Flies in the high for floral hike? Altitudinal variation in species diversity and composition of Diptera (Insecta) in the eastern Himalaya, India

: Species diversity and composition enable us to understand the conservation and management of an ecosystem. There is scarcity of knowledge in understanding the diversity change across the gradients of elevation, especially in the Himalaya. Here, we focused in the eastern Himalaya to investigate the patterns of taxonomic and functional diversity of true flies with relation to variation in altitude. The study was conducted in protected area (Neora Valley National Park) in the eastern Himalaya, India and the survey was conducted at five altitudinal zones (from 500 to 3,000 m). A total of 201 species of Diptera, with 105 genera and 33 families were recorded, of which 25 species are new to the state of West Bengal and seven species are new to India. The species diversity increased with elevation (maximum was near 2,500 m) and most of the flies preferred to be close to bushes with flowers, with a substantial percentage of them being pollinator species. Flies adapt to the various vegetation and climate patterns, which was evident by the abundance of fly species at high altitudes (1,500–2,500 m). Hence, it is very important to implement appropriate actions to protect the diversity of true flies in this Himalayan landscape.


INTRODUCTION
Patterns of species composition and diversity, along with environmental and elevational gradients, provide insights into our understanding of ecosystem conservation.Research trends have shifted toward a greater understanding of the elevation gradient and its impact on species diversity across various geographic regions (Terborgh 1977;Brown 2001;Sanders & Rahbek 2012;Acharya & Vijayan 2015;Marathe et al. 2021).Furthermore, changes in landscape physiology and climatic conditions due to the different gradients of elevation effects the species diversity (Sundqvist et al. 2013).Many studies have documented and described the mechanisms on patterns of diversity with respect to elevational gradient (Acharya et al. 2011a,b;Kraft et al. 2011;Sundqvist et al. 2013;Chun & Lee 2018).
In harsher environments at higher elevations, niche differences and relative fitness differences may drive the presence of fewer species (HilleRisLambers et al. 2012;Kraft et al. 2015).Understanding such patterns and their underlying mechanisms is important for understanding the implications of insect conservation, particularly in the Himalayan regions that are vulnerable to climate change.The Himalaya is unique for examining such gradients and their impact on a variety of habitats with steep altitudinal gradient and unstable climate.
Biogeographical studies of multiple taxa have increased in recent years in various parts of the Himalayas.Most of the studies are focused on birds, plants, and pollinating insects such as butterflies.In the eastern Himalaya, bird species richness is greatest at intermediate elevations (Acharya et al. 2011b), whereas low elevations (<2,000 m) are important for butterfly conservation (Acharya & Vijayan 2015).When it comes to plants, elevation and high temperature have a considerable influence on the distribution and growth of trees (Acharya et al. 2011a).The reduction in tree height and richness noticed beyond 2,300 m, allows herbs to dominate due to climatic constraints (Sharma et al. 2019).In this context, a comprehensive study of true flies (Diptera) is also useful for identifying habitats with conservation value in the Himalayan mountain landscape.
Studies also indicate that species diversity and richness of Diptera change with elevation for example, species composition changes along the altitudinal gradient (700-2,500 m) and partitioning between seasonally dry lowland and moist montane evergreen forests on the Doi Inthanon mountain in northern Thailand (Plant et al. 2012;Chatelain et al. 2018), species richness and distribution of Hemerodromiinae and Clinocerinae are changing with the elevational gradient on the Pieniny Mountains in central Europe (Słowińska & Jaskuła 2021).Therefore, it is important to investigate their community composition across different environmental and elevational gradients in the Himalayas.The objective of this study was to investigate the variation of species composition and distribution of Diptera fauna in the eastern Himalaya between 500 m and 3,000 m elevation gradient.

Study area
The study was conducted in the Neora Valley National Park which covers an area of 159.78 km 2 .The park has diverse ecosystems with a wide range of elevation gradients (183-3,200 m), located near the ecological trijunction of West Bengal, Sikkim (India) and Bhutan on the northeast (26.8675-27.1263 0N; 88.750-88.83330 E).It is considered as crowning glory of the state of West Bengal (Mallick 2010).The study area is an east Himalayan moist mixed deciduous forest (Champion & Seth 1968), with lower areas (up to 1,800 m) recognized as subtropical mixed broadleaf forest, lower temperate evergreen forest, and upper areas (1,800-3,200 m) recognized as upper temperate mixed broadleaf forest and Rhododendron forest (Mallick 2012).The study area was divided into five categories based on the vegetation composition-Lower Hill Forest (<762 m), Middle Hill Forest (762-1,676 m), Broad-leaved Forest (1,676-2,133 m), Oak Forest (2,133-2,500 m) and Rhododendron Forest (>2,500 m) (Figure 1).

Field methods
The survey was conducted at 14 different locations (forest camps) between March 2018 and September 2021 as part of the Biodiversity Assessment Programme (organized by the Department of Forest, Government of West Bengal), using pre-set representative trail transects in representative elevations (Table 1).In each camp sites, four to five surrounding areas were surveyed from 0800 to 1500 h (7 hours).The flies in the different habitats were observed and collected by the first author, which were then classified (Table 2).During the field survey, insect collecting hand nets and one malaise trap were used to collect true flies.Average hand net collection time was 3-4 hours and malaise trap was used accordingly to the suitability of the terrains.Insect hand net specimens were paralyzed by benzene vapour in a killing jar and stored in an envelope for future use.Specimens were also pinned (No. 2) in the field and stored in an insect box.Specimens collected by malaise trap were sorted by sub-family and stored in 70% alcohol.

Identification of species
In the laboratory, collected insects were placed in a wet chamber overnight before being pinned by inserting an insect-pin slightly laterally through the pro-thoracic segment.Pinned specimens were labeled with the location of collection, date, altitude, and substances on which the flies were found.The flies were taxonomically identified using chaetotaxy key (Senior-White et al. 1940;Emden 1965;Shinonaga & Kano 1971;Crosskey 1976;Nandi 2002;Scudder & Cannings 2006;Buck et al. 2009;Joseph & Parui 2012) under a stereoscopic binocular microscope, and genitalia of male individuals were dissected in some cases for confirmation of identification.The specimens of Culicoides were separated and stored in different microcentrifuge tubes (1.5 ml) containing 70% ethyl alcohol.After mounting the adults on a slide using the phenol-balsam technique mentioned by Wirth & Marston (1968), the midges were identified using the identification keys used by Wirth & Hubert (1989) under a compound microscope.Following the identification keys used by Borror & Delong (1970), specimens were identified up to the suborder level, Nematoceran flies were identified up to the family level, and rest of the flies were identified up to the order level.

Analysis
A map of the study area indicating all sampling sites was prepared using QGIS software (version 3.16.11).The normalized difference vegetation index (NDVI) was calculated using a December 2019 (Landsat 8) satellite image.This month was chosen for its peak forest vegetation as it is just post-monsoon and to minimize the effect of atmospheric load on remote sensing data due to lower moisture content in the air.The remote sensing data (Landsat 8 image) was obtained from USGS Earth Explorer (https://earthexplorer.usgs.gov/).In ENVI software, the captured image was radiometrically corrected and normalized.The NDVI was employed to determine vegetation on the ground.It is used to

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monitor and detect changes in vegetation and land cover.The image was classified based on the NDVI value.
The indices like α-diversity index, Simpson's index and Shannon-Weiner Index were measured to understand the species richness and species evenness of flies in the study area (Krebs 1999).The correlation between the diversity indices like Shannon-Weiner Index and Simpson's Index with the elevation of all sites were done.Pearson's correlation coefficients were estimated between altitude for all the study sites and the occurrence of fly species (Bhardwaj et al. 2012).IBM SPSS Statistics 20, PAST Version 4 software and Microsoft Excel were used for analyzing the data and preparing different diagrams.A QQ-plot was done to understand the distribution pattern of all species in 14 sites.This has been done using elevation and Simpson's Index.

RESULTS
Collection and identification of 201 species belonging to 105 genera and 33 families were enumerated (Table 3).Members of the Muscidae dominated the area with 66 species followed by Syrphidae (33), Calliphoridae (17), and Tachinidae (12).A total of 25 species are reported as new records to West Bengal and seven being new to India (Table 3).Within the newly reported species in West Bengal, 13 belonged to Muscidae.
When the total number of native species (201 species) was taken into account, the accumulation curve tended to stabilize after 12 sampling efforts (Figure 2).Spatial patterns of species distribution over various habitats were observed (Figure 3).It was found that the  In general, comparison of distribution of species and families across all 14 sites revealed that Chaudaferi (S13) and Alubari (S14) were high in diversity with respect to families, in the higher elevation (Figure 4).On the other hand, Mouchuki has the highest number of families, having moderate number of species.
A graphical representation is made with respect to centroid position of both the variables (indices and elevation) in Figure 5.The centroid is the intersection point of means of both Simpson's index and elevation.It is the same in case of Shannon-Weiner index and elevation.It shows that, the Simpson's indices of most of the sites are near the centroid, indicating it is in a normal distribution.Here, maximum number of flies are found within the range of 1,500-2,500 m.Likewise, the Shannon-Weiner indices of most of the sites are very near to the centroid and similarly, the maximum number of flies are found within the range of 1,500-2,500 m.So, Pearson's correlation test (Figure 6) between Simpson's Index and elevation was performed which reveals

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that, there is a negative correlation between them (r = -0.108).On the other hand, a correlation test between Shannon-Weiner Index and elevation reveals that there is a positive correlation but very less association between them (r = 0.092).Another correlation was done among the 14 sampling sites to find out what kind of association prevails on basis of abundance of flies.A QQ-plot showed that the observed values (estimated quantiles) were normalized (Figure 7).A rarefaction curve was generated on the basis of all 14 sites, which showed the abundance and species richness at high altitude sampling sites (Figure 8).Diptera are the primary potential pollinators at high altitudes and latitudes where bees are scarce.In the eastern Himalaya, the diversity of Syrphidae reflected the supremacy of these flies over other pollinator insects such as honeybees at the higher altitudes (Sinha et al. 2022).Studies found a similar pattern in the tropical region, such as Doi Inthanon mountain in northern Thailand (Plant et al. 2012;Chatelain et al. 2018).Even as we move farther north, the proportion of dipteran species in the total pollinator fauna grows with latitude, and they are the most common families of flower-visiting insects in the arctic (Elberling & Olesen 1999;Tiusanen et al. 2016;Lefebvre et al. 2018).In the light of this, we assessed the species richness and distribution pattern of Diptera at various elevations in the eastern Himalaya, with the highest number of flies found between 1,500 and 2,500 m.This is most likely because there are large amount flowering plants.On the contrary, in the lower elevation (1,500 m), there is dense forest with fewer fly species.Less fly species live in higher elevation areas comprising Maling bamboo forests.Furthermore, it becomes windy higher up (>2,500 m), and that area is covered with Rhododendron and wild rose plants, which reduces fly activity.Muscidae (32.83%) and Syrphidae (16.41%) were the most abundant families at all of our sample sites.This could be due to their ability to survive in environments ranging from extremely low to extremely high elevation.Members of these families can also be found at all of the sites in a habitat that is relatively bushy and densely populated with flowering plants.Muscidae is the most common family of flower-visiting insects in the Arctic region, and they are much more abundant and widespread than the insects of other dipteran families which like to visit flowers (Elberling & Olesen 1999).
Pollinator communities are changing dramatically as a result of climate change (González-Varo et al. 2013;Rafferty 2017).However, there are large gaps in our understanding of the role of Diptera in pollination networks in the Himalaya in relation to climate change.Although our findings suggest that more sampling is required to obtain a complete picture of the study area, plant-Diptera interactions also need to be examined.

Altitudinal variation species diversity and composition of Diptera in the eastern Himalaya
Sinha et al.

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Figure 1 .
Figure 1.Sampling sites in the Neora Valley National Park, West Bengal, India: a-area with elevational gradient categorized as different forest types | b-area with normalized difference vegetation index (NDVI).

Figure 2 .
Figure 2. Temporal variation in the frequency of Diptera species in all sites: Series 1-Number of new species found in each survey | Series 2-Total number of species.

Figure 3 .
Figure 3. Distribution of species of flies in different habitats of all sampling sites: S-Shade area | OS-Open spaces/ Rock surface | NS-Near stream | MS-Moist surface | HS-Human settlements | F-Flowering plants | B-Bushes | AD-Animal, human dung, decaying fruits.

Figure 4 .
Figure 4. Distribution of the species and families across all 14 sites.

Figure 5 .
Figure 5. Scatter-plots showing the Simpson's Index and Shannon-Weiner Index of all flies on basis of elevation where most of the flies were observed in elevation ranging from 1,500-2,500 m.

Figure 6 .
Figure 6.Correlation matrix displaying Pearson correlation analysis results.Data from the presence of fly species in 14 sampling sites were used in the analysis.Pearson correlation coefficient values and directions are color-coded: positive correlation-blue, light to dark | negative correlation-red, light to dark (see color-bar next to the matrix).
Taxa | www.threatenedtaxa.org| 26 November 2023 | 15(11): 24241-24254 24247 J TT DISCUSSION This is the first elaborate survey of dipteran fauna from the Neora Valley National Park along the elevation gradient.A total of 201 flies from 105 genera and 33 families were documented.Recently, Sinha et al. (2021) reported 31 species of family Muscidae from Neora Valley, including two species, Limnophora (Heliographa) ceylanica and Neomyia pacifica recorded for the first time in India.A new species, Heligmonevra paruii (family Asilidae) was described and illustrated from Neora Valley by Naskar et al. (2019), but it was not recorded in the present investigation.

Figure 7 .
Figure 7. QQ-plot showing the distribution pattern of all Diptera species across 14 sites.Figure8.Rarefaction curve (taxa vs. abundance) based on 14 sites.

Journal of Threatened Taxa | www.threatenedtaxa.org | 26 November 2023 | 15(11): 24241-24254 Altitudinal variation in species diversity and composition of Diptera in the eastern Himalaya Sinha et al. 24252 J TT
*First time recorded from the state of West Bengal (WB), or India(IND)