Erect long black setae scattered in brown area. While long yellow setae present on lateral sides. Meso and Meta thorax dominantly brown with some white markings. Prescutum divided by a furrow and have off-white spots on raised surface. Meso scutellum with two white spots on posterior edge. Meta thorax mostly dark brown. Wings Wings hyaline with brown maculation. Pterostigma basal part blackish brown and apical part white. Apex of wings acute. Fore wing have 8 cross veins before radial sector and RS divided into 10 branches. Radial sector have some veins with much dark stains to form box formations. Hind arm of cubitus and 1A union makes a recurrent vein like appearance with brown stain. Mostly veins, peripheral gradates and branches have brown staining. Hind wing little narrow than forewing. Hind wing have only one cross vein before radial sector start and RS divided into 11 branches. Legs Fore coxa yellow with only two small brown stains and have tufts of yellow hair like setae. Meso and Meta coxae dark brown in colour. Femora brown colour on yellow base Meta femur much dark. All femora have brown and yellow setae. Tibia yellow with blackish brown stains and black setae. Tibial spurs present. Fore and meso tibial spurs reaching to first three tarsal segments and meta tibial spurs only upto first two segment. Basal tarsal segment longer, apical tarsal segment longest and median three are smaller. Tarsal claws not much curved. Abdomen Dark brown in colour and have distinctive white band on tergits. Abdomen have dark stout black setae on basal region and they increase in length in apical region. Females have posterior gonapophysis cylindrical longer. Median gonapophysis small 58 swollen and contain digging setae. Ectoproct well developed and have digging setae on posterior lobe. Males have broad ectoproct with longer setae.

(a)

(b)

(c) (d) (e)

(f)

(g)

Fig.21. Neuroleon egenus habitus, wings and other parts (a) habitus (b) wings (c) face (d) pronotum (e) labial palpi (f) male gentalia (g) paramere

Material Examined 1♂ Kalam Swat, 02-vii-2010 light trap, Coll. Saleem Akhtar,

59

1♀ Kaghan, 18-Jvii-2013, sweep netting in dry grass, Coll. Saleem Akhtar Distribution Species well distributed in northern areas of Pakistan. Generally restricted in low temperature areas. Habitat The species occurs in the cold area and high rainfall zone. Also during winters areas remain snow covered. The soils are sandy and loam soils also vegetation includes grasses and alpine trees. 4.7.1.3 Neuroleon (Ganussa) leptaleus (Navás, 1912) Neuroleon algericus Navas, 1913 Diagnosis Face yellow with small setae, clypeus yellow with medium setae and labrum yellow with brown setae. Fore femur with row of white setae. Body cylindrical weak and wings much narrow. Description Measurements Based on ♂ length of forewing: 20 mm; length of hind wing: 18 mm and length of body: 23 mm. Based on ♀ length of forewing: 23 mm; length of hind wing: 21 mm and length of body: 21 mm. Head Face yellow with small setae, clypeus yellow with medium setae and labrum yellowish with brown setae. Labial palpai basal segment yellow and distal segment blackish brown. Sensory spot round and bifurcated narrow apex. Inter antennal mark and epicranial mark blackish brown. Vertex raised and base colour yellowish. Interior row have six distinct blackish brown spots and median row have six spots variable. Posterior row have one central spot. Thorax Pronotum length and width almost equal. Pronotum yellow with brown bands. One median wide band having a longitudinal yellow spot. Two lateral fine lines variable in shape sometime not reaching to extreme margins. Pronotum lined with brown setae and few setae scattered over surface. Long setae present on lateral sides. Meso and meta thorax dominated by dark brown colour. Meso scutum lined by a yellow line. Meso scutellum also light in colour. Meta scutum have three yellow spots.

60

(a)

(b)

(d) (e)

(f)

(c) Fig.22. Neuroleon (Ganussa) leptaleus habitus, wings and other parts (a) habitus (b) wings (c) thorax (d) face pronotum (e) labial palpi (f) male gentalia Abdomen Abdomen long almost equal to length of hind wing. Abdomen generally dark brown. Sternites completely brown and tergites have elongated yellow spots on either sides of median line. Females have very reduced gonapophysis. Ectoproct well developed and have digging setae. In male ectoproct rectangular with fine hairs. Pregenital plate elongate triangular. Apex of pregenital plate light in colour. Material examined 2♀ and 2♂ Kargah Nallah, Gilgit West, 20-vi-2011, light trap, Coll. Saleem Akhtar

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Distribution Species well distributed in northern areas of Pakistan. Habitat Species inhabits in high altitude low temperature and rainfed area. The area dominated by rocky mountain soils with sandy and loam soils. The vegetation dominated by silver oak and some alpine species. 4.7.1.3 Neuroleon (Ganussa) tenellus (Klug in Ehrenberg, 1834) Neuroleon naxensis Navas, 1915; Ganussa aegyptia Navas ,1935 Diagnosis Distal segment of labial palpi much swollen in basal 1/3. Sensory spot round and pointed apex forked. Prnotum has clusters of stout black setae in middle on either side of the median line. Pro and meso tibial spurs smaller than first tarsal segment while in meta leg its larger. Description Measurements (mm) Based on ♀ length of forewing: 24 mm; length of hind wing: 22 mm and length of body: 20 mm. Head Face yellow, clypeus yellow and labrum yellow with brown setae on lower margin. Labial palpi short almost equal to maxillary palpi. Distal segment of labial palpi much swollen in basal 1/3. Sensory spot round and apex pointed forked. Antennal base yellow and scape brownish yellow. Flagelomers segments brown on base and apex reddish yellow. Interantennal mark and epicarinal mark brown. Vertex base colour yellowish and interior row has six spots on raised surfaces. Posterior row has also brown irregular spots. Vertex has very small stout black setae on it. Thorax Pronotum base colour yellow. Two broad brown lines in center expanded at posterior base divided by yellow middle line. Two lateral brown lines. One brown spot on both sides starts from interior margin narrow down as moves to posterior to join median line. Pronotum has clusters of stout black and white setae. Prontum ends in triangular membranous fold over prescutum. Meso and Meta thorax brown with irregular median and lateral yellow spots. Lateral sides have white setae scattered. Wings Wings elongate hyaline narrower at the base and expanded towards apex.

62

(a)

(b)

(c) (e)

(d)

Fig.23. Neuroleon (Ganussa) tenellus habitus, wings and other parts (a) habitus (b) wings (c) thorax (d) face pronotum (e) labial palpi Pterostigma blackish brown. Wing venation colour less with brown patches. Fore wing have 7 cross veins before Radial sector and radial sector divided into 8 branches. Brown spots on union of 3A and posterior arm of CU. Another spot on second last gradate for branches of RS, median and upper arm of Cubitus. Cup fuses with 3A

63 after one cross vein. Posterior wing has 1 cross vein before Radial sector and RS divided into 8 lines. Legs Coxae brown at base and yellow at apex. Coxae have white setae on them. Profemur dark brown colour. Meso and meta femora have very less brown color. Femora have black and white setae. Pro tibia has brownish colour meso and Meta tibia colourless. Tbia have balck and white setae. Meso tibia have row of white setae arranged in comb formation. Tibial spurs present pro and meta tibial spur almost equal to first tow tarsal segments. Meta tibial spur equal to ¾ of first tarsal segment. Abdomen Abdomen brown in colour with yellow spots on either side of tergits. Abdomen has light colour setae on it upto 5 segment and from 6th segment to onward setae colour turns black and increased thickness. Female posterior gonapophysis smaller and have long seate. Ectoproct well developed and have short stout digging setae. Material examined 1 ♀ from Ziarat Chitral, 18-vi-2011, light trap, Coll. Saleem Akhtar. Distribution Species well distributed through Afghanistan, Iran (Hölzel 1987) and Pakistan. First time reported from Pakistan. Habitat The species inhabits in high altitude. High rainfall and green areas. The soils of area are early weathering soils. With mostly gravel and coarse sand. 4.7.2 Genus Indoleon 4.7.2.1 Indoleon vartianorum (Holzel, 1972) Diagnosis Body very week cylindrical and legs long cylindrical. Abdomen with loighter brown spots. The male genaltia paramers are Plate like distinctive in nature. Description Measurements (mm) Based on ♀ length of forewing: 30 mm; length of hind wing: 27 mm and length of body: 23 mm. Head Face brown, clypeus light yellow with long hair like setae, labrum yellow with small setae. Labial palpi with apical segment swollen in middle and sensory spot

64 rounded brown. Interantennal and Epicranial mark brown. Vertexes have brownish colour line on interior and posterior margin. Antennae scape brown and long cylindrical antennae having basal part of segments brown. Apical part lighter in colour and expanded.

(a)

(b)

(e) (c) (d) (f)

Fig.24. Indoleon vartianorum habitus, wings and other parts habitus (a) Habitus (b) wings (c) thorax (d) face (e) male genitalia (f) paramere Thorax Pronotum equal in width and length. Pronotum brown in colour with few brown and yellow setae scattered over it. No lateral strong setae present. Meso and meta thorax predominantly brown. The discs of meta scutum have dark spots.

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Wings Wings hyaline and veins have yellow colour. Few markings on veins present. RS rises beyond cubital fork. Forewing has 9 cross veins before radial sector and RS divided into 11 branches. Forewing has dominant brown, white marking on subcosta and cubitus. A brown spot formed by coloration of veins. Spot starts from joining point of 3A and lower arm of cubitus then moves upward 3-4 veins. Gradates in apical area have stains made by veins with brown colour. Hind wing much narrow than fore wing. Hind wing have 1 presectorial cross vein and RS divided into 9 branches. Legs Long cylindrical and very week. Coxae yellow with basal part brownish with few fine small yellow setae. Femora cylindrical yellow with brown maculation and blackish setae. Pro and meso femoral sense hair medium i.e. less than half length of femur. Tibia also yellow with black setae. Tibial spurs equal to first three tarsomers in pro and meso leg. While in hind leg upto first two segments. Tibial spurs not curved. Basal tarsi smaller than epical one and have tarsal claws gently curved. Abdomen Not much expanded. Abdomen brown has fine white setae in basal region and turn strong black beyond fifth segment.Also have lighter spots of large size. Male Terminalia post ventral lobe not much elongate. Longer setae present on postventral lobe and genetal plate. The paramers are plate like. Material examined 1♂ Ziarat Chitral, 20-vii-2011, light trap, Coll. Saleem Akhtar Distribution Species is distributed in northern areas of Paksitan, Afganistan and Iran (Hölzel, 1972) Habitat The species is found in high altitude dry mountain areas with sandy soils and rock bed areas.The vegetation includes Alpine, Salix and Oak trees as dominant with some other high altitude species prevail in the area.

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4.8 Tribe Myrmecaelurini Esben-Petersen, 1918 4.8.1 Genus Gepus Navas, 1912 4.8.1.1 Gepus invisus Navas, 1912 Gepus curvatus Navàs, 1914; Myrmecaelurus scutellatus Klapalek, 1914 Diagnosis Long ocular rim setae present. Proleg femora have setae comb.Prescutum much raised anal margin of fore wing has a convex bend at base. Meso secutellum clearly raised like swollen part. Description Measurement (mm) Based on ♂ length of forewing: 29 mm; length of hind wing: 26 mm and length of body: 24 mm. Based on ♀ length of forewing: 27 mm; length of hind wing: 25 mm and length of body: 23 mm. Head Face yellow and clypeus yellow with a median brown line. Labrum yellow with brown hairs. Epicarinal mark brown. Vertex with base colour yellow has three discontinuous brown lines. Vertex has black setae scattered over it. Long ocular rim setae present. Labial palpi distal segment relatively small and basal 1/3 swollen. Sensory spot in middle elongated brown and covered with fine hairs. Thorax Pronotum yellow with three brown stripes one median and two lateral stripes. Lateral borders brown and a longitudinal brown spot before border. Pronotum has thick whiter setae spread all over the pronotum. While setae on lateral sides are very strong and long. Meso thorax with yellow base colour have brown streaks. Meso scultellum much swollen. Meta thorax has base colour brown with yellow spots. Metascutum spots with orange tint. Wings Wings elongated hyaline with veins having brown colour spots. Forewing have basal part convex. Radial sector starts after 6-7 cross veins and RS has 9 branches. Hind wing narrow at base and not convex like forewing. Hind wing has 4 cross veins before Radial sector and RS divided into 9-10 branches. Pterostigma blackish brown in both wings. Wings have pilula axillaris elongated brown with hairs.

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(a)

(b)

(c) (d)

(f) (g) (e)

Fig.25. Gepus invisus habitus, wings and other parts (a) habitus (b) wings (c) thorax (d) face (e) female genitalia (f) male genitalia (g) paramere Legs Fore coxae yellow with brown patches and white setae. Meso and meta coxae brown with white setae. Fore femur much brown with white setae comb on ventral surface and hard white setae line on lateral surface. Meso and Meta femur have less brown colour with rows of black setae. Tibia yellowish brown with black setae on them. Fore and Meso tibia reduced as compared to hind tibia. Tibial spurs present. Fore tibial spurs equal to first tarsal segment. Shorter than first segment in meso and meta leg.

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Tarsi yellow with black setae. Tarsal claws gently arched and length equal to half of distal segment. Abdomen Abdomen brown with brownish spots on it. Tergits have brown spot in center encircled by yellow base colour. While sternites have yellow colour with scattered brown lines. First two segments have whiter setae. Small black setae present on 3rd segment to onward. Setae become thick and long in apical segment. In males pregenital plate keel like and have long thick setae. While ectoproct well developed and have long setae. In females interior gonapophysis elongated and have long thick black setae. Material examined 2♂ and 1♀ from Goath Rhimoon Sindh, 13-vi-2011, light trap, 2♂ from Sadiqabad10-v-2012, light trap, Coll. Saleem Akhtar Distribution Species have very wide distribution through Algeria, Egypt, Iran, Israel, Libya, Mauritania, Morocco, Saudi Arabia, Sudan, Tunisia (Stange, 2004) and is the first record from Pakistan Habitat Specie inhibits in dry arid areas with sandy soils and hot climate with scrub thorby forest dominated by acaciplants. 4.8.2 Genus Iranoleon Hölzel, 1968 4.8.2.1 Iranoleon vartianae Hölzel, 1968 Diagnosis Adults have long black setae on mesoleg femora extended straight to tibia. Males have hair pencils. Also the 8th tergite have a clear cleft like shape. Description Measurements (mm) Based on ♂ length of forewing: 17.5 mm; length of hind wing: 15.5 mm and body length: 16 mm. Based on ♀ length of forewing: 19.5-22 mm; length of hind wing: 17-19 mm and body length: 17-18 mm. Head Face, clypeus and labrum all yellowish brown. Face has small black setae reduced in size in case of clypeus. Labrum has longer brownish black setae. Inter antennal mark brown (prominent in males), antennal sclerite yellow and scape yellow

69 with brown spot on inner side. Flagellomers with segments brown at the base and yellow apex.

(a)

(b)

(c) (d) (e)

(f) (g) (g)

Fig.26. Iranoleon vartianae habitus, wings and other parts (a) habitus (b) wings (c) face (d) labial palpi (e) female genitalia (f) male genitalia (g) paramere Labial palpi off white with epical segment much dilated as in Nohoveus genus. Palmicula sensory spot long about half length of palpus. Palpi have long black setae on side about more than half length of palpai.

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Thorax Pronotum brownish yellow having three black lines.One median and two on each side. The lateral lines thick from base of posterior margin upto frontal furrow reduced beyond it. Prescutum has three black lines and mesocutum has seven black lines. Mesoscutellum have 3 black lines. Meta thorax has 5 lines which some time become three by fusion of two lateral lines. The sides of thorax have small stout white setae. Wings Narrow in males and relatively broad in females. Wing venation black and white patches on veins. A small mark on joining point of Cup + 1A and hind fork of CuA. Fore wings have 5 cross veins before RS and Rs divided into 7 branches. Hind wing has 5 cross veins before RS and Rs divided into 7-8 branches. Ending forks of veins brownish black. Legs Coxae yellow with brown splash and have stout white setae. Femora brownish yellow with white and black setae. Tibia yellow with light brown spots. Small setae white on pro and meso leg while only black setae on hind tibia. Meso tibia have long black setae dropped down to tarsi. Some setae may cross first tarsal segment. Tibial spurs present. Larger in pro and meso tibia more than length of first segment. Tibial spur little shorter in case of meso leg and almost equal to length of first tarsal segment. Abdomen Abdomen with black broad lines with yellow gaps. Sometime forming triangles of colurs. Abdomen covered small stout white setae in basal area and turn black in apex. In female posterior gonapophysis much swollen and small. Interior gonapophysis longer and swollen epically. In males 6th - 7th segment with hair pencils and post ventral lobe not much extended outward. Material examined 1♂ and 4♀, Lal Sonhara Narional Park, 07-v-2012, light trap, Coll. Saleem Akhtar. Distribution Species well distributed in eastern desert areas of Pakistan. The sepceies inhibits in scrubbed vegetation and Acacia plantations.

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Habitat Specie prefers dry sandy deserted areas. The area majorly occupied by the acacia plantation 4.8.3 Genus Myrmecaelurus Costa, 1855 4.8.3.1 Myrmecaelurus laetus (Klug in Ehrenberg, 1834) Diagnosis Almost similar to Myrmecaelurus major but size and color difference prominent. The body colour light brownish yellow, wing venations and all spots brown as compared to black in case of Myrmecaelurus major. Wings narrow in breadth. Epicarinial mark not indistinct. Male abdomen has hair pencils.

(a)

(b) (c) (d)

Fig.27. Myrmecaelurus laetus habitus, thorax and male genitalia (a) habitus (b) pronotum and thorax (c) face (d) male genitalia i.e. paramere Description Measurements (mm) Based on ♂ length of forewing: 23mm; length of hind wing: 21mm and length of body: 23mm. Based on ♀ length of forewing: 26mm; length of hind wing: 23.5mm and length of body: 24mm Head Face brown clypeus and labrum light brown. Clypeus have two to three strong black setae in middle of posterior margin. Labrum have setae on anterior boarder. While small setae scattered on face area. Antennal sclerite yellow and scape mild brown. Flagelleomer brown with light coloured dilated apex. Labial palpi yellow with distal

72 segment less swollen in middle and have oval Palmicula. Apex of palpi bifurcated. Vertex raised with two rows of spots. Anterior with two medium spots and middle line reaching to occiput. Two small posterior spots on each side of middle line. Thorax Thorax brownish yellow with three brown lines. One in middle and two parallel laterally. Median across borders while lateral two reach to anterior furrow. Colour become dark at end points of lateral strips. Boarders lined with setae. Three to four long yellow setae along lateral sides. Meso and Meta thorax with three black lines. One median and two lines on sides. Thorax has fine yellow setae. Wings Wings narrow with brown veins and yellow pterostigma. Forewing has 7 cross veins before radial sector. Radial sector divided into eight branches. In hind wing 4 cross veins before Rs and Rs divided into 8-9 branches. Wing venation black in colour. Banksian lines present. Legs Coxae yellowish except pro coxae with brown spot. Tibia yellow and tarsi yellow with black setae. Yellow setae present on femora. Tibial spurs present longer than first tarsal segment. Tarsal claws elongate and slightly curved at apex. Abdomen In female abdomen tergites brownish yellow with median brown line on tergites. Sometime in posterior region two lateral lines and tergites covered with white small setae. The sternite sclerites are completely brown in colour. Males have hair pencils on 6th and 7th segment. Postventral lobe not extended. Material examined 2♂ 2♀ Hatongo Khipro, Thar Sindh, 14-iv-2012, light trap, Coll. Saleem Akhtar Distribution Species is well distributed through south part of country and first time recorded from Pakistan. Habitat The species occurs in dry hot climate and sandy soils. The collection of species has been made from Thar Desert and Lal Sohnara Park. The vegetation in area includes sparse vegetation.

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4.8.3.2 Myrmecaelurus longiprocessus Iqbal & Yousuf, 1997 Diagnosis Ponotum with three brown line median across pronotum while lateral stop at anterior furrow. Abdomen with long yellow fine hair all along its length prominent in males. The hair peicls longer than all other species in genus. Description Measurements (mm) Based on ♂ forewing length: 28mm; hind wing length: 24 mm and body length 29 mm. Head Face, clypeus, labrum, interantennal mark, epicarinal mark all yellow. Labrum have setae on anterior margin. Vertex raised with three brown spots on anterior and posterior row. Labial palpi yellow. Distal segment swollen in middle and has pointed apex bifurcated. Palmicula brown and oval in shape. Very small brown setae on scape and white setae present on epicarinal mark. Thorax Pronotum elongated; length more than its width and yellow in colour. Pronotum has three brown lines one median solid line and two mild on sides reaching to anterior margin furrow. Pronotum lined with medium yellow setae and 4 long setae present on sides. Meso and meta thorax have three brown lines. One median continuous throughout length of thorax and two broken on sides. Wings Wings hyaline with brown pterostigma. Venation pale brown. Forewing not acute at apex. Five cross veins before RS and RS divided into 9 branches. Both banksian lines very distinct. Hind wing narrow. Four cross veins before RS and RS divided into 9 branches. Both banksian lines present. Legs Coxae yellow, femora yellow and tibia yellow. Meta femoral sense hair absent. Setae on femora yellow while on tibia and tarsi black in colour. Abdomen First segment tergite have distinctive black colour patch on each side and hairy. All other segment tergits have three longitudinal brown lines. In males longhair pencils present on 6th and 7th segments. Sternites yellow in colour.Whole abdomen covered

74 with very fine brownish yellow setae prominent in males. Post ventral lobe not extended.

(a)

(b)

(c) (d)

(e)

Fig.28. Myrmecaelurus longiprocessus habitus, thorax and male genitalia (a) habitus (b) wings (c) pronotum (d) face (e) male genitalia i.e. paramere Material examined 1♂ form Sadiqabad, 10-v-2012, light trap, Coll. Saleem Akhtar,

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Distribution Species well distributed through south eastern parts of Pakistan ( Iqbal and Yousuf, 1997). Habitat The species can be found in low vegetation covered sandy desert areas. The species generally prefer to be in hot arid climate zone. All specimens collected were from Cholistatn Desert and Lal Sohanra National Park. 4.8.3.3 Myrmecaelurus paghmanus McLachlan, 1875 Diagnosis Shining mustered yellow coloured insect. Mustered yellow pterostigma. Wings much broad, longitudinal veins black. The lateral lines on pronotum stop in halfway while miffle across pronotum. Males have hair pencils on 6th and 7th segment. Description Measurements (mm) Based on ♂ length of forewing: 26.50mm; length of hind wing: 23.53mm and length of body: 26mm. Based on ♀ length of forewing: 29mm; length of hind wing: 26mm and length of body: 28mm Head Face yellow, clypeus and labrum creamy yellow. Clypeus have two black setae in middle of posterior margin. Labrum have setae on anterior boarder. Antennal sclerite yellow. Scape with mild black colour smear and flagelleomer black with dilated yellow apex. Labial palpi yellow with distal segment swollen in middle and have oval Palmicula. Apex of palpi bifurcated. Vertex raised with two rows of spots. Anterior with two spots reaching to antennal area and middle line reaching to occiput. Two posterior spots on each side of middle line. Thorax Thorax yellow with three shining black lines one in middle and two laterally arranged. Median across pronotum length. While lateral two reach to anterior furrow. Pronotum borders lined with setae and three to four long yellow setae along lateral sides. Meso and meta thorax with three black lines. One median and two lines on sides. Thorax has fine yellow setae. Wigs Wings broad with black veins and mustered yellow pterostigma. Forewing 7 cross veins before radial sector. Radial sector divided into eleven branches. In hind wing 5 cross

76 veins before Rs and Rs divided into 11 branches. Wing venation black in colour. Banksian lines present.

(a)

(b)

(c) (d) (e)

(f)

(g)

Fig.29. Myrmecaelurus paghmanus habitus, wings, thorax and male genitalia (a) habitus (b) wings (c) pronotum (d) face (e) male genitalia (f) paramere different views (g) legs Abdomen In female abdomen tergites yellow with median brown line and two lateral lines. Tergites covered with white small setae. Sternites have yellow median strip upto 5th segment later turn complete black. Posterior gonapophysis longer and ectoproct well developed. Lateral gonapophysis have digging setae. Males have one median brown line in tergites and sternites black. Post ventral lobe not extending out ward. Material examined 6♂ Umerkot, 2♂, Naukot Thar, 28-viii-2010, light trap, Coll. Saleem Akhtar, Lal Sohanra, 15-v-2013, light trap, Coll. Saleem Akhtar 5♂ and 8♀, Dera Ghazi Khan, 20-vi-2010, light trap, Coll. Saleem Akhtar

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Distribution Afghanistan, Armenia, Romania, Russia, Turkey (Stange, 2004) and first time recorded from Pakistan. Habitat Species have been collected from arid areas of Pakistan. Dry hot area with sandy soils main part of species habitat. 4.8.3.4 Myrmecaelurus (Nohoveus) vanharteni (Ábrahám, 2012) Diagnosis Clypeus, face and labrum all yellow. Vertex raised. Pronotum have 3 brown lines. Median line starts from anterior margin and does not reaches posterior margin. While lateral line start from posterior margin and does not reaches anterior margin. Paramer much curved as compared to other species in genus. Description Measurements (mm) Based on ♂ length of forewing: 18.5 mm; length of hind wing: 16.5 mm and length of body: 20.5 mm. Based on ♀ length of forewing: 18 mm; length of hind wing: 15.9 mm and length of body 17.2 mm. Head Face clypeus and labrum all shining yellow without any marks. Clypeus has two black hairs like setae. Labial palpus yellow; last segment of palpi long and swollen. Sensory slit brown epically dilated and narrow at base. Antennal sclerite yellow, scape brownish yellow and fallgellomer yellowish brown. Vertex raised; with two spots on frontal ridge side and two small round spots along posterior ridge. Thorax Pronotum length more than width. Yellow colour with three brown lines. Median line starting from anterior margin but dissolve before reaching posterior margin. Lateral strips start form posterior margin and dissolve before reaching anterior margin. Pronotum lined with setae along margins. Lateral sides have 3-4 strong setae. Meso and meta thorax yellow with two discontinuous lines. The furrows dark brown colour while ridges and discs yellow in colour. Meso and Meta thorax without any setae. Wings Wings elongate with sub-acute at apex. Hyaline with yellow venation. Forewing 6 cross veins before RS and radial sector divided into 7 branches. Hind wing have 4

78 cross veins before radial sector and RS has 7 branches. Pterostigma light brown in colour.

(a)

(b)

(c) (f)

(e) (d)

Fig.30. Myrmecaelurus (Nohoveus) vanharteni habitus, wings, thorax and male genitalia (a) habitus (b) wings (c) pronotum (d) face (e) male genitalia (f) paramere Legs Coxae yellow except foreleg coxa has large brown mark. Femora and tibia yellow with scattered setae. Tibial spurs present and gently curved. Tibial spurs larger than one tarsal segment and smaller than two segments in length. Abdomen Abdomen has two lateral light brown lines on tergites in male and female. Abdomen covered with fine white setae. Setae less dense in female and much dense in

79 case of males. Males have hair pencils on segment 6 and segment 7. Post ventral lobe of ecotoproct extended outward. In females posterior gonapophysis short cylindrical in shape. Material examined 4 ♂ and 2♀, Naukot, Thar Sindh, 28-viii-2010, light trap, Coll. Saleem Akhtar. Distribution Species recorded first time from Pakistan but previously it was recorded from Oman (Abraham, 2012) Arabic region. Habitat The species inhabits in dry sandy areas. With sparse vegetation, the dominant species of area are some cactus and acacia species. 4.8.4 Genus Solter Navas, 1912 4.8.4.1 Solter felderi Navás, 1912 Diagnosis Vertex have triangle sahped spots. The wings elipical at the end. Abdomen white with triangular brownish black spots. Description Measurements (mm) Based on ♀ length of forewing: 40-30 mm; length of hind wing: 38-28 mm and length of body: 30-20 mm. Head Face yellow with median brown line. Clypeus yellow with erect yellow setae. Labrum yellow with brown setae. Labial plapi have distal part swollen and densely covered with setae. Sensory spot slit like and ¼ of swollen part in size. Interantennal mark brown. Scape yellow and flagellomers yellow with brownish tint in basal area of segments. Epicranial mark brown. Vertex raised, yellow base with black setae and have brown maculation on all rows. Thorax Pronotum longer than its width, pronotum without lines broken lines if present. Three spots on margin as lines bases and endings. Lines distributed in series of spots interior middle and posterior. Median line longer and prominent on both sides before it dissolves. Pronotum have hard black setae on interior row posterior rows and in the middle. While long brown setae clustered on ridges.

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(a)

(b)

(d) (e) (c)

(g) (f)

Fig. 31. Solter felderi habitus and other body parts (a) habitus (b) wings (c) legs (d) pronotum (e) face (f) labial palpus (g) female terminalia Prescutum indent, base yellow with two black spots on interior and posterior margin. It has large black setae on spots. Meso scutum have splashes of blackish brown spots and three distinct yellow lines. Median yellow line reaches to middle of meso

81 scutellum and meso scutum have cluster of white setae on posterior side. While scutellum without any setae. Meta thorax covered with splashes of blackish brown spots. Meta thorax has discs colour yellow forming an interconnected tetrad shape. Wings Wings much longer and broader. Wing venation yellow with brown maculation. Fore wing have 7 presectorial veins and radial sector divided into 10 branches. Hind wing narrower than fore wing. Radial sector starts after 5 cross veins and RS divided into 10 branches. No prominent banksian line in both wings. Legs Coxae yellow with brown spot on bases in meso and meta leg while in proleg it goes little higher. Femora yellow with sensory hair on pro leg and meso leg more than half length of femur. Profemur, mesofemur have yellow and black setae. While hind femur has only black setae. Tibiae yellow with some brown spots on pro tibia and meso tibia. Colour of setae on hind tibia black. While pro, meso tibia has yellow and black setae. Tibial spurs present slightly arched reaching third tarsal segment in pro and meso tibia while upto second in meta leg. Abdomen Abdomen shorter than wings. Sternites yellow with brownish black setae. Tergites yellow base with large brown marks and splashing on them. Tergites have black setae on them. Females have long broad gonapophysis having long hair like setae on them. Ectoproct well developed with digging setae on it. Material examined 5 ♀ Gheen Nala, Chilas, 21-vi-2013, light trap, Coll. Saleem Akhtar Distribution The species well distributed in northern areas of Pakistan gernrally around Gilgit Balitistan Habitat The species found in high altitude dry mountain areas with sandy soils and rock bed areas. The vegetation includes Oak trees as dominant and some other high altitude species. 4.8.4.2 Solter hardei Hölzel, 1968 or Gepella spp. Diagnosis Small stout body and wings relatively large. Face yellow with sides blackish brown triangular yellow spot. Clypeus yellow and labrum yellow with brown setae. 82

Labial palpai distal segment swollen and sensory spot round. Antennae anteriorly yellow and posterior side brown. Brown base and yellow apex. Long ocular rim setae present. Description Measurement (mm) Based on ♂ length of forewing: 20 mm; length of hind wing: 19 mm and length of body: 15 mm. Based on ♀ length of forewing: 20 mm; length of hind wing; 19 mm and length of body: 15 mm. Head Face yellow with sides blackish brown leaving triangular yellow spot. Clypeus yellow and labrum yellow with brown setae. Labial palpai distal segment swollen and round sensory spot. Inter antennal mark blackish brown. Epicranial mark indistinctive brown in colour. Antennae anteriorly yellow and posterior side brown. Vertex yellow with three blackish marks on anterior row. Five to six spots lined up on middle row of vertex and posterior row have three larger blackish brown spot. The two lateral spots on posterior row of vertex not much prominent. Ocular rim setae present and reaching middle of the eye. Thorax Pronotum broader than longer. Pronotum base colour yellow. Pronotum has one median brownish black stripe narrow in middle and expanded on either end. Three brown spot on either side of pronotum lined laterally. Pronotum have very thick white and blackish brown setae clustered. Prescutum bilobed and have long strong setae. Meso scutum also have a row of strong setae on anterior boarder. Rest of thorax much blackish brown with just patches of yellow colour. Wings Wings hyaline with brown maculation and pterostigma distinctively white. Forewing broad. Five cross veins before start of radial sector and RS divided into 9 branches. Fore wing with maculation on fusion point of cubital fork lower arm. Other small spots present in radial area of apex. Hind wing have one presectorial cross vein and RS divided into 9 branches. Abdomen Abdomen yellow in colour. Tergites have much blackish colour splashing. Sternites mostly yellow with very small brown spots scattered. Abdomen have yellow brown setae mixed in basal region. After beyond 4th segment only black setae which 83 become dense and long. Female posterior gonapophysis much swollen, shorter and have elongate setae. In males ectoproct broad and pregenital plate swollen triangular.

(a)

(b)

(c) (e) (f)

(d)

Fig. 32. Solter hardei habitus and other body parts (a) habitus (b) wings (c) face (d) pronotum (e) female terminalia (f) paramere Legs Legs cream whiter woth brown patches. Pro and meso femour litte short. Femora with thick white setae and few black setae. Tibial spurs present.pro and meso legs tibial spurs reaching uptomiddle of thirs tarsal segment. While in case of meta leg reaching upto second tarsal segment. Material examined 1♂ and 1♀, Lal Sohanra National Park, Bhawalpur, 07-v-2011, light trap,

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Coll. Saleem Akhtar Distribution Species well distributed in Iran (Hölzel, 1972), Saudi Arabia (Hölzel, 1982, 1988, 1998) and Pakistan (Iqbal & Yousuf, 1997) Habitat The species inhabits in arid desert area with scrub vegetation. The soil dominated by sand dunes with acacia as dominant vegetation. 4.9 Tribe Nesoleontini Markl, 1954 Fig. 32.(e) Legs

4.9.1 Genus Cueta Navas, 1911 4.9.1.1 Cueta lineosa (Rambur, 1842) Myrmeleon lineosus Rambur, 1842; Nesoleon lineosus (Rambur): Esben- Petersen, 1930; Cueta lineosa (Rambur): Kimmins, 1943; Cueta lineosa (Rambur): Hölzel, 1968; Myrmeleon morosus Walker, 1853 Myrmeleon ulianini McLachlan, 1875; Cueta trilineata Navas, 1911; Myrmeleon grammaticus Navas, 1912; Cueta syriaca Navas, 1914; Cueta triplicata Navas, 1914; Cueta natti Navas, 1914; Cueta anomala Navas, 1915; Cueta albanica Capra, 1945 Diagnosis Face, clypeus and labrum all yellow. Palmicula swollen in middle and pointed at apex. Sensory spot oval. Interantennal, epicarinaial mark connected and blackish brown in colour. Scape yellow and antennae yellow. Nine presectorial cross veins in fore wing and 8 presectorial cross veins in hind wing before RS. Description Measurements (mm) Based on ♂ length of forewing: 19 mm; length of hind wing: 15mm and body length: 25 mm. Based on ♀ length of forewing: 17 mm; length of hind wing: 15 mm and body length: 21mm. Head Face, clypeus and labrum all yellow. Labrum has brown setae. 85

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(e) (f)

Fig. 33. Cueta lineosa habitus and body parts (a) habitus (b) wings (c) face (d) pronotum (e) male terminalia (f) paramere

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Face has a mild brown black line in center. Scape brownish and flagellomers also brown. Inter antennal mark and epicarinal mark fused black. Epicranial mark has small setae. Vertex little raised. Anterior and posterior rows have marks on them. Three black spots on front. Two circular on side and one middle extending to posterior of vertex cross shape spot present on posterior row of vertex. Thorax Pronotum have three lines running from anterior to posterior margin of thorax. Meso, meta thorax discs filled by streaks and spots. Presutum filled by triangle spot and inlined by brown lines. Meso scutum with four lines. Meso scutellum have two shining black spots on front. Meta thorax mostly dark with discs yellowish brown. Wings Wing venation black and yellow patches. Pterostigma yellowish brown in colour. Forewing 9 cross veins before RS and 8 cross veins before RS in hind wing. Banksian line not prominent just makes formation on apex in both wings. Legs Coxae brown and yellow. Femora mostly brownish. Tibia and tarsi yellow with black spots on tibia. Tibial spurs present and smaller than first tarsal segment. Tarsal claws slightly curved. Abdomen Abdomen with three lines on tergites. While sternites have median yellow line. First two segments have small white hair like. Segment 6 to onward large thick black setae. In male postventral lobe extended more than twice of thickness. In females ectoproct well built and posterior gonapophysis thumb like swollen. Material examined 5 ♂ Naukot, Thar Sindh, light trap, 28-viii-2010, Coll. Saleem Akhtar Distribution Species well distributed in Pakistan and most parts of the world. Habitat Species can be found in arid areas of Pakistan and prefers fine soils. The larvae of the species make pits in open fields. Generally do not prefer much shady places.

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4.9.1.2 Cueta minervae Hölzel, 1972 Diagnosis Seven cross veins before radial sector in both wings. Wings much broad and have tinting on the longitudinal veins and cross veins junction. Tibial spurs smaller than first tarsal segment. Description Measurements (mm) Based on ♀ length of forewing: 29.5 mm; length of hind wing: 27 mm and length of body: 25 mm. Head Face, clypeus and labrum all blackish brown. Labial palpai have yellow basal segment with a brown mark at apex. Distal segment much swollen and sensory area long slit longer than half length of swollen part. Epicranial mark brown. Vertex much raised. Vertex yellow in colour with black setae. Frontal row of vertex have three black spots on frontal row. Middle row marking scattered in a line and posterior row have only one spot in middle. Thorax Pronotum yellow with three brown lines. Median line reduced in width at frontal furrow and near posterior margin. Two lateral lines throughout same width. Two dissolving brown spots on sides. Pronotum covered with long thick yellow setae. Lateral sides have 6 thick setae arranged towards middle of pronotum. Prescutum have long erect brownish yellow setae. Mesoscutum have five brown lines. Meso scutellum with small brown mark much yellow. Meta thorax much brown with yellow spots and yellowish setae tuft on brown Prescutum. Wings Wings much broad, hyaline and with suffusion of blackish brown colour on veins. Forewing has more suffusion as compared to hind wing. Radial sector starts after 8 cross veins and RS divided into 10 branches. Pterostigma white colour between brown spots and a brown spot at rhegma. Hind wing comparatively narrow. Hind wing have 7 cross veins before radial sector and RS divided into 9 branches. Legs Coxae yellow with brown spots and fore coxae without comb of hairs. Femora yellow with few long black setae and small white setae. Pro and meso femoral sense hair medium and thick. Tibiae yellow with black setae and tuft of golden yellow hairs 88 on pro-tibia. Tibial spurs present and not much curved. Tarsi yellow with basal and apical segment large. Tarsal claws not much curved.

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Fig. 34. Cueta minervae habitus and body parts (a) habitus (b) wings (c) face (d) pronotum

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Abdomen Brownish in colour and have white setae on basal segment. Sternites have much yellow colour while tergites have yellow spots. The setae colour turns black in epical 3 segments and longer in size. Posterior gonapophysis elongated and have long hair like setae. Anterior gonapophysis reduced and ecotoproct well-built having digging setae on lower side. Material examined 1♀ from Pokhin Jheel, Karchat National Park, Thana Bula khan, 08-iv-2013, light trap Coll. Saleem Akhtar Distribution Species distributed in southern part of Pakistan i.e. upper Sindh and Balouchistan zone. Habitat Species inhabits in the dryland areas with small mountain range. The dominant vegetation including Jujuba, Acacia and Tamarix species. Soils are hard rock soils at intial weathering process. Environment is very hot and dry. 4.9.1.3 Cueta plexiformia Krivokhatsky, 1996 Diagnosis Clypeus brown and labrum brown with 8 black setae. Labial palpai distal segment swollen and have sensory spot long slit like. Description Measurements (mm) Based on ♂ length of forewing: 30 mm; length of hind wing: 34 mm and length of body: 25 mm. Head Face, clypeus and labrum all blackish brown. Labial palpai have yellow basal segment with a brown mark at apex. Distal segment much swollen and sensory area long slit like longer than half length of swollen part. Epicranial mark brown. Vertex much raised. Vertex yellow coloured with black setae. Middle row marking scattered in a line and posterior row have only one spot in middle. Thorax Pronotum yellow and has three brown lines. Median line reduced in width at frontal furrow and near posterior margin. wo lateral lines throughout the same width. Two dissolving brown spots on sides. Pronotum covered with long thick yellow setae. 90

Prescutum have long erect brownish yellow setae. Mesoscutum have five brown lines, meso scutellum with small brown mark much yellowish. Meta thorax brownish with yellow spots and yellowish tuft of setae on brown prescutum.

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Fig. 35. Cueta plexiformia habitus and body parts (a) habitus (b) wings (c) face (d) pronotum (e) male terminalia (f) paramere Wings Much broad, hyaline and with suffusion of blackish brown colour on veins. Fore wing has more suffusions as compared to hind wing. Radial sector starts after 7 cross veins and RS divided into 10 branches. Pterostigman white colour between brown spots and a brown spot at rhegma. Hind wing comparatively narrow with 8 cross veins before radial sector and RS divided into 9 branches. Legs Coxae yellow with brown spots and fore coxae without comb of hairs. Femora yellow with few long black setae and small white setae with brown bases covering femora. Pro and meso femoral sense hair small and thick. Tibiae yellow with black setae and tuft of small golden yellow hair on pro-tibia. Tibial spurs present and not much curved. Tarsi yellow with basal and apical segment large and tarsal claws not much curved.

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Abdomen Brownish in colour and have white setae on basal segment. Sternites have much yellow colour while tergites have yellow spots. The setae colour turns black in epical 3 segments and longer in size. Posterior gonapophysis elongated and have long hair like setae, anterior gonapophysis reduced and ecotoproct well-built having digging setae on lower side. Material examined 1♂ from Pokhin Jheel, Karchat National Park, Thana Bula khan, 08-iv-2013, light trap, Coll. Saleem Akhtar Distribution Species distributed south region of the country especially in upper from southern Punjab, Sindh and Balochistan zone. Habitat Dry hot arid area with scrub vegetation and rocky area. 4.9.1.4 Cueta striata Kimmins, 1943 Diagnosis Almost same as in case of Cueta linosa. Except heavy coloration of few veins i.e. radius, upper arm of cubits and 6 cross veins before RS in both fore and hind wing. Basal segment of tarsi larger than apical segment. Medial region yellow and clear in both wings. Description Measurements (mm) Based on ♂ length of forewing: 20 mm; length of hind wing: 17 mm and body length: 30 mm. Based on ♀ length of forewing: 25 mm; length of hind wing: 21 mm and body length: 24 mm. Head Face, clypeus and labrum all yellow. Labrum has brown setae. Scape brownish and flagellomers also brown. Inter antennal mark and epicarinal mark fused with black colour. Epicranial mark has small setae. Vertex little raised. Anterior and posterior rows have marks. Three black spots on front. Two circular on side and one middle extending to posterior of vertex. Thorax Pronotum have three lines running from anterior to posterior margin of thorax. Meso and Meta thorax discs filled by streaks and spots. Prescutum filled by triangle 92 spot and inlined by brown lines. Meso scutum has four lines. Meso scutellum has two shining black spots on front. Meta meta thorax mostly dark with discs yellowish brown.

Fig. 36. Cueta striata habitus Wings Wing venation black and yellow patches. Pterostigma yellowish brown in colour. Forewings have 9 presectorial veinsand hind wings have 8 presectorial veins. Banksian line not prominent just start formation on apex in both wings. Legs Coxae brown and yellow. Femora mostly brownish. Tibia and tarsi yellow with black spots on tibia. Tibial spurs present and smaller than first tarsal segment. Tarsal claws slightly curved. Meta femur in male has a comb of setae on posterior surface. Abdomen Abdomen with three lines on tergites while sternites have median yellow line. First two segments have small white hair like setae. While 6 segment to onward large thick black setae. In male postventral lobe extended more than twice of its thickness. In females ectoproct well formed and posterior gonapophysis thumb like swollen. Material examined 1 ♂ Quetta, Sariab road, 17-vi-2010, light trap, Coll. Saleem Akhtar

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Distribution Species well distributed in Afghanistan (Zeleny 1972), Iran (HölzelHölzel, 1968), Kuwait (Aspöck et al. 2000), Saudi Arabia (Hölzel, 1998) and arid areas of Pakistan. Habitat This species found in south western region of Paksitan. Hard rocky mountains with many sandy valleis and caly soils. These areas usualy have very poor vegetation mostly barren lands. 4.9.1.5 Cueta thaliae Hölzel, 1972 Diagnosis Pronotum with three lines. one median straight and two from side froming “J” shape lines on sides. Yellow colour dominating with fine white bristles. Fore leg coxae have comb of setae. Meta femur in male has a comb of setae on posterior surface. Description Measurements (mm) Based on ♂ length of forewing: 21 mm; length of hind wing: 18 mm and body length: 28 mm. Based on ♀ length of forewing: 28 mm; length of hind wing: 23 mm and body length: 22 mm. Head Face, clypeus and labrum all yellow. Labial palpi yellow with palmicula brown. Antennal sclerite yellow, scape brownish and flagellomers yellow. Inter antennal mark and epicarinal mark connected. Vertex raised, spots on anterior and psterio row divided in to three marks. Middle line from front to end of vertex and two on each side. Spots on front reaching to epicedial spot on edges. Vertex has small setae on it. Thorax Pronotum yellow with five dark brown lines. Exterior one on each side of median stops at half length of pronotum. Pronotum has long pale yellow setae on lateral sides and few brown on posterior side. Meso thorax, Meta thorax have one median brown line and one fine on each side. Thorax has tufts of very fine setae. Wings Fore wing broad and little larger than hind wing. Indiscriminate yellow pterostigma. Nine 9 cross veins before RS and RS has 10 branches. Cup originates at the base of first cross vein and unites with 1A after a very short distance. Wing apex acute not round. Hardly distinguishable banksian lines present. 94

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Fig. 37. Cueta thaliae habitus and body parts (a) habitus (b) thorax (c) wings (d) face (e) male terminalia (f) paramere Hind wing little narrow as compared to the fore wing. Ten 10 cross veins before RS and Rs have 9 branches. Mild colour pterostigma and banksian lines present. Legs Coxae yellow, only a small brown mark and comb of setae on pro coxa. Femora yellow with little brown spots sprinkling with black and white setae. Meta femur in male has a comb of setae on posterior surface. Tibia pale yellow with a single brown ring after a short distance from base. Tibia have scattered long black setae and small

95 brown setae. Tibial spur present straight and less than length of first segment. Tarsi yellow with tarsal claw brown and curved apically. Abdomen Abdomen with one median dark brown line on tergits and sometime very narrow line spots on sides. Basal abdominal segment has fine long setae. While rest upto 5th segment; contain small yellow and brown setae. From segment 6 to apex setae become long and thick. Male abdomen with post ventricular lobe little extended. In female posterior gonapophysis smaller and swollen. Anterior gonapophysis broad and have digging setae. Material Examined 2 ♂ Khushab, 18-xii-2009, 1♀ Dera Ghazi Khan, 01-ix-2010, 2♂, Kanju Swat, 08-i-2010, 1♀ Samundri Faisalabad, 04-x-2010. All were collected as larvae and laboratory reared, Saleem Akhtar Distributions Species well distributed thorough southern country. The specimen records collected from Sawt, Mianwali, Khushab, Bhakar, Dera Ghazi Khan, Bhawalpur, Sadiqabad and Thar Desert of Sindh. Habitat It generally lives in sandy soils. But have very diverse range of habitate. I have been collected from very hot south Thar Desert of Sindh to very cold region of Swat. 4.10 Tribe Dendroleontini Banks, 1912 4.10.1 Genus Epacanthaclisis Okamoto, 1910 4.10.1.1 Epacanthaclisis alaica Krivokhatsky, 1998 Diagnosis Forewing costal area biareolate. The tibial spurs as long as the basal segment. Sensory setae three in fore leg and two in meso leg. Fore wing presectoral area with five cross veins and hind wing with two cross veins. Description Measurements (mm) Based on ♀ forewing length: 37-35 mm; hind wing length: 36-3 mm and body length: 29-27mm. Head Face black, clypeus and labrum yellow. Labial palpi basal segment white while distal segment black from base to sensory opening. Epicarinal mark black with thick 96 white setae. Antennal sclerite yellow and scape brown with small white setae. Flagellomere black with white on apex of segments. Vertex raised dark brownish black and very small yellow marks on posterior row of vertex near occiput.

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Fig. 38. Epacanthaclisis alaica habitus and body parts (a) habitus (b) thorax (c) face (d) face (e) female terminalia (f) female terminalia Thorax Pronotum longer than its width and brownish yellow. Pronotum has one thick median strip dark brown and lateral sides also brown. Within yellow part very week elbow like brown streaks. Pronotum lined with white and dark brown setae. Meso and meta thorax dark brown with yellowish brown colour prominent along discs ridges.

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Discs of meta scutum yellow. The ridges of discs have few scattered setae with coinciding colour. Wings Fore wing costal area biareolate and RS starts before cubital fork. Five presctorial veins. CuP moves free a little distance equal to 1A and fuses with 3A. Ptero stigma very small white and with some black area. Venation white and dark in patches, radius and cubitus upto fork are more dark and prominent. Hind wing little smaller than fore wing, costal area simple. Two 2 cross veins before radial sector. Bankisian lines present but not much prominent. Legs Coxae brown, femora dark brown to black. Tibia brownish black in pro and meso leg with white spots. In Meta leg tibia mostly white in colour. Tibial spurs present; slightly curved as s long as basitarsus. While upto second segment in meta leg. Abdomen Abdomen blackish brown with small hair like setae on tergites and sternite. Membrane along the sides yellow in colour. Ectoproct well developed. Lateral gonapophysis small and with setae. Anterior gonapophysis much larger than posterior. Material examined 2♀ Chitral, Ziarat, 2-vi-2011, light trap, Coll. Saleem Akhtar Distribution Afghanistan (Hölzel, 1972), India, Tadzhikistan (Krivokhatsky, 1998), Tibet (Yang, 1987). First time reported from Pakistan. . Habitat Species can be found in very high altitude low temperature zone with alpin forests. The dominating species also include Oak, Salix and Tamarix. 4.11 Tribe Nemoleontini Banks, 1911 4.11.1 Genus Pseudoformicaleo Van der Weele, 1909 4.11.1.1 Pseudoformicaleo gracilis (Klug, 1834) Diagnosis Face yellow and clypeus yellow with long black setae. Labrum yellow with brown setae. Interantennal mark black. The tibial spurs much straight. Basitarsus longest and tarsal claws toothed in middle.

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Fig. 39. Psudoformicaleo gracilis Habitus and body parts (a) habitus (b) wings (c) labial palpus (d) face (e) vertex & pronotum (f) female terminalia Description Measurements (mm) Based on ♀ length of forewing: 24-22 mm; length of hind wing: 20-18 mm and length of body: 24-20 mm. Head Face yellow, clypeus yellow with long black setae and labrum yellow with brown setae. Labial palpai basal segment yellow and distal segment shiny blackish brown. Labial palpai basal ¼ of distal segment swollen. Sensory spot round present at apex. Inter antennal mark black and epicarinal mark black. Scape yellow with brown maculation and have white setae more than black. Flagellomers have dominating colour black and turns light apically. Vertex little raised and have two lines of brownish black spots. Vertex base looks greyish in colour.

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Thorax Pronotum majorly dark brown with grey ash colour appearance. Pronotum have 3 yellow spots on interior margin but end before interior furrow. Pronotum have very few setae on surface. Long strong setae present on lateral sides. Meso and meta thorax predominantly dark brown with greyish appearance. Mesocutum have a dark transverse line in frontal area. Mesoscutum have long black and white setae intermingled. Discs of meta scutum have dark pads. Meta scutellum have fine small white setae on posterior margin. Wings Wings hyaline with few black spots in fore wing. Wings veins mostly colorless with some blackish maculation on cross veins. Wings broad upto hypostigmatal cell and suddenly reduce in to pointed ends. Fore wing have 7 cross veins before radial sector and RS divided into 11 branches. Fore wing have brownish black spots in mediocubital area. First above cubital fork extending upwards to origin of radial sector. Second one cell spot four cells beyond origin in radial sector. Other spots scattered along width of wing below peterostigma. Hind wing have only 1 cross vein before radial sector and RS divided into 10 branches. Banksian lines absent in both wings. Legs Coxae dark blackish brown having tufts of white setae. Only forecoxa sometime light colour. Femora dark brown to shiny black. Fore femur with much white setae. Meso femur with mixed setae while hind femur dominated with black setae. Tibiae yellow with brown spots and apex blackish in all legs. Tibae have long black and few white strong setae. Tibial spurs present almost equal to first tarsi in length. Tarsi have blackish colour and basal segment longest most with some colour less part. Epical tarsus second long segment rest small. Tarsi have some fine hair like setae on them. Tarsal claws present and have a medial tooth. Abdomen Abdomen mostly dark brown; apex of sternites and tergits yellowish appearance. Basal segment have long white hairs and apical segments have blackish setae. Female posterior gonapophysis much reduced. Ectoproct well developed and have stout digging setae. Material examined 2♀ Lal Sonhara National Park, Bhawalpur, 07-v-2012, light trap, Coll. Saleem Akhtar 100

Distribution Species found in southern areas of Pakistan i.e. Cholistan desert. Habitat This species is found in dry hot arid climate sandy desert area. The vegetations in area contain scrub sparse thorny trees and acacia as dominant species. 4.12 Tribe Neuroleontini Navas, 1912 4.12.1 Genus Delfimeus Navas, 1912 4.12.1.1 Delfimeus intricatus (Hölzel, 1972) Pignatellus intricatus Hölzel, 1972 Diagnosis Face brown; darker upper half while lower has blots of brown and yellow colour. Clypeus has two long black setae on each side. Labrum yellow with brown setae. Antennae small and clubbed. Labial palpai have apex of basal segment swollen and basal part of apical segment swollen. Apex of labial palpi narrow flat. Post ventral lobe extened. Description Measurements (mm) Based on ♂ length of forewing: 12.5-13 mm; length of hind wing: 12-12.5 mm and length of body: 12 mm. Based on ♀ length of forewing: 21 mm; length of hind wing: 19 mm and length of body: 18 mm. Head Face brown darker upper half while lower has blots of brown and yellow colour. Clypeus has two long black setae on each side. Labrum yellow with brown setae. Antennae small and lubed. Labial palpai have apex of basal segment swollen and basal part of apical segment swollen. Apex of labial palpi narrow flat. Antennal sclerite yellow, scape brown with small stout black and white setae on it. Pedicel brown with white setae on it. Flagellomers have brown bases and yellow apex. Interantennal spot brown and epicarinal mark brown. Vertex has brown lines on interior and posterior row. Vertex also have scattered black and white setae. Thorax Pronotum wider than its length. Pronotum ground colour yellow with one central wide dark brown stripe and two lateral brown lines. Near anterior end of the apex a large brown dot between central and lateral brown line. Pronotum lined with yellow setae and hard setae present on the sides. Meso and meta thorax entirely brown 101

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Fig. 40. Delfimeus intricatus habitus and body parts (a) habitus (b) wings (c) face (d) vertex & pronotum (e) female terminalia (f) male terminalia with some light colour area. Especially border of prescutum has bright yellow line encircles black area. Prescutum-2 has white stout setae cluster on each side.

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White spots on discs of meso scutellum. Wings Wings elongated oval and rounded at apex. Veins colorless with some having brown colour. Fore wing have 5 cross veins before radial sector and RS divided into 7 branches. Pterostigma black at base and white at end. Brown colour spread over veins. Cubital fork base, cross veins of lower arm braches, gradates of radial sector branches median and cubitus all brown. Cup merges with 1A right at its origin. Hind wing have 1 cross vein before RS and RS divided into 7 branches. Banksian lines and pilula axilaris absent in wings. Legs Legs coxae brown and fore coxae have long apically swollen setae. Femora dark blackish brown and colorless patchy. Fore femora have very long white setae. Tibae interiorly brown and exterior colourless. Except two dark brown rings. First at base and second at apex of tibia. Tibial spurs present straight and short in length almost equal to half of basal tarsal segment length. Abdomen Abdomen dark brown in colour with yellow patches on tergites and sometime sternites also. Abdomen covered with fine white setae. Setae become thick after 6th segment and turn stout black. In female posterior gonapophysis very reduced and have long setae. Ectoproct well build and have digging setae. Material examined 2♂ and 1♀, Ziarat Chitral, 20-vi-2011, light trap, Coll. Saleem Akhtar, Distribution Species well distributed through Afghanistan (Hölzel, 1972) and Iran (Hölzel, 1972) and first time recorded from Pakistan. Habitat The species inhabits in high altitude. High rainfall and green areas. The soils of area are early weathering soils. With mostly gravel and coarse sand. 4.12.2 Genus Distoleon Banks, 1910 4.12.2.1 Distoleon cyrus Hölzel, 1972 Diagnosis The ends of cross viens in the forks shaded. The abdomen colour r browninsh. Pronotum with central fine yellow line and 2-3 blurred small bright spots laterally arranged also long white setae on sides. 103

Description Measurements Based on ♂ length of forewing: 25.5 m; length of hind wing: 24.5 mm and length of body: 22.5mm. Based on ♀ length of forewing: 30-27 mm; length of hind wing: 28-26 mm and length of body: 24-23 mm. Head Face yellow with few splashes of brown colour on the upper margin. Clypeus yellow with yellow erect setae on it. Labrum yellow with brownish yellow setae on lower margin. Labial palpi distal segment swollen and sensory spot oval in shape Interantennal mark and epicarinal mark brown. Scape yellow with white setae, falgellomers segments brown at base and yellow at apex. Vertex not much raised, divided by suture and have two brown lines. Yellow colour base area has black stout setae. Thorax Pronotum have base colour predominantly brown with three yellow lines. One median and two lateral discontinuous fine lines. Pronotum has white setae scattered over it and lateral sides have comb like arrangement of long setae. Meso and meta thorax have predominantly brown colour. One median yellow fine line with two irregular lines on notum discs. Discs of meta scutum have yellow spot with black tint. Sides yellow with brown patches irregularly spread and have tufts of yellow hair like setae. Wings Hyaline with veins colourless and blackish brown maculation. Wings narrow, elongate hind wing narrower than forewing. Forewing have eight cross veins before radial sector RS divided into 12 branches. In fore wing apex have oblique brown line due to brown maculation of gradates. Less prominent banksian lines present. Hind wing much narrow in basal region. Fore wing with one pre sectorial cross vein and RS divided into 10 branches. In forewing upper arm of cubitus, median and first branch of Radius have brown maculation make turn into brown spot. Legs Coxae yellow with basal brown mark and tufts of white setae. Femora swollen, femoral sense hair longer than femur. Femora yellowish except fore femur with brown spots at outer face. Tibia yellow in colour with black setae and spots on bases of setae. Meta tibia longer than femur. Tibial spurs present, strongly curved reaching to third 104 tarsi in pro and meso legs. While in hind leg tibial spur hardly reaches second tarsal segment. Tarsi distal segment largest and tarsal claws much curved.

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Fig. 41. Distoleon cyrus habitus and body parts (a) habitus (b) wings (c) face (d) thorax (e) male terminalia Abdomen Abdomen majorly brown with yellow maculations. Sternites have yellow strips and tergits have diamond shape spot on tergites. Abdomen covered with small white setae which increase in length from 5th segment to onward in males. Beyond 5th segment endings of tregites and sternites have brown setae. In females posterior gonapophysis

105 much small swollen and have setae. The ectoproct have digging setae. In males ectoproct well developed. Material examined 1♂ from Faisalabad 20-vi-2010, light trap, Saleem Akhtar, 1♀ from Sadiqabad 10-v- 2012 light trap, Saleem Akhtar and 1 ♀, from Chitral, KPK, 20-vi-2011, light trap, Saleem Akhtar Distribution Species well distributed in northern parts of county but not recorded in southern areas. Habitat The species occurs in plane areas with loam soils and mostly cultivated lands and riverian areas. 4.12.2.2 Distoleon nefandus (Walker) 1853 Diagnosis Pronotum with brown lines on the sides. Wings tapering to the end hind wing lanceolate. Hind wing have distnict brown spot in apical region near hinf margin of cubital area. Description Measurements (mm) Based on ♀ length of forewing: 33 mm; length of hind wing: 35 mm and length of body: 24 mm. Head Face yellow with brown maculation on margins. Clypeus yellow with upper margin having brown maculation. Labrum yellow with brown setae on lower margin and erect yellowish setae on face. Labial palpi with basal 1/3 swollen with sensory spot rounded and apical portion pointed cylindrical. Interantennal spot brown and epicarinal mark lower half light brown than upper. Antennal scelrite yellow and scape yellow. Flagellomers segments have basal part brown while epical part yellow. Vertex not much raised with brown line on interior row and have two brown spots on posterior row. The yellow base area have large black setae. Thorax Prnotum broad than length at its wide most spot. Pronotum with one broad median yellowish stripe and two dark brown lines on sides. Another yellow line before brown lateral margin. Pronotum have hard blackish brown setae scattered over it and

106 lateral setae thick longer. Meso and meta thorax have broad median yellow stripe thorough out length. The area around yellow stripe brown in colour.

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Fig. 42. Distoleon nefandus habitus and body parts (a) habitus (b) wings (c) face (d) vertex (e) pronotum

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Back side of scuteum discs have tufts of hairs and border of scutellums lined with yellow hair like setae. Sides yellow with irregular brown spots, dominating in pro and meso thoracic region. Wings hyaline with brown venation and some dark spots. Wings apex acute. Forewing much broader as compared to hind wing. Fore wing convex beyond medial fork and hind wing concave in apical region. Fore wing have 7 cross veins before radial sector and radial sector divided into 12 branches. Fore wing have a line of brown spots near border (formed by brown maculation of end forks of veins). Forewing have indistinct bakisian line. Hind wing have 1 cross vein before radial sector and radial sector divided into 12 branches. Hind wing a one cell wide brown spot in lower part of epical region. Spot starts from median and expands in radial sector about 5-7 branches at last gradate. Hind wing also have brown maculation on end forks of veins. Legs Coxae yellow with only fore coxae with a brown spot. Coxae have tufts of hair like setae. Fore femur with much brown colour and rest femora yellow. Fore and meso femoral sense hair longer then femur. Tibia also brownish yellow except hind tibia yellow. Tibial spurs much curved. Tibial spurs reach beyond basal tarsal segments except last longest segment. Tarsal claws also curved. Femoral hard setae yellowish brown. Tibial setae hard blackish in colour. Abdomen Abdomen brown in colour and covered with yellow fine setae. In female setae in apical segments become stout and black. Posterior gonapophysis cylindrical long. Anterior gonapophysis much swollen and smaller with digging setae. Ectoproct medium sized and have thick digging setae on lower end. Material examined 1♀ from Kirthar National Park Sindh, 08-iv-2013, light trap, Coll. Saleem Akhtar. Distribution Species occurs in southern part of the country. Not very common collected species. Habitat The species found in arid areas with dry mountains of south west region. The soils are shallow rocky with some sandy areas. The vegetations include Tamerix spp and Acacia spp as most dominant.

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4.12.2.3 Distoleon obducens Walker, 1845 Myrmeleon mustelinum Fabricius, 1798; Distoleon pugnax (Walker, 1853); Myrmeleon perplexus Walker 1860 Diagnosis Prnotum have three fines yellow lines. Hind wing narrower than forewing. Fore wing have brown spots before half way length of the wing at ends of fork. Abdomen coulr uniform brown. Description Measurements Based on ♂ length of forewing: 33 mm; length of hind wing: 30.5 mm and length of body: 30 mm. Based on ♀ length of forewing: 33-25.5 mm; length of hind wing: 30-25 mm and length of body: 30-24 mm. Head Face yellow with median brown line. Clypeus yellow with erect yellow setae and labrum yellow with fine setae on lower margin. Labial palpi swollen near base and have oval sensory spot. The width of distal segment beyond sensory spot reduces gradually into pointed end. A distinct ridge above face with black colour very prominent. Antennal sclerite yellow, scape brown, flagellomers have basal part of segments brown and lateral part yellow. Interantennal and epicarinal mark brown. Vertex little raised and have brown lines on rows of margins. Thorax Pronotum brown with three yellow lines. One median fine line and two wider yellow lines on sides. Pronotum have clusters of erect blackish and brown setae. Lateral setae comparatively very long. Prescutum indent, scutums and scutellum dominantly brown. Discs of meta scutum have blackish spots. Wings Wings hyaline, peterostigma brown at base and brown in apical area. Apex acute and hind win much narrow. Fore wing have 10 cross veins before Radial sector and RS divided into 11 branches. Fore wing have small markings in apical area. First at recurrent vein from lower fork of cubitus. Posterior bankisian line present in posterior area of cubitus. Hind wing have 1 cross vein before Radial Sector and RS divided into 11 Branches. Banksian lines absent in hind wing. Legs Coxae brown; except fore coxa with some yellow colour. Fore femur swollen with some yellow brown setae. Meso and meta femora less swollen.

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(c) (d) (e) (f)

Fig. 43. Distoleon obducens habitus and body parts (a) habitus (b) wings (c) face (d) pronotum (e) male terminalia (f) Paramere different views Tibia all brownish yellow and tibial spurs not much curved. Tibial spurs reach upto basal 4 tarsal segments in fore and meso leg. While in meta leg it only reaches to third tarsal segment. First tarsal segment larger while epical segment longest. Tarsal claws slightly curved. Abdomen Abdomen mainly brown in colour. Abdomen has light brown in colour setae at base. Medially setae become stout and black. While in apex become little longer. Females have posterior gonapophysis shorter with setae. Ectoproct well developed and have digging setae on its lower part. In males ectoproct well developed. Pregenital plate elongate and pointed.

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Material examined 1♂ & 2♀ Garhi Habib Ullah, 02-x-2010, light trap, Coll. Saleem Akhtar Distribution Species has been collected from northern areas of Pakistan. Habitat Specie occurs in rainfed araes with broad leaf vegetation and alpine trees canopy area. The soils are rich in organic matter and mostly with some sandy profile. 4.12.2.4 Distoleon tesselatus Yang, 1986 Diagnosis Abdominal pattern distinctive shapes of blocks on third and fourth segment. Fore wing have brown spots on the epical region of fore wing on apical part at the endings of RS branches on cross veins. Description Measurements (mm) Based on ♀ forewing length: 37-36 mm; hind wing length: 35-34 mm and body length: 32-31 mm. Head Face yellow, clypeus yellow and labrum yellow brownish setae. Clypeus has erect long yellow setae. Scape brownish yellow surrounded by white setae. Flagellomers segments brown at base and yellowish in apical half. Interantennal spot brown and epicarinal mark brown. Vertex yellow with brown strips on frontal, median and posterior furrow. In middle a checker pattern with one yellow in center and surrounding all corners. Thorax Pronotum yellow dominated by brown lines. Two median broad brown lines separated by yellow line. Margins have brown colour. A brown dissolving spot in between yellow field between median brown lines and border. Pronotum have erect brown setae in middle and long brown setae on lateral sides. Meso thorax have prescutum indent with median and lateral brown lining. Meso scutum has yellow spots on disc area. Meso scutellum with median brown line much brown. Meta thorax with median brown line strike through to end. Discs of meta scutellum have yellow spot with orange tint. Wings Narrow at the base and elongate. Wing venations clear with blackish brown spots.

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Fig. 44. Distoleon tesselatus habitus and body parts (a) habitus (b) wings (c) face (d) thorax (e) male terminalia (f) Paramere different views

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Fore wing has 8 cross veins before radial sector and RS divided into 11 branches. Hind wing has 1 cross vein before radial sector and RS divided into 11 branches. Pterostigma white in between black part. Abdomen Abdomen predominantly yellow with specific check patterns. Lower check expended laterally. Abdomen has black setae on it. In first two segment little longer and in middle very stout. While in apical two segments again longer setae. In female posterior gonapophysis very short with long setae. Ectoproct well developed with digging setae on lower end. Material examined 1♂ from Shah Kot, 15-v-2011, light trap, Coll. Saleem Akhtar, 1♀ from Sharda AJK, 11-vii-2012 , light trap, Coll. Saleem Akhtar. Distribution Species well distributed in southern Punjab first time repoeted from Pakistan. Habitat The species is found in plan areas with loam soils and mostly cultivated lands and riverian areas. 4.12.3 Genus Ganguilus Navas, 1912 4.12.3.1 Ganguilus pulchellus (Banks, 1911) Macronemurus pulchellus Banks, 1911; Neuroleon pulchellus (Banks): Hölzel 1988; Nelees princeps Navás, 1936; Hölzel, 1936 Diagnosis Colour bright yellow with black markings on body and wings. Hind wing little falcate and pointed at apex. Brown marks on hind wings. Legs long; slender with tibia much long. Pronotum have erect setae. Description Measurement (mm) Based on ♀ forewing length: 29 mm; hind wing length: 27 mm and body length: 25.40 mm. Head Face, clypeus and labrum all yellow. Upper part of clypeus has yellow and small black setae. Pedicel brown and flagellomers yellow with black setae. Interantennal mark and epicarinial mark brown. Vertex raised and have light brown marks on

113 posterior row. Vertex has setae on dome. Palpai distal segment dilated at base area hosting round sensory spot and gradually narrowing to bifurcated apex. Thorax Pronotum yellow with very small basal area brown. Anterior end have brown colour spots not reaching to posterior margin. Pronotum have erect setae. Meso thorax mostly yellow with groves and anterior part black. Wing base areas black. Meta thorax Meta scutum black with distinct black mark on disc. Prescutum and scutellum yellow. Wings Wings have black and brown markings on them. Fore wing has 3 black dots in mediacubital area upto fork and 3 spots in posteror area. A spot on first division of RS and second below it. All other spots arranged in lower part of wing in radial area. Radial sector starts beyond cubital fork 5 cross veins before RS, Rs divided into 10 branches. Venation white and few branches have black spots. Hind wing little falcate and pointed at apex. Hind wing has 2 per sectorial cross veins and RS divided into 10 branches. Hind win have some brown dots on gradates and apex.

(a)

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Fig. 45. Ganguilus pulchellus habitus and body parts (a) habitus (b) face (c) thorax (d) female terminalia Legs Coxae, femora, tibiae and tarsi all yellow except hind femora brownish black.

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Tibial spurs present equal to first two tarsal segments. Hind tibia much longer than femora. Abdomen Abdomen yellow and black. Sternites predominantly brownish black and tergits have variable colour. Frist tergite black second and third yellow with black marking. Forth to onward tergite predominantly black with yellow spots. Abdomen covered with fine white setae. Material Examined 1 ♀ Naukot, Thar Sindh, 28-viii-2010, light trap, Coll. Saleem Akhtar; Distribution Species well distributed from northern Kenya to Pakistan (Michel and Mansell, 2010) Habitat Specie inhabits in deserted areas of Pakistan. The species have been collected from Nau Kot Thar desert with huge sand dunes and acacia vegetation. 4.13 Tribe Myrmeleontini Latreille, 1802 4.13.1 Genus Myrmeleon Linnaeus, 1767 4.13.1.1 Myrmeleon assamensis Ghosh, 1984 Diagnosis Pronotum pale with two light brown lines. Vertex little raised. Vertez all black except “H” shaped mark at posterior row. Description Measurements (mm) Based on ♂ length of forewing: 28 mm; length of hind wing: 27 mm and body length: 26 mm. Head Face black, interantennal mark black, epicarinal mark black. Clypeus brownish black. Antennal sclerte yellow, scape interiorly with brown spot and flagellomers black. Vertex little raised and most of it black except “H” shape mark at posterior row of the vertex. Distal segment of palpai dilated at basal part and narrow at apex. Sensory spot rounded and present at apical part. Thorax Pronotum with outer margin pale yellow, two oblique lines and darker spot at furrow. Pronotum densely covered with thin brown setae.

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Meso and meta thorax also brown with yellowish end of meso and meta scutellum. Lateral sides of thorax brown. Wings Hyaline, longer and narrower in shape. Hind wing much reduced in width. Veins white in colour. Black colour tint in forewing subcostal where cross veins land. A small blackish brown Pilula axilaris present. Fore wing 6 cross veins before Rs and Rs divided into 9 branches. Legs Coxae, tibiae and femora all yellow but brown from inner sides. Long thick black setae on femora and tibia. Tibial spurs larger than first tarsal segment.

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Fig. 46. Myrmeleon asamiances habitus and body parts (a) habitus (b) face (c) thorax (d) male terminalia different positions

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Abdomen Abdomen elongate slender and chocolate brown in colour. Abdomen covered with fine yellowish brown setae. In males ectoproct well developed but not elongated. Material examined 1♀ and 2♂ Laboratory reared, 14-viii-2010, Larave were collected from Jehlum and Islamabad Koral area. Distribution Species have been reported from Iran (Hölzel, 1972) also well distributed in central areas of Pakistan i.e. Islamabad, Jhelum and Bhakkar. First time recorded from Pakistan. Habitat The species inhabits semi arid areas of Pakistan. The collected specimens were from Jehlum. The topograpgy of the area is undulating with well dariend soils and some sandy areas too. 4.13.1.2 Myrmeleon bore (Tjeder, 1941 ) Enza otiosus Navas 1912 Diagnostics Face brown, clypeus brown and labrum brownish yellow. Clypeus with four black setae and labrum has a row of setae on lower boarder. Antennal socket yellow, scape brown on anterior side and yellow posterior side. Pedicel brown and flagellomer black in colour. Descriptions Measurement (mm) Based on ♀ length of forewing: 24 mm; length of hind wing: 21 mm and length of body: 20 mm. Head Face brown, clypeus brown and labrum brownish yellow. Clypeus has four black setae and labrum has a row of setae on lower boarder. Antennal socket yellow scape brown on anterior side and yellow posterior side, pedicel brown and flagellomer black in colour. Three dark lines in face one central and two on lateral sides. Interantennal mark brown and epicranial mark dark brown with a transverse black line. Vertex raised brown spots on anterior and posterior row of vertex. The marks similar to Myrmeleon hyalinus.

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Fig. 47. Myrmeleon bore habitus and body parts (a) habitus (b) face (c) thorax (d) female terminalia different positions Thorax Pronotum brown and anterior margin lined with white colour. Pronotum has two clusters of long setae on either side of the middle line. Pronotum has long black setae and small brown setae. Meso and meta thorax uniformly brown without any mark or spot. Black setae tufts on prescutum. Fine yellow setae on disc endings of scutums and scutellums. Wings Wings long narrow and acute at ends. Wing margins have fringes of setae. Wing venation colour clear with patches of brown colour. Fore wing has 7 cross veins before

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Radial sector and RS divided into 7 branches. Hind wing has 5 cross veins before RS divided into 7 branches up to hypostigmatic cell. Posterior banksian line very clear in both wings fromed by lower arm of cubitus. Abdomen Dark brown in colour abdomen has small black setae which turn thick and long from apex of sixth segment to abdomen tip. Posterior gonapopgysis elongated and very fine cylindrical with setae on apex. Setae longer than length of gonapophysis itself. Ectoprcot well-built and have small stout digging setae. Material Examined 4♀ from Malot Jhelum, laboratory reared specimens adult emerged 05-iv-2010, larvae scoped from sand. Coll. Saleem Akhtar Distribution Species have been collected from central Punjab and some areas of Sindh also Habitat The species habitat limited to dry sandy soils of arid climate with very sparse vegetation. 4.13.1.3 Myrmeleon hyalinus Olivier, 1811 Diagnosis Male have small pilula axilaris. Description Measurements (mm) Based on ♂ length of forewing: 18 mm; length of hind wing: 16.5 mm and length of body: 16 mm. Based on ♀ length of forewing: 21.5 mm; length of hind wing: 19 mm and length of body: 20.5 mm. Head Face black and yellow lower margin. Clypeus yellow and have two to four black setae. Labrum yellow and have setae on lower border. Palpai basal segment yellowish and distal segment shining black well swollen. Sensory spot in apical region. Apex pointed ant tip have two lobes. Antennal sclerite yellow, scape yellowish brown, pedicel black antennae black only a mild yellowish appearance in apical part. Interantennal and epicarinal mark black. Vertex raised. Frontal row black and middle row yellow but has a black spot in middle. Interior and posterior rwo field yellow area has black setae. Posterior row has 3 blackish brown spots. Two lateral spots on posterior row get triangular shape by expansion as they proceed toward occiput. 119

Thorax Pronotum yellow base with brown colour spread starts from posterior margin and narrows down into a line beyond frontal furrow. Colour expands in groves of furrow but not in middle forming concave blank areas. Ultimatly pattern looks like flying squirrel/fox shape. Pronotum has pale setae on it absent in furrows. Strong setae present on lateral sides: Meso and Meta thorax dominated with brown colour except some yellow spots along central row on discs. Bright orange yellow spot on Meta scutum discs prominent in males.

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(b) (c) (d)

Fig. 48. Myrmeleon hyalinus habitus and body parts (a) habitus (b) face (c) pronotum (d) male genitalia Wings Wings narrow, hyaline and sub-acute at the apex. Wing venation pale yellow only little dark appearance in subcostal vein. Fore wing has 9 cross veins before radial sector and radial sector divided into 9 branches. Hind wing has 5 cross veins before radial sector and RS divided into 9 branches. Veins and cross veins have hair liker black and white setae. Pilula axilaris small and white in colour.

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Legs Prolegs swollen as compared to others. Coxae brown at base and yellow beyond half. Fore coxa have black stout setae on inner face. Femora yellowish at base and epically brown. Pro tibia much brown while Meso and Meta tibia on ventral side has brown tint while dorsal side yellow. Tibial spurs brown and do not reach the apex of first tarsal segment. The apical tarsal segment much enlarged. Abdomen Dark brown in both male and female. Tergites and strintes have yellow hair like setae On them. Setae longer on first two segments. Apex of sternits and tergites yellow transversely. In female posterior gonapophysis small cylindrical and thin. Anterior gonapophysis thick and pointed. Ecoproct well build. The lower lobe of ectoproct and lateral gonapophysis have digging setae. In males epical abdominal segments yellow in colour. Male ectoporoct rectangular shape. Pregenital plate elongated and pointed and have long hair like setae. Material examined 2♂ Thar Sindh 14-viii-2012, 3 ♀ Lal Sohanra National Park 18-ix-2014, light trap, Coll. Saleem Akhtar. Distribution Species have wide distribution Afghanistan (Hölzel, 1972) Algeria (Navas, 1918), Ethiopia (Navas 1936), Morocco, Tunisia, Senegal, Gambia, Egypt (Navas 1926), Gambia, Iraq (Hölzel, 1972), Iran (Hölzel, 1972), Israel, Lebanon (Hölzel 1972), Libya (Navas, 1914), Oman, Senegal, Sudan (Esben-Petersen 1931), Saudi Arabia and Pakistan. Habitat The species inhabits in the arid climate with sandy soils. The vegetation cover includes thorny shrubs and acacia plantations. 4.13.1.4 Myrmeleon inconspicuus Rambur, 1842 Myrmeleon incertus Rambur, 1842; Myrmeleon erberi Brauer, 1868; Myrmeleon ariasi Navàs, 1913 Diagnosis Face black with round yellow spot in middle. Clypeus yellow with two blackish brown spots in lower area containing one black setae. Labrum yellow lined with

121 yellowish brown setae. Palpi yellow basal segment. Distal segment shiny black with round sensory spot in apical protion and ends into bifurcated tip. Description Measurements (mm) Based on ♂ length of forewing: 18 mm; length of hind wing: 17 mm and length of body: 18 mm. Based on ♀ length of forewing: 20 mm; length of hind wing: 18 mm and length of body 21mm.

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Fig. 49. Myrmeleon inconspicuus habitus and body parts (a) habitus (b) face (c) pronotum (d) thorax (e) male genitalia Head Face black with round yellow spot in middle. Clypeus yellow with two blackish brown spots in lower area containing one black setae. Labrum yellow lined with

122 yellowish brown setae. Palpi yellow basal segment but distal segment shiny black with round sensory spot in apical protion and ends into bifurcated tip. Antennal sclerite yellow. Scape brownish black on front side while yellow on back side. Antennae have yellowish appearance on anterior and posterior side. Thorax Pronotum dark blackish brown predominantly and along anterior border have two yellow spots. Pronotum have setae all over it. Setae bases brown and little raised. Borders of pronotum lined with setae. Meso and Meta thorax dark brown in colour with few erect setae on presecutum. The colour gets lighter on discs. Meta scutum discs have prominent orange yellow spot on them. Pilula axilaris present and white at base with golden brown brush towards apex. Wings Wings narrow littler longer abdomen. The wing venation have white colour with few splashing of brownish black colour. Colouring much prominent in subcostal and cubitus. Pterostigma blackish at base and end part white. In hind wing an indistinct white stegema present. Fore wing has 7 cross veins before RS and Rs divided into 7 branches. The hind wing has 5 cross veins before RS and divided into 9 branches. Legs Procoxae yellow with outer yellowish brown spot. While meso and Meta coxae blackish brown with silvery appearance. Pro and meso tibia with irregular brown and yellow. Meta femur basal half yellow and apical half black. Tibia variant colour; inner side blackish and outer side whitish distinctive in hind tibia. Tibial spurs brown and equal to first tarsal segment while little smaller in hind tibia. Last tarsal segment too much long and black. Abdomen Dark brown in colour with fine white setae on it. In females cylindrical small posterior gonapophysis and well developed ectoproct. The lower end of ectoproct have digging setae. In males ectoproct broad with long apical setae. Pregenetal plate elongated. Material examined 1♂ from Thar Sindh, 01-ix-2010 and 2 ♀ (larvae collected) from Sahiwal Punjab, 11- viii-2010, and 1♂ Lal Sohanra National Park, 15-v-2013, light trap, Coll. Saleem Akhtar

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Distribution Species well distributed through Pakistan and first time recorded from. Habitat The species generally can be found in arid areas with sandy soils. The species have been collected from Bhakar and Layyah district. The soils of area are sandy and topology contains large fine sand dunes with tall grasses.

4.13.1.5 Myrmeleon noacki Ohm, 1965 Diagnosis Bright yellow ring formation around eyes with black frons and interantenal and epicrinal mark. Fine yellow line in middle of pronotum and sides yellow. Description Measurement (mm) Based on ♂ length of forewing: 28.50 mm; length of hind wing: 26.50 mm and length of body: 26 mm. Based on ♀ length of forewing: 28mm; length of hind wing: 27 mm and length of body: 25.5mm. Head Face black with lower boarder yellowish. Clypeus yellow with two large black spots containing one seta. Labrum yellow with setae on lower border. Labial palipai basal segment yellowish and epical segment shining brownish black. Sensory slit present in apical area of dilated part. Antennal sclerite yellow, scape brownish black, flagellomers brownish to black in colour and dialed apically. Interantennal mark and epicarinal mark black. Vertex raised and black. Thorax Dark colour in males and light colour in females. Pronotum dark brown with one light colour median strip centrally dilated. Lateral sides of pronotum also light yellowish in colour. Pronotum boarder lined with setae. Lateral sides have strong thick 4-5 setae. Meso and Meta thorax dark brown in colour. Prescutum has erect setae rest thorax have few scattered fine setae. Lateral sides also brown in colour. Wings Wings narrow, pointed at apex and elongate. Wings little longer than abdomen. Black and brown patches on the veins. Venation much darker in males as compared to females. Pterostigma brownish at base and white at apex. Forewing has 10 cross veins

124 before Radial sector and RS divided into 10 branches. Hind wing has 5 cross veins before radial sector and radial sector divided into 10 branches. Legs Coxae blackish brown. Femora, tibia, tibial spurs all yellowish brown except hind tibia with inner side brown and outer whitish. Coxae have small fine yellow setae. Femora and tibia have strong setae. Tibial spurs almost equal to first segment length. Last segment of tarsi much enlarged as compared to basal segments.

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Fig. 50. Myrmeleon noacki habitus and body parts (a) habitus (b) wings (c) face (d) thorax (e) male genitalia Abdomen Brown colour dark in males and light in females. In males stout setae while in female little small and fine setae present on abdomen. Setae colour much darker in males.Females have a well build ectoproct and very small thick gonapophysis.

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Material examined 1♂ from Gilgit West, 20-vi-2013, light trap, Coll. Saleem Akhtar. 1♀ from Bagh, 13- vii-2014, light trap, Coll. Saleem Akhtar Distribution The species has been recorded from Swat (Hölzel, 1972) and Bagh. The species speread may be restricted to northern areas because of its low temperature zone living habit. Habitat The species occurs in lush green areas hilly areas. Generally larvae are found under rock overhangs and bulidings. 4.13.1.6 Myrmeleon trivialis Gerstaecker, 1885 Myrmeleon zanganus Yang, 1987 Diagnosis Larger species. Yellow line in middle of pronotum which disappears before hind margin. Sides of pronotum much yellowish and have black setae. Descriptions Measurements (mm) Based on ♂ length of forewing: 36 mm; length of hind wing: 34 mm and length of body: 30 mm. Based on ♀ length of forewing: 40 mm; length of hind wing: 38 mm and length of body: 32 mm. Head Face black, clypeus upper half blackish brown with two long setae one on each side. Labrum brownish yellow with setae on lower border. Labial palpai yellow with distal end swollen at the base and pointed at the apex. Sensory spot beyond 1/3 of palmicula and circular. Interantennal sclerite yellow. Scape black on front and posterior yellow. Flagellomers, interantennal mark and epicarinal mark all black. Thorax Pronotum blackish brown and have one central yellowish line. Lateral sides yellow. Pronotum has fine scattered yellow setae all over it and 4-6 long thick black setae on lateral sides. Prescutum black has erect setae. Discs of meso and meta thorax have fine yellow setae. Sides along the wing bases yellow. Wings Wings elongate with acute at ends. Wing venation yellow and pterostigma white. Fore wing uniformly convex and hind wing little concaved inward in apical ¼. 126

Fore wing has 7-8 cross veins before radial sector and RS divided into 13 branches. Hind wing has 5 cross veins before RS and RS divided into 11 branches. Male has small pilual axillaris

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Fig. 51. Myrmeleon trivialis habitus and body parts (a) habitus (b) thorax (c) male terminalia (d) paramere Legs Basal part of coxae brown rest yellow, Femora, Tibia and tarsi all yellow except apex of tibia and tarsi black. Tibial spurs present in pro and meso leg tibial spur longer than first tarsal segment and in hind tibia almost equal to basal tarsal segment. Abdomen Abdomen brown in males with fine brown setae. Last two segments have apical part yellow in colour. While ectoproct and pregenital plate yellow. In female abdomen much darker and have black and yellow setae. While on 7 and 8th segment there are only stout black setae. Posterior gonapophysis cylindrical and reduced. Ectoproct well developed and have digging setae. Material Examined 1♂ Garhi Habib Ullah, 02-ix-2010, 1♀ Pir Chanasi, 30-vii-2010, light trap, Coll. Saleem Akhtar Distribution Species is well distributed in northern areas of the country.

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Habitat The species occurs in medium rain fall high alititude areas. The larvae are unknown. The vegetation in the area includes hebaceous shrubs with broad leaf and Alpine trees 4.14 DNA Based identification The DNA sequence for each species was generated. Then the species sequences were tested for similarity with availbel sequences in the GENEBANK by using nBLAST (http://www.ncbi.nlm.nih.gov/blast/). The Barcode sequences for species were compared with the existing data in BOLD using The BOLD identification System (IDS) for COI (http://v4.boldsystems.org/index.php/IDS_OpenIdEngine). The BOLD system uses Barcode Index Number (BIN) system to cluster species data into operational taxonomic units (OTUs) (Ratnasingham and Hebert, 2013). DNA squece of all antlion species in this study were assigned a BIN. Neucleotide sequence alignments were perfomed by using BOLD aligner and MEGA7. Nearest neighbor clustering was done by using MEGA7. The analysis was performed by using phlogentic reconstruction and the statistical method used was neighbor joining. Test apllied was used boots rap and 500 replications were selected. While Kimora 2-parameter model was used. Similarity pattern was obtained by selecting uniform rated transition and transvertions in homogeneous lineages. The missing data was treated as complete deletions. All codon postion were selected for the analysis. Whiele sequence from ascalaphidae family member was used as out group for neighbor joing clustering. Th e results revealed that all individuals of single species occupied single BIN. All species sequence was blasted in NCBI, BOLD and other databases to compare the species with the existing data base. It was observed that most of the species do not have any data from other countries. While some species have been processed by other researchers around the globe those have been listed in the table below. The interpeciefic and itera specifid distance was calculated by BOLD.

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4.15 Barcode Index Numbers (BINs) Analysis Table No. 2. Barcode Index Numbers (BINs) and maximum intraspecific distances for Antlion species of Pakistan and other countries. The species specific BIN numbers were obtained and area listed in table below

Max intraspecific Countries S.No Average Species BIN Distribution with Distance % matches (individuals) Palparellus AAP9945 n/a ------0 1 astutus Indopalpares ACJ6331 n/a ------0 2 pardus Palpares ACI8805 0.55 1.22 (5) ---- 0 3 rajasthanicus Palpares AAN8602 1.9 2.29 (27) ---- 0 4 trichogaster Parapalpares AAZ4141 n/a ------0 5 solidus 6 Palpares astarte No BIN n/a ------0 Acanthaclisis AAZ4142 0.46 0.46 (2) ---- 0 7 neozelandica Acanthaclisis ABX6854 0.2 0.31 (4) ---- 0 8 obscura Centroclisis thari AAQ3533 0.4 0.76(5) ---- 0 9 n.sp Myrmecaelurus AAZ4149 1.16 1.8 (3) ---- 0 10 longiprocessus Myrmecaelurus AAN8605 .82 1.38 (11) ---- 0 11 paghmanus Nohoveus AAN8596 .98 1.68 (17) ---- 0 12 vanharteni Iranoleon ABX7053 0.89 1.68 (6) ---- 0 13 vartianae

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Myrmeleon ABX7022 0.93 1.68 (4) ---- 0 14 trivialis 15 Myrmeleon bore AAN8606 1.07 1.07(2) ---- 0 Greece, Italy, Myrmeleon AAQ3532 0.81 1.71 (3) ---- Morocco, hyalinus Israel, 16 Egypt Myrmeleon ACR1404 N/A ------0 17 noacki Myrmeleon AAN8604 18 inconspicuus Myrmeleon AAN8600 0.24 0.61 (5) ---- 0 19 assamensi 20 Cueta thaliae AAN8599 1.03 2.14 (80) ---- 0 21 Cueta lineosa AAN8603 1.29 2.69(34) ---- 0 Distoleon ACI8886 n/a ---- 22 nefandus 23 Distoleon cyrus AAN8597 0.79 1.22 (4) ---- 0 Distoleon AAP9943 0.41 0.46 (3) ---- 0 24 obducens Distoleon AAZ4148 0.92 0.92 (2) ---- 0 25 tesselatus Neuroleon AAZ4145 n/a ---- 26 tenellus Neuroleon (Ganussa) ACI8944 0.45 0.61 (7) ---- 0 27 leptaleus Neuroleon AAP9944 28 egenus Neuroleon ACB8966 N/A ---- 0 29 asiricus

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Delfimeus AAZ4144 0.24 0.61 (6) ---- 0 30 intricatus Echthromyrmex ACI8808 n/a ---- 0 31 orientalis Epacanthaclisis 32 AAZ4143 0.4 1.07 (7) ---- 0 alaica Ganguilus AAN8594 1.35 2.14 (5) ---- 0 33 pulchellus

Gepus invisus AAZ4150 0.2 0.46 (6) ---- 0

34 Indoleon AAZ4147 n/a ---- 0 35 vartianorum Iranoleon ABX7053 0.89 1.68 (6) ---- 0 36 vartianae Pseudoformicaleo ABX7054 n/a ------0 37 gracilis 38 Solter felderi ACI8806 n/a ------0 39 Solter hardei ABX7131 n/a ------0 40 Cueta lineosa AAN8603 1.29 2.69(34) ---- 0 41 Cueta minervae ACI8884 n/a ------0 42 Cueta plexiformia ACI8807 n/a ------0

131

4.16 Nighbour joining cluster analysis for species 4.16.1 Nighbour joining cluster analysis for species in subfamily Palprinae Neighbour joining clustring was done for all species presented in this study. The results reveald that COI gene discriminated all spcies from each other. The individuals for same species were clusterred on same clade. With the very high to 100 bootsrap value for same species individuals .While results also revealed that congenric species tend to be more close neighbour as compared to transgenric species. As here in the nighbour joining cluster presented below. There is no BIN assigned to species Parapalpres solidous because there was a very short sequnce recoverd for the species i.e. 407bp long so ot does not qualify for the standard 658bp lenght for COI gene DNA barcode region.

100 Palpares trichogaster (27) AAN8602 50 74 Parapalpares solidus (1) AAZ4141 76 Palpares astarte (1)

Palpares rajasthanicus (5) ACI8805 100 47 Indopalpares pardus (1) ACJ6331 Palparellus astutus (1) AAP9945

Ascalaphidae

0.02 Fig. 52. Nighbour joing cluster for species in subfamily Palprinae The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.80879767 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 37 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 402 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4].

132

4.16.2 Nighbour joining cluster analysis for sub-family Acanthaclisini

100 100 Acanthaclisis obscura (5) ABX6854

Acanthaclisis neozelandica (2) AAZ4142 100 Centroclisis spp (5) AAQ3533 100 Ascalaphidae

0.020 Fig. 53. Nighbour joing cluster for species of sub-family Acanthaclisini The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.37425016 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 13 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 551 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4]. 4.16.3 Nighbour joining cluster analysis for genus Neuroleon 100 71 Neuroleon (Ganussa) leptaleus (7) ACI8944 84 Neuroleon tenellus (1) AAZ4145

Neuroleon egenus (5) AAP9944 100 Neuroleon asiricus (1) ACB8966

Ascalaphidae

0.020 Fig. 54. Neighbour joining cluster for species of genus Neuroleon The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.42274006 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with

133 branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 654 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4]. 4.16.4 Nighbour joining cluster analysis for genus Myrmelecerus and other close taxa

100 34 Nohoveus vanharteni (17) AAN8596

64 Iranoleon vartianae (6) ABX7053 100 Myrmecaelurus paghmanus (13) AAN8605 100 Myrmecaelurus longiprocessus (3) AAZ4149 100 Ascalaphidae

0.020 Fig. 55. Neighbour joining cluster for species of genus Myrmelecerus and other close taxa The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.57461825 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 40 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 570 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4].

134

4.16.5 Nighbour joining cluster analysis for genus cueta

Ascalaphidae

91

Cueta thaliae (81) AAN8599 100

100 Cueta lineosa (52) AAN8603

Cueta plexiformia (1) ACI8807 100 Cueta minervae (1) ACI8884

0.020 Fig. 56. Neighbour joing cluster for species of genus cueta The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.62564217 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 113 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 593 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4].

135

4.16.6 Nighbour joining cluster analysis for genus Distoleon

100 80 Distoleon cyrus (3) AAN8597

76 Distoleon nefandus (1) ACI8886

Distoleon tesselatus (2) AAZ4148 100 Distoleon obducens (3) AAP9943 100 Ascalaphidae

0.020 Fig. 57. Neighbour joing cluster for species of genus Distoleon The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.44398911 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 10 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 654 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4]

136

4.16.7 Nighbour joining cluster analysis for Genera reprsented by single species

41 Ascalaphidae 19 Delfimeus intricatus (6) AAZ4144 100 Pseudoformicaleo gracilis (1) ABX7054

Echthromyrmex orientalis (1) ACI8808 27 30 35 Ganguilus pulchellus (3) AAN8594 100 Gymnocnemia variegata (1) AAZ4147 45 Gepus invisus (4) AAZ4151 100100 Solter felderi (4) ACI8806

Epacanthaclisis alaicus (7) AAZ4143 100

0.020 Fig. 58. Neighbour joing cluster for species of Genera reprsented by single species The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 1.00654609 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 28 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 597 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4].

137

4.16.8 Neighbour joining cluster analysis for genus Myrmeleon 100 65 Myrmeleon inconspicuus (12) AAN8604 37 Myrmeleon noacki (1) ACR1404

27 Myrmeleon trivialis (4) ABX7022 100 Myrmeleon assamensi (5) AAN8600 100 100 45 Myrmeleon hyalinus (4) AAQ3532 Myrmeleon bore (2) AAN8606 100 Ascalaphidae

0.020 Fig. 59. Neighbour joing cluster for species of genus Myrmeleon The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.67450004 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 29 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 452 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [4].

138

4.17 Antlion’s species Barcode Gap Analysis (BGA) using BOLD System

Fig. 60. Maximum intraspecific desistance versus Nearest Neighbor distances

Fig. 61. Mean intraspecific distance versus Nearest Neighbor distances

139

Fig. 62. Number of individuals per species verses maximum Intra-specific distance

Fig. 63. Frequency histogram of distance to nearest neighbor.

140

4.18 Pairwise distance analysis of Antlion species generated by Automatic Barcode Gap Discovery (ABGD).

Fig. 64. Histogram of Pairwise distance

Fig. 65. Ranked Pairwise distances

141

V. Discussion The antlion species collected from different areas of Pakistan subjected to morphological and DNA based taxonomic studies. COI 3-prime region was used as marker for species discrimination and as a support to morphological studies. The studies revealed that Pakistan have a very diverse fauna of Antlions. Previously 28 species have been reported from Pakistan by various workers out of which few were still not exactly reported from country but presumable presence was mentioned by Navas (1912), Hölzel (1972), Gosh (2004) Saji and whitington (2008) on basis of their studies conducted on fauna of regions in Iran, Afghanistan, China, UAE and India. While some species have been validated from country by Iqbal and Yousuf (1990, 1991, and 1997) and Michel and Mansell (2010). The exact number of species remained debatable. Current studies revealed species count of country may reach beyond. Current studiy presents 44 species collected from various regions of country where collection expeditions were possible to conduct. Two subfamilies Palparinae and Myrmeleontinae were collected from various parts of country. Sub family Palparinae 8 species have been recorded and presented with detailed description and Pictures. The species include Indopalpares pardus previously reported by Iqbal and Yousuf (1990) as Palpares pardus from Faisalabad. Current study reports its new locality record from Chakwal. The species descriptions are similar to descriptions made by Ghosh (1984) Insom and Carfi (1988) Iqbal & Yousuf (1990). The species have much similarity with few close species in terms of wing spots. But male genitalia are very unique and have long setae on the inner side of ectoproct. Although there was some controversy over presence of two long setae on inner side of male ectoproct. The species was collected on light trap during late hours around 22:00hours time. Palparellus astutus first time reported as from Pakistan previously it was reported from India. The species description was similar to description presented by Mansell (1996). Only female specimen was observed. Author have seen pictures of male from collection of Jhon O Dell; Ukrainian amateur’s personal collection. The male females are similar in wing pattern and body size has very little variation. Males have a little larger body. Species was collected from Makhi Belo, Thar, and Sindh. The specimen was collected at time of dusk and early evening. Palpares rajasthanicus first record from Pakistan previously reported from India by Ghosh (1991). Species description was similar to Ghosh’s description. Recent 142 collection was made from Cholistan desert, Bhawalpur. The species seems to be rare and distribution may be limited in India and Pakistan only. The species remains active for long hours but during early summer. No collection records have been made during late season. Palpares astarte previously reported by Iqbal and Yousuf (1990) from Rajanpur recent collection was made from Dera Ismail Khan as new locality record. The species description was similar to Iqbal and Yousuf (1990) and Banks (1913). The species seems to be rear not much collection was made regardless of repeated efforts in same area. Species was collected on low light tungsten bulb light during early hours of night. Palpares trichogaster was first reported by Navas (1913) from Minora Karachi. It has been presumed that species might be Palpares cepholates; later reported by Iqbal and Yousuf (1990) as Palpares cephalotes. Palpares trichogaster and Palpares cephalotes are not same. The Palpares cephalotes from Senegal (Author observed pictures kindly provided by Andres Prost via email) was found different on basis of abdominal hairs. In case of Palpares cephalotes there are white hairs at first three basal abdominal segments and in P.trichogaster there are small thick setae. Hence the both species must stand valid and to remove confusion genomic data comparison should be made for any firm decision making. Parapalpares solidus was previously reported as Palpares solidus by Iqbal and Yousuf (1990) from Rawalpindi, from Pasni Balochistan by Hölzel (1972). Recent collection was made from Ziarat Quetta and Rhamboor Chitral near Afghan border indicating wide distribution of species but limited to high elevation and low temperature zones. The species description were similar to Iqbal and Yusuf (1990) Insom and carfi (1988) and little different from Koçak and Kemal (2008) in wing spots. It may be because of environmental or species independent geographical evolution. The female abdomen does not have any long hairs or setae instead little rough appearance due to very small setae. The species activity occurs during early evening hours. Collection was made by using low light tungsten bulb. Stenares clarus collected and presented as first record from Pakistan. The species description was compared to the previously described species Stenares hyaena clara by Ghosh (1990) from India described as a sub species of Stenares hyaena. Stenares clarus have lot of morphological differences and distinctive black lines were not observed on face of species. As species name was not properly selected as per rules of International Commission on Zoological Nomenclature. The clara 143 feminine cannot be used in combination with Stenares. Hence combination Stenares clarus should be used for the new species and should be considered as a valid species. Though the species have been collected from Chakwal a relative low land area. Author had news that same species have collected by someone from Astore, Gilgit Baltistan area. As author have not observed the species by himself. So genric existence in both ecological situations stands valid but not species distribution may be confirmed. Stenares improbus first reported from Pakistan was previously reported by Ghosh (1990) from India. Current study collection was made from Islamabad. But I have also seen one specimen from Dadar, Abottabad in (Jhon O Dell’s UK, personal collection). The description of the specimens were similar to Ghosh (1990). The specimen used in current study belongs to National Insect Museum, NARC Islamabad. As the abdomen was already damaged so no observation were recorded in thins study. It was observed that Palparinae were present in various environmental and ecological conditions. But they were mostly present in less disturbed and woodland ecologies. They exist in trees dominated ecologies. Also it has been observed that species mostly existed in isolated ecologies no two species were collected from same ecology at same time it may be due to their independent evolution to avoid competition and other biological complexities. Subfamily myrmeleontidae represented by 37 species recorded. Genus Acanthaclisis has two species. Acanthaclisis neozelandica was first reported by Navas (1912) as Cosina neozelandica mistakenly from New Zealand but later Wise (1963) corrected about its occurrence in Quetta Baluchistan Pakistan. Current collection was from new locality. First recorded from Ziarat Chitral Himalayan range. The species descriptions were similar to Navas (1912). Although the species looks lot more like Acanthaclisis occitanica. That’s why researchers say there are much chances that Acanthaclisis neozelandica and Acanthaclisis occitanica are synonymous. But if as per description of Mirmoyedi (2006) Acanthaclisis occitanica pale colour while tillers (2013) images show a pinkish brown colour while in most cases it have been observed as greyish white. But in case of Acanthaclisis neozelandica its similar to the Tillers (2013) and different form Mirmoyedi (2006) also it has 4 cross veins before radial sector. Also male ectoproct is long and have two lobes before end lobe. So its still hard to say that two species are same. Hence Acanthaclisis neozelandica still considered as valid species. 144

Acanthaclisis obscura first time reported from Pakistan. Previously species described and reported from Afghanistan (Hölzel, 1972) and from Tadzhikistan (Krivokhatsky, 1998). The species exists in two morphs. One has wings with clear wings with brown colour splashes while second morph has a clear black stripe under the costal area and above medial area. Currently species have been collected from Lal Sohanra National Park, Bhawalpur, Punjab. The species collection was made during late summer. Species more activity have been observed late summer and early winter. Two species from Genus Centroclisis were recorded during this study. Centroclisis indica previously recorded by Iqbal and Yousuf (1997) there were no figures presented by them. But descriptions made by Ghosh (1984) were similar to the species described here. The species exists in very hot region. But the adults are active during late summer till October. Specie common in east desert part of Pakistan ranging from Bhawalpur to Thar Desert end near Karachi. Centroclisis thari n.sp. First recorded from Pakistan Althogh much similar to Centroclisis cervina (Gerstaecker) and C.bandrensis (Navás) but does not satisfy description of species described till now by previous workers like (Navas, 1912; Gosh, 1984 and Iqbal and Yousuf, 1997). Hence presented as possibly new species. Current name must be considered as tentative and not for permanent Zoological record. The Article 8.2 of the International Code of Zoological Nomenclature applies till publication of proper name. Genus Echthromyrmex represented by single species in this study Echthromyrmex orientalis. The species first time reported from Pakistan. The species descriptions are similar to Ghosh (1984) and Krivokhatsky (2011). The species occurs in lush green forest areas of Kohistan area. The species was collected from bank of a water stream coming downhill from mountain. General topology was mixed kind with large stones to sand and loam soils. Vegetation included acacia trees and some alpine forest trees. The species remains active in mid-summer. The abdomen of the specimen damaged so no observation on genitalia and other structures discussed. Genus Neuroleon has four species occurring in Pakistan the species in genus have a significant size variation. Neuroleon asirensis species descriptions are similar to Holzel (1983) the species is first time reported from Pakistan previously species was reported form Saudi Arabia. This species only exists in northern parts of the country. Not commonly collected. Only single specimen was collected after multiple collections from same spot. The specimen 145 collected was not pin by author himself. So there were no tibial spurs present may be due over pressing of the specimen on pining board or block. If the situation is otherwise then species identification becomes doubtful and may be species will need revision. Neuroleon egenus species descriptions are similar to the descriptions of Monserrat and Acevedo (2013). The species occurs in northern areas of Pakistan. The species remains active at night. During the day time may be hunted through grasses near water streams and sandy pathways. The species not common very few specimens collected till now. Neuroleon (Ganussa) leptaleus species descriptions are similar to the Krivokhatsky (1995). The species occurs on very high altitude and cold areas. Adults area active at night. Species occurs in abundance in its habitat. Collections were made during early hours of the night using light trap. Neuroleon (Ganussa) tenellus species descriptions are similar to Krivokhatsky (1995) the species occurs in northern areas of Pakistan. Adults are active in early hours of night. Species have abundant population in its habitat. Genus Indoleon only one species presented here. Indoleon vartianorum (Holzel) 1972. Although species morphology much similar to Gymnocnemia variegate. But species body colour difference and presence of tibial spurs in Indoleon differentiate it form Gymnocnemia variegate. Also plate like structures of paramere in male distinctive character in case of Indoleon. The species exists in the north western areas of the country. Adults remain active from early simmer to the end summer. Collection can be made by using low light source or Mercury vapour bulb with low watts. Species active during early hours of night. Species not commonly collected. Species Gepus invisus two morphs (normal and black stripped) of species were collected as first report from Pakistan. The species detail descriptions have been presented by Saji and Whittington (2008) and details about different morphs have been presented by Ábrahám and Harten (2014) according to them all morphs are same species. The results from COI gene have shown that two morphs don’t have any genomic difference. The species distributed through warmer climate and low land zone i.e. south eastern part population of the species is very high. While in high altitude and low temperature areas no collection records were made during this study. Genus Iranoleon presented by single species. Only species reported Iranoleon vartianae was previously reported by Hölzel from Quetta Pakistan new locality

146 recorded for the species have been provided. The species descriptions are similar to description provided by Hölzel (1968, 1969). Genus Myrmecaelurus four species are collected from Pakistan during this study. Myrmecaelurus laetus species descriptions are similar to Banks (1913) and Kimmins (1943) much abundant in its ecology. Genrally distributed through Thar Desert ranging from Bhawalpur to the end areas of Nagar Parker. Myrmecaelurus longiprocessus species descriptions area similar to Iqbal and Yousuf (1997). Species much abundant in its ecology. But restricted to deserted areas of south part of country. Adults are active during the night. the species have larger body and longer hair like setae on abdomen. The species is largest amongst all other species of the genus. Myrmecaelurus paghmanus species descriptions are similar to Holzel 1969. Species much similar to M. laetus but with distinctive colour difference. The species bright yellow colour and bright black colour lines on body make species very distinct. Species widely distributed in the country from north to south. Species remains sitting against the grass stems. Sometime found on open grounds and sunny places. Species also exhibits day flying that may be cause for wide distribution. Along with much voracious predatory nature. Even if wings held in hand and offered any insect it will just munch of food without caring about captivity. Species present in much abundant and commonly collected Myrmecaelurus (Nohoveus) vanharteni the species descriptions are similar to the Abraham (2012). The generic differentiation based on some reasons seems to be logical. Nohoveus is diffrent from Myrmecaelurus in terms of its wings male genetalia. Myrmecaelurus have some broader wings and also the straight paramer. While in nohoveus wings are narrow and paramer much curved. Also Nohoveus implexus from India by Ghosh (1981) and N. Implexus by Iqbal and Yousuf (1991) seem to be confused. They are same as Myrmecaelurus (Nohoveus) vanharteni so may be considered as synonyme of the species. Species Solter hardei was previously reported by Iqbal and yousuf (1997) from Pakpatan. The species descriptions were much similar to descriptions of Hölzel (1968). There were some differences observed from Iqbal and Yousuf (1997). The species not commonly collected from the area. Generally adults active at night. Solter felderi species descriptions are similar to Navas (1929) and Holzel (1969). Navas, has reported species from Kashmir route. Which may the same locality 147 as reported in this study. The species abundant near water springs with wide fields of sandy lands. The species adults remain active during early hours of night. Species distribution restricted to the northern areas of Pakistan. Four species from genus cueta were collected and reported in this study. Cueta lineosa (Rambur, 1842) was previously reported by Iqbal and Yousuf (1997) from Muzafar Garh. In recent study many new locality records have been added. The species description are similar to Krivokhatsky (1998), Gosh (1981), Mirmoayedi (2006) and iqbal and Yousuf (1997) while the specimen presented by Devatak (2013) have little lighter colour. The species seems to be very abundant. The species is commonly collected in its habitat. Adults are active at night till late hours. Cueta minervae the species descriptions are similar to Holzel (1972). The species have relativively broader wings. Also larger body size. The species not much spread. Actually found in south east part of the Pakistan. Species not commonly collected. Cueta plexiformia first reported another larger species. Not commonly collected occurs in northern areas of Pakistan. Much satisfy the description of Krivokhatsky (1996). This species is morphologically similar to C. minervae with just more brighter yellow colour and little narrow wings. But when DNA was compared for two idividial. It was observed that both are different species. There are many chances that both have a common ancestor and recent split. So not much morphological separation have occured. While other possibility may be that one species may be the cryptic species of other. In this situation new name should be assigned to one while other can stay same. Cueta striata Kimmins (1943) Cueta striata being reported first time from Balochistan Pakistan. The species have a very little difference from Cueta lineosa. Cueta striata usually have veins much tinted with brown color as compared to Cueta lineosa along with some colour of body. The species was descriptions were similar to Kimins (1943), Hölzel (1969) and Saji and Whittington, (2008). Cueta thaliae Hölzel, 1972 species has been collected first time from Pakistan. previously it was only reported from India. The species descriptions are similar to Holzel (1972) with minor colour variation. The species is well distributed through southern parts of Punjab province and most of the Sindh province. Species commonly collected species in its region. Species Epacanthaclisis alaica new report from Pakistan. Though species descriptions are much similar to Epacanthaclisis continentalis but since the tibial spurs 148 as long as the basal segment. Hence species becomes Epacanthaclisis alaicus not Epacanthaclisis continentalis. The species descriptions are much similar to description made by the Krivokhatsky (1998b). The species inhabits in low temperature and high altitude area i.e. northern areas of Pakistan. Rarely collected species. Collection was made by using light trap. Species adults are active in early hours of night 20:00hours to 22:00hours. Genus Pseudoformicaleo represented by only onse species in this study. Pseudoformicaleo gracilis first reported from Pakistan in this study. Species descriptions are similar to Khabiev and Krivokhatsky (2014). The species is not very common only three specimens collected after many expeditions. Species occurs in dry sandy desert with thorny scrub vegetation with acacia as dominating species. All specimens collected from man made forest reserve i.e Lal Sohanra National Park. Genus Delfimeus only species recorded Delfimeus intricatus was previously reported by Hölzel (1968). He described species under genus Pignatellus genus was renamed as Delfimeus. The species much smaller in size. Also inhibits in sloppy areas. Generally lives in an area with loose soils made by land slides and fast flowing stream banks. Adults are active at night during early hours much abundant in its habitat. Genus Distoleon four species have been recorded from Pakistan. Distoleon cyrus species descriptions are similar to Holzel (1972) Although species type specimen was not available. But the distinctive marking defined by Holzel (1972) is unmistakable. While there is a little difference in case of mark on frons. The abdomen colour is also same as described by Holzel (1972). The species have a little size variation based on collection sites. The areas with regular vegetation have larger size specimens while traditionally cultivated zone specimens have smaller size. Distoleon nefandus species descriptions are similar to Ghsoh (1984). The characters like brown lines on either side of pronotum are unmistakably same as described by Ghosh (1984). Although Ghosh have mentioned abut wings over all but did not mentioned about the brown mark presence. Distoleon obducens species descriptions are similar to walker (1853). The species have the spots on front and hind wings which are exactly described by Walker (1853). While the pronotum lines are also same as described by Walker hence species is considered to be same as described. Distoleon tesselatus Yang 1986 the species descriptions are similar to the Yang (1986) the species has a very unique abdominal pattern. The pattern geometrical shape 149 is unique with a pattern made by four different blocks arranging one on bottom two on sides and one on top leaving center hallow. The blocks are bit yellowish brown while center remain brown. Genus Ganguilus presented with single species Ganguilus pulchellus was previously reported by Michel and Mansell (2010) from Mauripur some where near Krachi. In fact it’s Mirpur Khas District. The real ecology of the species occurs even further east area known as Thar Desert. Specimen presented in current study was collected from The Thar Desert. Recent new locality records have been added. The species descriptions satisfy descriptions by Michel and Mansell (2010). The species adults are collected during late summer. Not very common but fairly abundant population of species exists in its habitat. Genus Myrmeleon have shown a broad distribution and a good species richness 6 species have been collected and identified. Myrmeleon assamensis species descriptions are similar to the Gosh (1984) and Iqbal and Yousuf (1992). Species occur in the northern Punjab usually highland areas. The species population well established in its habitat. Myrmeleon bore first time reported from Pakistan. species descriptions are similar to Tiller (2013) with a very little coulour differences. The species have a very much existence in many parts of Europe and other countries. But there are no record of species in Pakistan. Although species have been reported from Iran. This study represents its distribution to further south than previous described distribution. Myrmeleon hyalinus first time reported from Pakistan species are similar to the Holzel (1986), Krivokhatski (2011) and Devetak et. al. (2013). The species is the most abundant species in the genus. It is widely distributed from Punjab to Sindh province. The species can be found active during dusk. Though five (Hölzel (1986) and Abraham 2010) subspecies have been described for the species but in this study no subspecies were observed. The species is much closer to Myrmeleon valentine but presence of pilula axilaris differentiates the species as distinct taxon. Myrmeleon inconspicuus new record from Pakistan the species descriptions are similar to Tillier et. al. (2013) and Pantaleoni et. al. (2010). Species larvae have been collected from fine sand and loose clay soils. Specie have a fair population in the south parts of the country major part in the Punjab province. Myrmeleon noacki species first time reported from Pakistan the species morphology similar to descriptions provided by Ohm (1965) the illustration provided 150 by Ohm clearly match with the species observed in current study. The pronotum and genitalia morphology is typical and same in both cases. The species have been reported in western regions i.e. Bulgaria, Greece, Macedonia, Turkey and Yugoslavia. However current evidence proves that species is not limited to the west. Its widely distributed to the eastern part of the world very well. Species have been collected on eastern part of Kashmir so may be its present in India too. Myrmeleon trivialis species first time reported from Pakistan. The descriptions are similar to Zhan et. al. (2011). The male genitalia are typical of species. The species was mistaken as new species by Yang (1987) as Myrmeleon zanganus which was later corrected by Zhan et. al. (2011). The species occurs on high altitude areas specially in green mountainous ecology. Species is not commonly collected. Occurs in northern areas of Pakistan. Collection has been made from Murree hills and Kashmir Hills. Species was not collected in valleys. Only high altitude collection records are observed. Adults are attracted to light but net sweeping or vegetation disturbance can initiate day flying in species. The Barcode Index Number (BIN) using BOLD Systems Version 3.0 Assigned 41 BINs to total 359 Antlion DNA sequences recorded for DNA barcode region. To find maximum distance distribution in species Barcode Gape Analysis and Automated Barcode Gap Analysis was performed using BOLD and online resource respectively. Results showed that maximum intera specific distance was 0 to 2.69%. While comparison with other countries was not possible as there was no data available for same species from other countries. This may be due to non-processing of specimens from other countries or species endemism. The analysis showed that no species have been already sequenced and added to BOLD. So it has been added first time at least for local fauna. Value of maximum interspecific distance was less than nearest neighbor distance (Ratnasingham & Hebert 2013). DNA based or molecular techniques have been used for species identification. DNA based identification can provide reliable results. However it may not be considered exceptional from ambiguity in some circumstances. If previous record does not exist in the database then result may be misleading (Virgilio et al., 2010). But such situation does not overcome perspective of a reference library. The controversies and confusions may be eliminated by comparison of both species comparison. Hence DNA based library can help taxonomic works globally. In case of Pakistan antlions fauna about 90% species from our species collection were barcoded and added to reference 151 library i.e. BOLD. DNA bar-coding have been used for several Insect groups for species discrimination and reference library establishment (Ashfaq et al., 2014a, Burns et al., 2007; Deng et al., 2012; Hebert et al., 2004; Park et al., 2011). Species tree/Cladograms were generated but using BOLD server with a presets species discrimination value at 2% difference. It was observed that all 42 species have been discriminated from each other by used marker gene at 2% difference. The neighbor joining cluster revelaed that there was a highest similarity index btween individuals of same species in most cases. The root support i.e. boots rap vaule was 100 in most cases in such situation. When nearest neighbor joining technique was applied fro the clustering of sub family palprinae members by keep Ascalaphidae member as out group. It was observed that all of species gathered on common clade as compared to ascalaphid in separate clade. It was observed that species with common morphological character or morphological homologies were gathered together e.g Palpares rajasthanicus, Palpares trichogaster, Parapalpares solidus and Palpares astarte were more closer and have strong boots rap value as compared to other individuals i.e. Palparellus astutus and Indopalpares pardus. While ascalphidae individual was pushed to edge as its member from paraphyletic group. Hence it infers that COI gene can not only decriminate species but can also perforeme the phylogenetic analysis to somewhat extent in a better way. Also similar trend was observed when species from other genra were analyzed by using COI gene DNA barcode region. The similar results have been reported by Asfaq et. al. (2013) in and Ashfaq et. al. (2014a) in case of mosquito. Similarly when Acanthaclini member were tested. There was a descrimated gathering of members of samer genra in same clade. Acanthaclisis neozelandica and Acanthaclisis obscura grouped together while Centroclisis thari sp-SA01 pused away while Ascalaphide was further distant. Which is clearly evident from there morphological homologies difference too. Also the root support boots rap value was maximum possible i.e. 100 also within species value was also 100. The similar trends were observed in almost all further analysis made. There was also an interesting case recorded in case of Cueta minervae and Cueta plexiformia. When we look at morphological basis then both species preseted in ths study seem to be the same. But when we look at DNA makeup of both species then it seems to be different. Both species have more that 2% genetic difference. That may lead to cryptic 152 species occurance with in one species. Also there is verymuch possible that two species are monophlyletic in recent past and morphological distinction have not established yet. Also there is pretty much chance that the genus cueta have its own speciallity of confusing species. For example if you look at Cueta striata and cueTa lineaosa . They are almost same but just one veinn colour variation in wings have lead to establishment of two different species. So the reason may be either one. Presence of cryptic species or may be over estimation of species by DNA barcode (which has very sleek chances).

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SUMMARY The present study was conducted during 2008-2014. This study initiate molecular taxonomy and reference library build up for future studies of antlion. The results revealed that Pakistan has a rich antlion fauna. Still lot of it needs to be explored. The current study have increased the species count from 28 species to 44 species belonging to 2 subfamilies 8 tribes and 20 genera. Current study has added 17 species to the local fauna out of which 2 are expected new species. Current study have not only revised the number of species but also indicated that there are so many genera which inhibit this country which have been neglected or missed somehow. Current study also synonymized some species like Palpers cepholate as Palpres trichogaster. Also two new species Stenares clarus and Centroclisis thari. Current study also report either Tomatares genus does not exists in this region or may have been raised due to mistaken identification of any other species. This study has established a reference library with specific Barcode Index Number (BIN) for each of 39 species consisting 703 COI DNA barcode sequences. While for one case Palpares astarte sequence length was less about 447bp so no Barcode index number was assigned to it. The integrity of sequences was tested using BLAST. Sequence having similarity to close relatives in Neuroptra were considered right and rest were discarded. The data regarding specimen’s collection, morphology (pictures) and DNA sequences were added to BOLD. Species discrimination was tested using online tool from BOLD and using MEGA6 (free tool). Specimens indicating more than 2% difference were considered different species. The species differentiation results from molecular data were similar to morphological data results. The analysis revealed that antlion species area not completely monophyletic. Still lot of data needs to be added for resolving antlions phylogeny. The sequence from DNA barcode region was very useful for species discrimination at 2% difference. But there was confusion in phylogenetic tree construction. When large number of taxa were used. Results showed that DNA barcode region can lead to some transgenic taxon arrangement as nearby neighbor. Hence it can be inferred that antlions morphological taxonomic hierarchy needs improvement. Also DNA barcode region alone may not be a good option to be used for phylogenetic analysis.

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RECOMMENDATION FOR FUTURE RESEARCH The identification performed on basis of most authentic taxonomic keys available today are not sufficient. Hence available literature needs improvement. Most of genera need revisions. Because there are so many complexities in some genera. There is an over estimation of number of species internationally in some cases. It may be due to very less communication by previous authors during working process. Limited communication resources may have played major role in this regard. Recent 43 species record as compared to previous records i.e. 28 species shows huge potential in species exploration for Pakistan. Also some of previously reported species were not recorded during this study. While some species have been synonymized. This again highlights need for research update. Keeping in view findings of current study in future research should focus on morphological studies. The fauna of antlions in Pakistan still needs exploration. Extensive area specific explorations should be done to know complete number of species. The knowledge about taxonomic identification needs improvements. Different generic reviews should be made. Now a day’s technology advancement has benefited most fields of biology. Taxonomic studies are also using modern techniques. The most reliable and considered valid today include DNA based molecular analysis. Hence there is a need to make some advancement in local fauna DNA based profile generation. The international museums have established their digital record system and digital libraries for museum specimens. Hence it’s much necessary that such libraries must be established in Pakistan too. The research must not be stopped at basic level it must proceed to applicable level. The antlioins are very good predator both at larval and adult stage. Hence there is a need to explore predatory potential of each species to know about their role in ecology. Antlions have been reported to have a very diverse microbial fauna. The gut fauna (bacteria) have been reported to have insecticidal properties. Hence local fauna must be used for exploring insecticidal bacteria. Due to heavy use of pesticides, logging of forests and intensive cropping. A vast habitat for antlion have been lost. Hence there is need to develop strategies to conserve this novel natural resource.

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SPECIES CHECKLIST MYRMELEONTIDAE OF PAKISTAN Subfamily Palparinae Genus Indopalpares Insom and Carfi, 1988 1. Indopalpares pardus (Rambur, 1842) Genus Palparellus Navas, 1912 2. Palparellus astutus (Walker, 1853) Genus Palpares Rambur, 1842 3. Palpares astarte Banks, 1913 4. Palpares rajasthanicus Gosh, 1991 5. Palpares trichogaster Navas, 1913 Genus Parapalpares Insom and Carfi, 1988 6. Parapalpares solidus (Gerstaecker, 1894) Genus Stenares Hagen, 1866 7. Stenares clarus n.sp 8. Stenares improbus (Walker, 1853) Subfamily Myrmeleontinae Tribe Acanthaclisini Navas, 1911 Genus Acanthaclisis Rambur, 1842 9. Acanthaclisis neozelandica (Navas, 1912) 10. Acanthaclisis obscura Hölzel, 1972 Genus Centroclisis Navas, 1909 11. Centroclisis indica (Banks, 1911) 12. Centroclisis thari sp-SA01 Tribe Dendroleontini Banks, 1912 Genus Epacanthaclisis Okamoto, 1910 13. Epacanthaclisis alaica Krivokhatsky, 1998 Tribe Nemoleontini Banks, 1911 Genus Pseudoformicaleo Van der Weele, 1909 14. Pseudoformicaleo gracilis (Klug, 1834) Tribe Neuroleontini Navas, 1912 Genus Delfimeus Navas, 1912 15. Delfimeus intricatus (Hölzel, 1972) Genus Distoleon Banks, 1910 16. Distoleon cyrus Hölzel, 1972 17. Distoleon nefandus (Walker) 1853 18. Distoleon obducens Walker, 1845 19. Distoleon tesselatus Yang, 1986 Genus Ganguilus Navas, 1912 20. Ganguilus pulchellus (Banks, 1911)

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Genus Indoleon Banks, 1913 21. Indoleon vartianorum Hölzel, 1972 Genus Neuroleon Navas, 1909 22. Neuroleon (Ganussa) leptaleus (Navás, 1912) 23. Neuroleon (Ganussa) tenellus (Klug in Ehrenberg, 1834) 24. Neuroleon egenus Navás, 1914 25. Neuroleon asirensis Hölzel, 1983 Tribe Myrmecaelurini Esben-Petersen, 1918 Genus Gepus Navas, 1912 26. Gepus invisus Navas, 1912 morph typica Genus Iranoleon, Hölzel, 1968 27. Iranoleon vartianae Hölzel, 1968 Genus Myrmecaelurus Costa, 1855 28. Myrmecaelurus longiprocessus Iqbal & Yousuf, 1997 29. Myrmecaelurus laetus (Klug in Ehrenberg, 1834) 30. Myrmecaelurus paghmanus McLachlan, 1875 31. Myrmecaelurus (Nohoveus) vanharteni Ábrahám, 2012 Genus Solter Navas, 1912 32. Solter felderi Navás, 1912 33. Solter hardei Hölzel, 1968 Tribe Nesoleontini Markl, 1954 Genus Cueta Navas, 1911 34. Cueta thaliae Hölzel, 1972 35. Cueta lineosa (Rambur, 1842) 36. Cueta striata Kimmins,(1943) 37. Cueta minervae Hölzel, 1972 38. Cueta plexiformia Krivokhatsky, 1996 Tribe Myrmeleontini Latreille, 1802 Genus Myrmeleon Linnaeus ,1767 39. Myrmeleon assamensis Ghosh, 1984 40. Myrmeleon bore Tjeder, 1941 41. Myrmeleon hyalinus Olivier, 1811 42. Myrmeleon inconspicuus Rambur, 1842 43. Myrmeleon noacki Ohm, 1965 44. Myrmeleon trivialis Gerstaecker, 1885

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