Trophic Associations of Ants with Aphid Partners and New Distribution Records of some Ants in Pothwar Region of Pakistan

A nts are the economically important and metropolitan insects on earth. They act as soil engineers, scavengers, decomposers, predators, pollinators and herbivores. The mutualistic relationship between ants and aphids is a very common and widespread phenomenon. It has been studied in various parts of the world. In this relationship, aphids provide food to the ants, while in return ants provide them protection from natural enemies. Ants also protect aphids from different diseases. Aphids (serious crop pests) can be divided into myrmecophilous and non-myrmecophilous. The main objective of this study was to determine trophic associations of ants associated with aphids on various host plants in Pothwar. For this purpose, seven ant species were selected, identified as Camponotus compressus (Fabricius, 1787), Formica


INTRODUCTION
A nts (Hymenoptera: Formicidae) are the economically important and metropolitan insects on earth. Formicidae is divided into 20 subfamilies (Bolton, 1994;Bolton et al., 2006;Ward, 2007). Ants are good in seed dispersal (Hanzawa et al., 1988). They improve soil aeration (Holldobler and Wilson, 1990). They are known as 1991). Ants also protect aphids from different diseases e.g. Formica podzolica protect aphids, Aphis asclepiadis, from lethal fungal infections (Nielsen et al., 2010). Flatt and Weisser (2000) reported that aphids associated with ants lived longer, had higher rate of reproduction and greater number of progeny (Yoo and Holway, 2011).
Among mutualisms, ant-aphid interactions are among the most variable in terms of their outcomes whether or not the ant aphid interaction is beneficial to the aphids often depends upon both the spatial and ecological context. Aphids, the serious insect pests of various crops, fruits, vegetables etc. are myrmecophilous (tended by ants) and non-myrmecophilous (unattended). The great majority of ants taking part in such associations belong to phylogenetically advanced sub-families Dolichoderinae, Formicinae and Myrmicinae (Delabie, 2001). The mutualistic relationship between ants and aphids has been the subject of many studies on various aspects of this phenomenon in various regions of the world. In different regions like Iran, 21 ant species associated with 26 aphid species from 37 host plant species (Mortazavi et al., 2015), 18 ants species associated with 34 species of aphids from Florida (Nielsson et al., 1971), 10 species of ants associated with 24 aphid species from Russia (Addicott, 1979), 23 species of ants associated with 11 aphid species from Oceania (Idechiil et al., 2007), 16 species of ants in association with 19 species of aphids from Ankara Province of Turkey (Özdemir et al., 2008), 17 species of ants associated with aphids in Indonesia (Herwina et al., 2013), 20 species of ants associated with 3 species of aphids in India (Kataria and Kumar, 2013) etc. But as far as Pakistan is concerned, recently a lot of work on both aphids (Bodlah et al., 2013Amin et al., 2017a, b;Maryam et al., 2019) and ants (Bodlah et al., 2016 has been done but on mutualistic associations are still missing. Recently  reported first association of a psyllid, Trioza fletcheri minor crawford, 1912 with two ants species in Pothwar, there is no report on aphids and their mutualistic ants in Pakistan. To fulfill this gap and provide a base for further studies on interactions between aphids and ants, the present study was conducted.

Ant-aphid collection and preservation
Surveys of Attock, Chakwal, Rawalpindi, Islamabad and Jehlum were conducted for the collection of ants and aphids from different host plants during 2015-17. Ants were collected from aphid infested crop plants, grasses, trees, weeds, ornamentals, fruits, vegetables etc. Only those ant-aphid pairs were collected where we observed ants stroke the aphids with their antennae, stimulating them to release the honeydew. Ants were collected with aspirator and through hand picking, were killed and preserved in 75 % ethanol in small glass vials. Aphids were collected and preserved in 75 % ethanol in small glass vials. Longitude and latitudes of the surveyed areas were determined with the help of GPS device.

Identification of ant-aphid partners
Ants were mounted on triangular cards and pinned. Large ants were preserved by using entomological pins. These specimens were identified by using compound microscope and identification keys by Bingham (1903). Aphids were identified using keys by Eastop (1994, 2000). Host plants inhabiting ants and aphids were also collected and identified to develop the mutualistic association among ants, aphids and host plants of aphids. The voucher specimens have been deposited in the Museum collection of Department of Entomology, PMAS-AAUR.

RESULTS AND DISCUSION
Seven different species of ants belonging to seven different genera were associated with different aphid partners on various host plants in different areas of Pothwar region. All associations are new. New distribution records for ants have also been added.

Comments on ant-aphid associations
This ant has been reported in association with 5 aphid species namely Aphis craccivora (Kataria and Kumar, 2013;Rakhshan and Ahmad, 2015), Aphis fabae (Kataria and Kumar, 2013), Aphis gossypii (Verghese and Tandon, 1987;Kataria and Kumar, 2013;Lokeshwari et al., 2015), Aphis nerii (Kataria and Kumar, 2013) and Myzus persicae (Kataria and Kumar, 2013)  Rosa indica (Rose) from areas of district Jhelum. It was found on the tree trunks and barks tending aphids for honey dew secretion. All the collected specimens were identified and found similar to published description of Bingham (1903). Camponotus compressus is reported for the first time in association with any aphid species from Pakistan, so it is reported as new country record.

Comments on distribution of camponotus compressus in Pakistan
Umair et al. (2012) mentioned the distribution of this species only from Islamabad and Rawalpindi. We have added new distribution records in various localities of Pothwar.

Formica fusca Linnaeus, 1758 Aphid partners in association reported during these studies in Pakistan
Pine aphid (Cinara orientalis Takahashi

Comments on ant-aphid associations
This species has been recorded to be associated with 57 aphid species from various parts of the world as reviewed by Siddiqui et al. (2019). During our studies, 1 new trophic association of this species with aphid, Cinara orientalis was observed. This association is new country record from Pakistan. This ant species was observed and found in abundance on the pine tree Pinus wallichiana (Himalayan pine) in association with Cinara orientalis. Both the ants and aphids were present in the lenticels of the tree. Aphids were sucking sap from tree trunk and ants were getting honey dew from aphids. All the collected specimens were identified and found similar to the published description of species by Bingham (1903). Menozzi (1939) reported this species from Karakorum (Province Gilgit-Baltistan). During our surveys, new distribution pattern in Murree (Province Punjab) has been added.  Siddiqui et al. (2019) mentioned this ant species to be in association with 8 aphid species from the world. During these studies, new associations with 2 aphid species have been added.

Comments on ant-aphid associations
Formica clara was found in association with two aphid species namely, Cinara confinis and Cinara orientalis on Abies pindrow (Pindrow fir) and Pinus wallichiana (Himalayan pine) trees. Both aphids and ants were present in the lenticels on the tree trunks. All the collected specimens were identified and found similar to the published species description of Bingham (1903). Formica clara is reported for the first time in association with any aphid species from Pakistan.

Comments on distribution of Formica clara
Seifert and Schultz (2009) mentioned only its distribution in Pakistan but not the exact localities were reported. Here we have added exact localities of its distribution in Pothwar region of Pakistan.

Comments on ant-aphid associations
According to Shiran et al. (2013), this species has trophic association with 11 different aphid partners. Siddiqui et al. (2019) mentioned this ant species to be in association with 2 aphids.
In our studies, 9 new ant aphids' trophic associations have been added. This ant was most abundantly found in association with aphids on a large number of host plant species. It was found associated with 11 aphid species on different plant species including Mentha longifolia (Mint), Chrysanthemum indicum (Chrysanthemum flower), Lagerstromia indica (Crepe myrtle), Malus pumila (Apple), Eriobotrya japonica (Loquat), Psidium guajava (Guava), Eugenia jambolana (Jaman), Ficus sp. (Fig) and Salix sp. (Willow) from different areas of district Rawalpindi, Jhelum, Chakwal, Attock, Murree and Islamabad. It was the most active species found in association with aphids. All the collected specimens were identified and found similar to the published description of species by Bingham (1903). Lepisiota frauenfeldi is reported for the first time in association with any aphid species from Pakistan, so it is added as new country record. Umair et al. (2012) recorded this species only from Rawalpindi and Islamabad. New locality records have been added during these studies. Menozzi, 1939 Aphid partners in association reported during these studies in Pakistan

Comments on ant-aphid associations
This species has not been recorded from the world in association with aphids. During our studies 4 novel association of this species were observed for the first time in different localities of Pakistan.
Myrmica aimonissabaudiae was found in association with Aphis fabae, Chaetosiphon (Pentatrichopus) glabrum, Cinara confinis and Cinara orientalis on a number of host plant species including Pinus wallichiana (Himalayan Pine), Abies pindrow (Pindrow Fir), Rosa brunonii (Himalayan Musk Rose) and Rumex dentatus (Jangli Palak) from different areas of Chara Pani, Kuldana, Aliote, Hill Dhuloo and Osia from district Rawalpindi. All the collected specimens were identified and found similar to the published description of Bingham (1903). Myrmica aimonissabaudiae is reported for the first time in association with any aphid species from Pakistan as well as from the whole world, so it is new country as well as new to science association. Menozzi (1939) reported this species only from 2 localities, Karakorum (Gilgit Baltistan Province), Gund (Sindh Province) of Pakistan. Here we reported it for the first time from Pothwar region of Pakistan.

Comments on ant-aphid associations
This ant species has been recorded in association with 4 aphid species namely, Aphis gossypii, Hysteroneura setariae, Myzus persicae and Pentalonia nigronervosa as reviewed by Siddiqui et al. (2019). Here we added 11 new trophic associations of this ant with different aphids on different host plants. Tapinoma Bingham (1903). Tapinoma melanocephalum is reported for the first time in association with any aphid species from Pakistan, so it is added to the country's fauna as new record.

Comments on distribution of Tapinoma melanocephalum in Pakistan
Wetterer (2009)

Comments on ant-aphid associations
This species has been reported in new association with aphid for the first time in the world. Previously it was not reported with any association with aphids. Tetraponera allaborans was found associated with Willow aphid (Chaitophorus sp.) on Salix sp. (Willow plant) from Neela Sand area of district Rawalpindi. This ant along with aphid was present on the branches of willow plant. Ants were collecting honey dew from aphids. All the collected specimens were identified and found similar to the description of Bingham (1903). Tetraponera allaborans is reported for the first time in association with any aphid species from Pakistan, so it is added as new country record.

DISCUSSION
Mutualistic association of ants and aphids has been a topic of interest by various ecologists and taxonomists of the world in the history and recent years. Mutualism is a fundamental element in aphid-ant communities, found within various aphid-ant groups and formulates the species richness, abundance and distribution (Styrsky and Eubanks, 2007). Understanding of the aphid-ant associations generally provides patterns of aphid-ant species complexes in different ecological zones. There is a need of more studies related with taxonomy and ecology for better understanding of aphid-ant associations .
Camponotus compressus with 12, Formica fusca with 1, Formica clara with 2, Lepisiota frauenfeldi with 9, Myrmica aimonissabaudiae with 4, Tapinoma melanocephalum with 11 and Tetraponera allaborans with 1 new associations on various host plants have been reported for the first time from Pakistan. Prior to our studies, only faunal work on aphids and ants separately has been done. These studies will provide baseline for further studies on patterns of antaphid mutualistic associations in various ecological zones of Pakistan leading towards their interactions with other communities of ecosystem. New distribution of studied ant species have been added in Punjab province, previously recorded from other provinces.

ACKNOWLEDGMENTS
We are grateful to Pakistan Science foundation for funding under the project PSF/NSLP/P-UAAR (313) for these findings.

Statement to conflict of interest
The authors declare there is no conflict of interest.