Houseflies speaking for the conservation of natural areas: a broad sampling of Muscidae (Diptera) on coastal plains of the Pampa biome, Southern Brazil

ABSTRACT

The Brazilian Coastal Plain of the Pampa Biome (CPPB), has suffered fragmentation caused by resource extraction and cattle raising. In turn, conservation proposals are needed to prevent the anthropisation of Pampa natural areas. The first step towards conservation proposals by using insects is fauna inventories, providing data support for legislators. Thus, we undertook a regional and broad-scale sampling survey to investigate the diversity of Muscidae flies in protected and non-protected areas of CPPB. In addition, we carried out an ecological guild diversity analysis as a metric approach of bioindication. The Muscidae sampling resulted in 6314 specimens, 98 species taxa in 31 genera. Based on diversity estimators, our sampling represents 70-86% of all muscids of CPPB. The highest diversity occurs in Pelotas streams (non-protected) and Taim Ecological Station (a huge protected area). Despite the fact these areas are more diversified and present more predatory muscid species than others, invasive species associated with livestock were observed at a higher level, providing evidence of the impact of livestock proximity to protected areas. Based on biological characters of Muscidae species and ecological guild analysis, we were able to identify: (i) high diversity of carnivorous species associated with forested and more preserved areas and (ii) a high level of a few saprophagous species as indicator of anthropisation process. In general, our results represent a significant step towards understanding Muscidae in Southern Brazil, and we demonstrate how the population ecology of muscid flies supports data to conservation proposals.

Keywords
Atlantic forest; Diptera; Ecology; Guilds; Grasslands; Muscidae

Introduction

The conversion of tropical forests, natural fields, and grasslands into agricultural and urban areas is the primary driver of the biodiversity crisis currently observed (Foley et al., 2005). Approximately 43% of the terrestrial world surface has been disturbed, and the original vegetation has been converted into anthropogenic new habitats (Barnosky et al., 2012). Tropical countries, such as Brazil, follow the same pattern showing an increasing trend as agricultural frontiers are still expanding (Ferreira et al., 2012). Extinction rates in this century are estimated to be more than double in comparison to previous ones (Pereira et al., 2010).

In the state of Rio Grande do Sul (RS), the Pampa biome is subjected to high anthropic pressure (Pillar et al., 2009), and knowledge on biodiversity is particularly neglected (Overbeck et al., 2007). Among areas of biological importance, we can highlight the Coastal Plain of the Pampa Biome (CPPB) which has a great diversity of habitats (wetlands, floodplains, riparian forests, and sands) (Overbeck et al., 2007).

Fragmentation process in the CPPB mainly occurs via the replacement of native grasslands with agriculture and exotic pastures and the destruction of wetlands by drainage or barring. Additionally, frequent fires, mischaracterisation of habitat by livestock overgrazing and trampling, hunting, and biological invasions are also significant (Bencke, 2009; Fontana et al., 2003; Machado et al., 2008; Mikich and Bérnils, 2004). In recent years, habitat loss has intensified (Bencke, 2009; Crawshaw et al., 2007), influencing some groups of birds and mammals (Bilenca et al., 2012; Filloy and Bellocq, 2006; Gonzalez and Merino, 2008; Krapovickas and Di Giacomo, 1998). However, knowledge about the impact on insect communities in Pampa biome is incipient, despite the efforts to survey the diversity of Lepidoptera (Paz et al., 2013), Coleoptera (Da Silva et al., 2013), Formicidae (Hymenoptera) (Rosado et al., 2012, 2013), and Diptera. Among studied flies diversity we can list the following families: Drosophilidae (Hochmüller et al., 2010; Poppe et al., 2012, 2013) Muscidae (Krüger et al., 2010), Sciomyzidae (Kirst et al., 2015), Syrphidae (Kirst et al., 2017) and Tabanidae (Krüger and Krolow, 2015).

The Pampa biome is a highly significant area for biodiversity conservation (MMA, 2004), but among the regions of CPPB, data on insect biodiversity are scarce, with the exception of the municipality of Pelotas and Capão do Leão, for Muscidae, Calliphoridae, and Tabanidae flies (Azevedo and Krüger, 2013; Krüger and Krolow, 2015; Krüger et al., 2010). Studies on the species richness of Sciomyzidae (Kirst et al., 2015) and the diversity of Syrphidae (Kirst et al., 2017) were the first to report data on insect fauna in most protected areas in the CPPB.

The first step towards insect conservation proposals is to increase knowledge on fauna, mainly by species inventories. Thus, this study involved a regional and broad sampling survey to determine the diversity of Muscidae on CPPB (including the transition zone to the Atlantic forest in the north of RS). Furthermore, this study aimed to establish measures of diversity (Magurran, 2004) to evaluate the success of sampling in protected and non-protected areas. A limitation of these estimates is the high dependence on sampling effort, mainly in the measures using abundance and richness (Bunge and Fitzpatrick, 1993). Therefore, for conservation proposals, determination of species richness is as essential as the number of species to be discovered (Santos et al., 2010). In this regard, nonparametric estimators of diversity were used to extrapolate species richness in the studied habitats (Gotelli and Colwell, 2001; Willie et al., 2012).

The importance of Muscidae to the CPPP habitats is that this family presents an evolutionary history in the Pampa biome, its Southern forests were an area of high diversification of this family (Löwenberg-Neto and Carvalho, 2009), and today along with the Atlantic forest, it has many areas of endemism (Carvalho et al., 2003; Nihei and Carvalho, 2005). Furthermore, the Muscidae show excellent taxa properties, which make them suitable for diversity studies in this biome. They present broad occupancy, high species richness (Carvalho et al., 2005) and wide habitat range (Ferrar, 1987; Skidmore, 1985). Due to these reasons, one can consider muscids flies assemblages comprising many species with fundamentals of bioindicator taxa. The expression ‘bioindicator' is an aggregate term referring to all sources of biotic and abiotic reactions to ecological changes. Instead of merely working as gauges of natural change, taxa are utilised to show the impacts of surrounding natural changes, or environmental changes (Parmar et al., 2016). In this sense, the following may be expected from Muscidae: the presence of synanthropic species as indicator of disturbance in native forested areas (e.g. Musca domestica Linnaeus, Hydrotaea Robineau-Desvoidy species, Stomoxys calcitrans (Linnaeus), among others) (Greenberg, 1971; Krüger et al., 2010; Skidmore, 1985; Zafalon-Silva et al., 2014). On the other hand, the presence of species that do not tolerate habitat anthropisation could be an indicator of forested "healthy" environments (e.g. Cyrtoneurina Giglio-Tos, Limnophora Robineau-Desvoidy, Neomuscina Townsend, Neodexiopsis Malloch, among others) (Krüger et al., 2010; Skidmore, 1985; Werner and Pont, 2006).

We can argue towards the ecological guilds as a bioindicator approach in Muscidae as the efficiently discrete delimitation of trophic levels are mainly based on data of larval morphology (Skidmore, 1985), and by the discussion gathered in Rodríguez-Fernández et al. (2006) and Krüger et al. (2010). This survey aims at complementing the guild descriptions by analyzing the number of species per guild in each region.

It also aims at answering the following questions: (i) which is the diversity of Muscidae assemblages in protected and non-protected areas of CPPB? (ii) Is it possible to use ecological guilds of Muscidae as a tool to check environmental aspects?

Material and methods

Study sites

The forested areas surveyed included the Grasslands of the Pampa biome, and forests in the northeastern Coastal Plain of the Pampa Biome (CPPB) in the transition zone to the Atlantic forest of the Rio Grande do Sul state (RS), Brazil. The sampling areas in the CPPB (Table 1 and Fig. 1) were chosen according to the high priority indicated for the conservation of invertebrates (MMA, 2000). The Ministry of Environment (MMA) indicates areas around the coastal lagoons of the Patos, Mirim-Mangueira complex, and the lagoons of North Coast of RS (MMA, 2000).

Table 1
Description of the 35 points sampled in five regions of the Coastal Plain on Pampa Biome (CPPB). Locality: sampled areas (four Malaise trap per area) inside the regions; LP: law-protected area; coordinates: central coordinate of the sampled area; site description: a general description of the locality, including it phytophysiognomy and geology; climate: average temperature and relative humidity during the sampling period. APL, Pelotas stream; ACS, Corrientes stream; ATU, Turuçu stream; LAMI, Lami Biological Reserve; RCMQ, Camaquã river; RPPN, private natural heritage reserve Barba Negra farm; TAIM, Taim Ecological Station; ITPA, Itapuã State Park; PMTY, Tupancy Natural Municipality Park; PEJL, José Lutzenberger State Park; PEVA, Itapeva State Park; LPXE, Lagoa do Peixe National Park; points 1–7, region 1 (Oct–Nov, 2011); points 8–14, region 2 (Nov, 2011); points 15–21: region 3 (Dec, 2011); points 22–28, region 4 (Jan, 2012); points 29–35: region 5 (Feb, 2012).

Point	Locality	LP	Latitude and longitude		Regional description
1	APL	No	−31.72175	−52.25422	Atlantic forest remains with pioneer vegetation and fluvial influence. The general classification is a semideciduous seasonal forest. Sandbank, riparian, and gallery forests predominate. Abundant marshes and river streams from Patos Lagoon system.
2	APL	No	−31.67118	−52.21764
3	APL	No	−31.62781	−52.35285
4	ACS	No	−31.55675	−52.14568
5	ACS	No	−31.56435	−52.13951
6	ATU	No	−31.43295	−52.12173
7	ATU	No	−31.49678	−52.00834
8	LAMI	Yes	−30.23630	−51.09601	Riparian forests with shrubby marshes, herbaceous marshes, sandbanks, and wet fields.
9	LAMI	Yes	−30.25632	−51.10126
10	RCMQ	No	−31.12159	−51.79237	Transition border between the Atlantic forest and Pampa biomes. Abundant riparian forests. The general classification is a semideciduous seasonal forest.
11	RPPN	Yes	−30.43135	−51.23704	Farming forests covered by abundant non-indigenousEucalyptus species. Heavy groves of native species covering dunes, internal beaches, and all borders of RPPN.
12	RPPN	Yes	−30.41323	−51.21834
13	RPPN	Yes	−30.42993	−51.14240
14	RPPN	Yes	−30.39651	−51.12725
15	TAIM	Yes	−32.55568	−52.50060	Several internal lagoons and forested marshes. The predominant phytophysiognomy is a coastal field with savanna-like grasses. The landscape is composed of a mosaic of lagoons, marshes, sandbank shrubberies, dunes, and riparian forests. Farming and cattle raising in the surrounding park area.
16	TAIM	Yes	−32.53865	−52.53732
17	TAIM	Yes	−32.56059	−52.50975
18	TAIM	Yes	−32.53347	−52.52538
19	TAIM	Yes	−32.53845	−52.53713
20	TAIM	Yes	−32.59684	−52.56837
21	TAIM	Yes	−32.63511	−52.48655
22	ITPA	Yes	−30.38372	−51.02095	Several wooded foothills with herbaceous vegetation and shrubby fields. The phytophysiognomy varies from shrubby/wooded fields to dense forests. The mosaic landscape is composed of internal lagoon beaches, riparian forests, and mobile dunes.
23	ITPA	Yes	−30.38291	−51.03008
24	ITPA	Yes	−30.36819	−51.02620
25	PMTY	Yes	−29.48945	−49.84442	Psammophile forested area, low-height wood profile. Highest trees are about 10-m tall. General physiognomy is characterised by a small riparian forest, three internal lagoons, one dune of 9 m height, and urban areas surrounding the park.
26	PEJL	Yes	−29.35508	−49.73529	Dense ombrophilous forest influenced by sea proximity. Paleodunes fixed by dense forest cover. Mobile dunes are covered only by herbaceous and shrub vegetation. Several marshes with swamp-like vegetation.
27	PEVA	Yes	−29.37978	−49.76215
28	PEVA	Yes	−29.37819	−49.75909
29	LPXE	Yes	−31.05366	−50.80873	High seasonal variation of salinity in soil, causing seasonal patterns in phytophysiognomy. Lagoon complex of developed riparian forests, savanna-like fields, and xerophytic shrubberies on fixed paleodunes. Farming and cattle raising in the surrounding park area.
30	LPXE	Yes	−31.05613	−50.81078
31	LPXE	Yes	−31.06581	−50.81776
32	LPXE	?	−31.21367	−50.96057
33	LPXE	?	−31.21715	−50.96621
34	LPXE	?	−31.21911	−50.97516
35	LPXE	?	−31.43412	−51.17502

Fig. 1
Partial map of South America, with details of the Coastal Plain of Pampa Biome, Rio Grande do Sul (Brazil). The marked points refer to all 35 collection areas (groups of four traps). Descriptions of each area are provided in Table 1. Stars: region 1; squares: region 2; pentagons: region 3; circles: region 4; triangle: region 5.

Fig. 1 and Table 1 show the study sites. Region 1 is made up of areas around the Pelotas stream (points 1-3), Corrientes stream (points 4 and 5), and Turuçu stream (points 6 and 7). Region 2 contains the Lami Biological Reserve (points 8 and 9), Camaquã river (point 10), and the private natural heritage reserve (RPPN) Barba Negra (points 11-14). Region 3 contains the Taim Ecological Station (points 15-21), which is a significant area of national protection. The Itapuã State Park (points 22-24), Tupancy Natural Municipality Park (point 25), José Lutzenberger State Park (known as Guardhouse Park, point 26), and Itapeva State Park (points 27 and 28) make up Region 4 on the northern coast of CPPB. Region 5 is the Lagoa do Peixe National Park (points 29-35).

The characterizations of the sampled areas and their protection status by law are summarised in Table 1. For a complete characterisation of all areas, including conservation unities, the reader is referred to Waechter (1985, 2002), IBGE (1986, 2004), Mello-Filho et al. (1992), SEMA (1996, 2013), Meira and Porto (1998), ICMBIO (1999), Burger (2000), Leite (2002), Celulose Riograndense (2012), Venzke (2012), and Witt (2013).

Sampling design

The Muscidae flies were collected in five regions of the CPPB using 140 Malaise traps, model Townes (1972) with a modified collection container (Duarte et al., 2010). Seven areas were sampled in each region (Table 1 and Fig. 1) and four traps were installed in each area. The traps remained in the field for eight days. On the eighth day, collector containers carrying material preserved in 70% alcohol were sent to triage and pinning in the laboratory. The periods of collection campaigns were the following: (i) region 1 between October and November of 2011; (ii) region 2 in November of 2011; (iii) region 3 in December of 2011; (iv) region 4 in January of 2012; (v) region 5 in February of 2012.

The flies were identified with specific keys of Neotropical Muscidae (Carvalho and Couri, 2002; Costacurta and Carvalho, 2005; Costacurta et al., 2005; Couri and Carvalho, 2002; Marques and Couri, 2007; Nihei, 2005; Nihei and Carvalho, 2007; Pereira-Colavite and Carvalho, 2012; Schüehli and Carvalho, 2005), and from the original descriptions of some species. Identification of many specimens was confirmed by comparing with material from the Coleção de Entomologia Padre Jesus Santiago Moure, Universidade Federal do Paraná (DZUP).

The vouchers are deposited at DZUP and Coleção de Diptera do Laboratório de Ecologia de Parasitos e Vetores, Universidade Federal de Pelotas (LEPAV).

Data analysis

All analyses were performed using the statistical program R (R Development Core Team, 2015) and with the statistical packages vegan and iNEXT (Hsieh et al., 2016; Oksanen et al., 2015). The species with specimens which were much damaged to identify or to recognise as morphospecies (e.g. Coenosia sp., Polietina sp., Pseudoptilolepis sp.) were removed from data analyses.

Dissimilarity in the southern assemblages of flies

The dissimilarity of fly communities between regions (1-5) and between Southern Brazil muscid assemblages (Costacurta et al., 2003; Krüger et al., 2010; Rodríguez-Fernández et al., 2006; and the present study) were subjected to multivariate analysis with agglomerative hierarchical classification by unweighted pair-group method using arithmetic averages (UPGMA), with complete linkage (Borcard et al., 2011; Sneath and Sokal, 1973).

The UPGMA clustering method was selected by the best Pearson's correlations index (Table 2) for verification between regions (1-5). The index was obtained comparing the cophenetic matrices of various hierarchical clustering methods. The abundance was standardised by Hellinger's transformation, and dissimilarities between pairs of samples were calculated using the Bray-Curtis dissimilarity coefficient (Borcard et al., 2011). UPGMA clustering of Muscidae assemblages from studies in Southern Brazil was performed by Jaccard index of dissimilarity using the binary matrices, as not at all the analysed studies contained data on abundance. This cluster was generated by comparing only species with specific names with morphospecies which are not considered in all studies.

Table 2
Results of the cophenetic matrices test. The choice of cluster method was based on the highest score in the Pearson's correlations index. UPGMA, unweighted pair-group method using arithmetic averages; UPGMC, unweighted pair-group method using centroids; WARD, Ward's minimum variance clustering; WPGMA, weighted pair-group method using arithmetic averages; WPGMC, weighted pair-group method using centroids; PCI, Pearson's correlation index.

Cluster method		PCI
UPGMA		0.8846567
WPGMA		0.8842323
WARD		0.8827867
WPGMC		0.7437148
UPGMC		0.6925564

Sampling effort

Sampling sufficiency was verified using a species accumulation curve (SAC), which was built using the vegan package, with the specaccum function. This function produces the SAC or the number of species at a given number of sampled points (Malaise trap). Within the specaccum function, the "exact" method was used, which obtained the expected (mean) species richness. The "exact" method finds the expected SAC using the method independently developed by Ugland et al. (2003), Colwell et al. (2004), and Kindt et al. (2006).

To estimate species richness (S), which is specific for each treatment, Rarefaction, and Chao2 were used (Chao, 1987; Chao et al., 2009; Chiu et al., 2014; Colwell and Coddington, 1994; Gotelli and Colwell, 2001). These are non-parametric methods, used to estimate S based on the incidence of species (presence/absence). The individual-based rarefaction computes the expected number of species in a subsample of "n" individuals drawn at random from a single representative sample from an assemblage. The values of Rarefactions and Chao2 were obtained using the function "Poolaccum," which estimates the extrapolated richness index, as well as the number of species for the random ordination of sampling units.

Ecological guilds in Muscidae

Before defining the ecological guilds, it is essential to determine how muscid flies explore environmental resources. The most notable study on Muscidae biology was Skidmore (1985), in which the author indicated larval habit mainly by the morphology of the cephalo-pharyngeal skeleton and the anal spiracles. Based on the anatomical features of larvae, we can suggest three definitions for the biology of Muscidae. The first one is the saprophagous larvae, characterised by well-developed suction mechanisms, lacking oral accessory sclerites, and large anal spiracles. The second one is the saprophagous larvae with predatory facultative behaviour in the third instar or parasitic behaviour, as in some genera (e.g. Charadrella Wulp, Philornis Meinert), characterised by well-developed oral hooks, the presence of oral accessory sclerites, the presence of a suction mechanism, and large anal spiracles. The third is the obligate carnivorous larvae, characterised by well-developed and well-sclerotised oral hooks and accessory sclerites, the absence of suction mechanisms, and small anal spiracles. For additional details of the species, morphology, and substrates used by immatures see Skidmore (1985) and Ferrar (1987).

The habits of adults could be identified by the hematophagous with labium modified for penetration and suction. The second type is lickers in saprophagous species, with labella that are not reduced and with a more considerable lateral expansion membranous. The last one is the predators, with reduced labella, developed prestomal teeth, and sclerotised prementum to penetrate the exoskeleton of their prey (Coelho, 1997; McAlpine, 1981).

Therefore, we defined an ecological guild as a group of species which exploit the same class of resources in a similar manner (Root, 1967). In Muscidae, we recognise four ecological guilds or functional groups (sensuBlondel, 2003) combining trophic roles of larvae and adult stages (Krüger et al., 2010). The species of CPPB are classified according to the following: saprophagous larvae and saprophagic/hematophagous adults species (SS); facultative predators/parasitic larvae and saprophagous adults species (SPS); predatory larvae and saprophagous adults species (OS): predatory larvae and adults species (PP).

Region-guild diversity proportionality

We tested the influence of region and guild of Muscidae on the proportional richness and proportional abundance using ANOVA and F test, considering P < 0.05 as significant.

Results

Muscidae of the Coastal Plain of Pampa Biome

We collected 6314 specimens, 98 species in 31 genera of Muscidae on the CPPB (Table 3). The most abundant species were Sarcopromusca pruna (Shannon and Del Ponte) (n = 934), Neodexiopsis sp. 9 (n = 641), Bithoracochaeta calopus (Bigot) (n = 503), Stomopogon argentina (Snyder) (n = 388), Limnophora sp. 5 (356), Phaonia sp. 2 (n = 338), Neodexiopsis sp. 4 (n = 309), Phaonia nigriventris (Albuquerque) (n = 300), Limnophora sp. 4 (n = 298).

Table 3
List of Muscidae (Insecta, Diptera) species collected in five regions of the Coastal Plain of Pampa Biome (CPPB), Rio Grande do Sul, Brazil. Sampled CPPB areas include R1: region 1, where: APL: Pelotas stream; ACS: Corrientes stream; ATU: Turuçu stream. R2: region 2, where: LAMI: Lami Biological Reserve; RCMQ: Camaquã River; RPPN: private natural heritage reserve Barba Negra farm. R3: region 3, where: TAIM: Ecological Station of Taim. R4: region 4, where: ITPA: Itapuã State Park; PMTY: Tupancy Natural Municipality Park; PEJL: José Lutzenberger State Park; PEVA: Itapeva State Park. R5: region 5, where LPXE: Lagoa do Peixe National Park.

Taxa	R1				R2			R3	R4				R5	(∑)
	APL	ACS	ATU		RPPN	RCMQ	LAMI	TAIM	ITPA	PEVA	PMTY	PEJL	LPXE
Biopyrellia bipuncta (Wiedemann, 1830)	0	0	0		0	4	0	13	8	0	0	0	3	28
Bithoracochaeta annulata Stein, 1911	0	0	0		0	0	1	0	0	0	0	0	33	34
Bithoracochaeta calopus Bigot, 1885	120	44	98		7	43	6	160	0	1	0	0	26	505
Bithoracochaeta flavicoxaa Malloch, 1934	1	12	2		0	10	1	4	1	0	3	0	0	34
Bithoracochaeta plumata Albuquerque, 1955	0	0	0		0	0	0	0	1	0	0	0	0	1
Bithoracochaeta sp. 2	0	0	0		0	0	0	0	1	0	0	0	6	7
Coenosia sp.	1	0	0		0	0	0	0	1	0	0	0	0	2
Coenosia sp. 1	1	0	0		0	0	0	1	0	0	0	0	0	2
Coenosiini Genus A	3	0	0		0	0	0	2	0	0	0	0	0	5
Coenosiini Genus B	0	0	0		0	0	0	1	0	0	0	0	0	1
Cyrtoneurina costalis (Walker, 1853)	5	3	0		0	10	0	2	2	0	0	0	1	23
Cyrtoneuropsis brunnea (Hough, 1900)	1	0	0		0	2	1	1	0	0	0	0	1	6
Cyrtoneuropsis maculipennisa (Macquart, 1843)	10	4	4		11	0	10	0	0	0	1	11	0	51
Cyrtoneuropsis pararescita (Couri, 1995)	0	0	0		0	0	0	1	0	0	0	0	9	10
Cyrtoneuropsis incognitaa (Snyder, 1954)	1	0	0		2	0	17	0	18	4	0	0	0	42
Cyrtoneuropsis sp. 2	0	0	0		2	0	6	0	0	0	0	0	0	8
Dolichophaonia cacheutaa (Snyder, 1957)	0	0	0		0	0	1	0	0	0	0	0	0	1
Dolichophaonia plaumanni (Carvalho, 1983)	0	0	0		0	0	0	0	0	0	0	0	1	1
Dolichophaonia trigona (Shannon and Del Ponte, 1926)	0	5	1		0	0	0	1	1	0	0	0	0	8
Dolichophaonia elongataa (Albuquerque, 1958)	0	0	0		0	0	0	1	0	0	0	0	1	2
Graphomya auriceps Malloch, 1934	2	0	1		0	0	0	21	0	0	0	0	0	24
Gymnodia delecta (Wulp, 1896)	0	0	0		0	0	0	15	0	0	0	0	0	15
Gymnodia quadristigma (Thomson, 1869)	0	0	0		0	0	0	9	1	0	0	0	0	10
Gymnodia sp.	0	0	0		0	0	0	1	0	0	0	0	0	1
Haematobia irritans (Linnaeus, 1758)	0	0	0		0	0	0	20	0	0	0	0	11	31
Helina sp. 1	1	5	2		0	0	0	2	0	0	0	0	0	10
Helina sp. 2	26	4	3		0	2	0	6	0	0	0	0	0	41
Helina sp. 3	8	0	1		0	0	1	4	1	0	1	0	0	16
Helina sp. 4	0	6	1		0	0	0	4	0	0	0	0	0	11
Helina sp. 6	2	5	1		5	0	4	1	5	0	0	0	24	47
Limnophora aurifacies Stein, 1911	14	7	7		3	5	15	19	0	0	0	0	0	70
Limnophora sp. 1	4	0	0		0	1	1	12	0	0	0	0	1	19
Limnophora sp. 2	13	2	2		0	1	0	50	1	0	0	0	0	69
Limnophora sp. 3	3	0	0		0	0	0	7	0	0	0	0	0	10
Limnophora sp. 4	99	11	8		6	5	5	155	2	0	1	4	2	298
Limnophora sp. 5	59	36	22		8	9	3	216	1	1	0	1	0	356
Limnophora sp. 6	0	1	0		0	0	0	2	0	0	0	0	0	3
Lispe serotina Wulp, 1896	1	0	0		0	0	2	0	0	0	0	0	0	3
Micropotamia minuscula (Albuquerque, 1955)	7	8	0		2	0	0	16	0	0	0	1	0	34
Morellia humeralis (Stein, 1918)	0	0	0		0	0	0	2	17	0	0	0	0	19
Morellia paulistensis Pamplona and Mendes, 1995	0	0	0		8	58	5	40	6	1	0	0	1	119
Morellia sp. 1	0	2	1		0	8	0	3	0	0	0	0	0	14
Morellia sp. 2	1	1	0		1	2	0	1	0	0	0	0	0	6
Musca domestica Linnaeus, 1758	0	0	0		0	0	0	0	0	0	1	0	0	1
Mydaea plaumanni Snyder, 1941	29	20	5		2	3	0	1	2	0	0	0	0	62
Myospila obscura (Shannon and Del Ponte, 1926)	1	7	0		1	6	0	13	5	1	0	2	0	33
Myospila sp. 1	4	1	0		0	0	0	4	0	0	1	1	1	12
Neodexiopsis erecta Costacurta, Couri and Carvalho, 2005	0	0	0		0	0	0	0	0	0	1	0	0	1
Neodexiopsis rustica Albuquerque, 1956	2	20	0		1	0	0	1	0	0	0	0	1	25
Neodexiopsis sp.	0	0	0		0	0	0	0	1	0	0	0	0	1
Neodexiopsis sp. 1	3	2	0		0	0	0	75	0	0	0	0	0	80
Neodexiopsis sp. 2	0	1	0		0	2	0	2	0	0	0	0	0	5
Neodexiopsis sp. 3	1	0	4		0	0	1	108	0	0	0	1	0	115
Neodexiopsis sp. 4	98	29	79		0	1	1	101	0	0	0	0	0	309
Neodexiopsis sp. 5	2	13	3		5	0	0	18	0	1	0	0	0	42
Neodexiopsis sp. 6	0	0	0		0	0	0	1	0	0	0	0	0	1
Neodexiopsis sp. 7	28	14	5		6	9	3	7	5	0	0	1	0	78
Neodexiopsis sp. 8	0	0	0		0	0	0	2	1	0	0	0	0	3
Neodexiopsis sp. 9	154	88	86		4	31	8	259	3	5	0	2	4	644
Neodexiopsis sp. 10	0	0	0		0	0	3	1	0	0	0	0	0	4
Neodexiopsis sp. 11	12	5	1		0	3	2	0	0	0	0	0	4	27
Neodexiopsis sp. 12 affvulgarisa Couri and Albuquerque, 1979	0	0	0		1	0	0	0	0	0	0	0	0	1
Neodexiopsis sp. 13	0	0	1		0	0	0	0	0	0	0	0	0	1
Neomuscina affdouradensisa Lopes and Khouri, 1996	1	0	0		0	0	0	0	0	0	0	0	0	1
Neomuscina aff.Goianensisa Lopes and Khouri, 1995	0	0	0		0	1	0	0	0	0	0	0	0	1
Neomuscina inflexa (Stein, 1918)	0	0	0		0	0	0	0	0	0	0	0	1	1
Neomuscina neosimilis Snyder, 1949	0	0	0		0	0	0	0	1	0	0	3	0	4
Neomuscina pictipennis pictipennis (Bigot, 1878)	1	0	0		0	2	0	0	2	1	0	4	0	10
Neomuscina sanespra Snyder, 1949	0	0	0		0	1	0	0	0	0	0	0	0	1
Neomuscina zosteris (Shannon and Del Ponte, 1926)	33	89	4		0	4	0	9	1	0	0	0	4	144
Neomuscina sp. 1	0	0	0		1	0	0	0	0	0	0	0	0	1
Neomuscina sp. 2	2	1	0		1	0	0	0	0	0	0	0	0	4
Neomuscina sp. 3	1	0	0		1	1	0	0	0	0	0	0	0	3
Neomuscina sp. 4	1	1	0		0	0	0	0	0	0	0	0	0	2
Neomuscina sp. 5	0	0	0		0	0	0	0	1	1	0	1	0	3
Neurotrixa felsina (Walker, 1849)	3	0	0		0	1	0	21	0	0	0	0	0	25
Neurotrixa marinonii a Costacurta and Carvalho, 2005	1	9	2		0	0	0	5	2	0	0	0	4	23
Neurotrixa sulina Costacurta and Carvalho, 2005	1	3	0		1	0	0	8	1	0	0	0	0	14
Phaonia advena Snyder, 1957	0	0	0		4	0	0	0	3	2	1	0	0	10
Phaonia annulata (Albuquerque, 1957)	9	21	0		0	0	0	0	4	0	0	1	0	35
Phaonia bigotia (Albuquerque, 1957)	0	0	0		0	0	0	0	1	0	0	0	0	1
Phaonia grajauensis (Albuquerque, 1957)	6	13	1		10	2	7	1	10	2	0	5	0	57
Phaonia nigriventris (Albuquerque, 1954)	18	18	0		8	3	1	160	20	1	0	2	69	300
Phaonia praesuturalis (Stein, 1904)	0	0	0		0	2	0	0	9	0	0	0	0	11
Phaonia similata (Albuquerque, 1957)	1	0	0		8	4	3	1	3	2	0	3	1	26
Phaonia trispila (Bigot, 1885)	7	20	18		2	50	0	64	0	0	0	0	7	168
Phaonia sp. 1	8	10	1		2	5	5	24	5	11	2	10	9	92
Phaonia sp. 2	0	4	1		1	0	3	316	0	0	0	0	13	338
Phaonia sp. 3	22	0	6		1	9	2	0	0	0	0	0	0	40
Phaonia sp. 4	0	0	0		0	0	1	0	0	0	0	0	0	1
Philornis seguyia Garcia, 1952	1	0	0		0	0	0	0	0	0	0	0	0	1
Philornis sp. 2	0	0	0		0	1	0	0	0	0	0	0	0	1
Plumispina sp. 1	0	0	0		0	0	0	1	0	0	0	0	0	1
Polietina orbitalis (Stein, 1904)	21	16	5		3	9	0	16	4	2	0	3	17	96
Pseudoptilolepis nudapleuraa Snyder, 1949	0	0	0		5	0	0	0	0	0	0	0	0	5
Pseudoptilolepis sp.	2	0	1		0	0	0	0	0	0	0	0	0	3
Sarcopromusca pruna (Shannon and Del Ponte, 1926)	5	2	3		0	43	1	903	0	0	0	0	18	975
Stomopogon argentina (Snyder, 1957)	10	30	3		0	1	1	398	0	0	0	0	0	443
Stomoxys calcitrans (Linnaeus, 1758)	0	0	0		0	0	0	2	0	0	0	0	3	5
Trichomorellia seguyia (Pamplona, 1983)	0	0	0		0	1	0	22	0	0	0	0	0	23
(∑)	871	593	383		123	355	121	3337	151	36	12	56	276	6314

• ^a
  New record for RS state.

Among the 98 identified species of Muscidae, 41 did not fit descriptions of valid species, indicating that there may be many new species in the studied regions. Other data show that 34 taxa were unique to at least one location (Table 3). The geographical distribution of 13 species of Muscidae was expanded, and they were registered for the first time in the state of Rio Grande do Sul, based on the distributional catalogue of South American muscids (Löwenberg-Neto and Carvalho, 2013).

Regarding the number of species as a function of the samples (areas) in CPPB, the SAC exhibited an upward trend (Fig. 2), although the Chao2 diversity estimator report was collected between 70% and 86% Muscidae of CPPB (Table 4).

Fig. 2
Sample coverage curves based on the number of individuals of Muscidae per region in the Coastal Plain (CPPB) of Rio Grande do Sul, Brazil. The grey shaded area represents the confidence intervals. (a) region 1; (b) region 2; (c) region 3; (d) region 4; (e) region 5; (f) CPPB total.

Table 4
Richness estimators by location and region of collection. Non identified specimens were pruned from data matrices before analysis. R1: region 1, where: APL: Pelotas stream; ACS: Corrientes stream; ATU: Turuçu stream. R2: region 2, where: LAMI: Lami Biological Reserve; RCMQ: Camaquã river; RPPN: private natural heritage reserve Barba Negra farm. R3: region 3, where: TAIM: Taim Ecological Station. R4: region 4, where: ITPA: Itapuã State Park; PMTY: Tupancy Municipality Natural Park; PEJL; José Lutzenberger State Park; PEVA: Itapeva State Park. R5: region 5, where LPXE: Lagoa do Peixe National Park. CPPB, Coastal Plains on Pampa Biome; Traps, number of traps per location; S, species richness; Abund, abundance; Chao2, Chao2 non-parametric diversity estimator; Srar, rarefaction; SE, standard error. In the rarefaction data by location, the standard number of individuals was 12 (PMTY), and by region the standard number of individuals was 254 (R4).

	Traps	S	Abund	Chao2 ± SE	Srar ± SE
R1
APL	12	55	870	81.96 ± 17.6	8.08 ± 1.36
ACS	8	42	592	46.49 ± 4.79	8.80 ± 1.34
ATU	8	34	383	51.95 ± 14.35	6.22 ± 1.35
Total R1	28	62	1845	104.23 ± 38.5	38.40 ± 2.65
R2
LAMI	8	31	121	54.8 ± 20.03	9.07 ± 1.27
RCMQ	4	39	355	46.12 ± 5.88	8.04 ± 1.36
RPPN	16	32	123	40.26 ± 6.85	9.62 ± 1.17
Total R2	28	60	599	72.78 ± 8.36	46.15 ± 2.60
R3
TAIM	28	62	3333	74.5 ± 8.46	30.11 ± 2.59
R4
ITPA	12	37	150	72.88 ± 25.7	9.01 ± 1.30
PMTY	4	9	12	31.45 ± 28.54	9.00
PEVA	8	15	36	22.77 ± 7.29	7.62 ± 1.26
PEJL	4	18	56	26 ± 8.13	8.08 ± 1.26
Total R4	28	46	254	86.34 ± 28.44	46
R5
LPXE	28	29	276	78.82 ± 59.37	28.19 ± 0.84
CPPB total	140	98	6307	181.65 ± 58.58	–

The diversity of muscid flies through CPPB regions and dissimilarity with Southern Brazil fauna

The species richness was the highest in regions 1 and 3, where there was a greater abundance of flies. When rarefaction by region was applied, computing the expected number of species in a subsample of n individuals (n = 255, corresponding to region 4), regions 2 and 4 had the highest estimated richness (Table 4). Rarefaction by area was computed to a subsample of n = 8 individuals, and the highest rarefied richness was observed in areas of regions 2, 4, and 1 (Table 4).

The most abundant species were found to vary in each region. In region 1, the most abundant species were Neodexiopsis sp. 9, Neodexiopsis sp. 4, and B. calopus, which are small predators in larvae and adult stages, and their combined abundance represented 38% of the collected specimens from this region. Singletons and doubletons accounted for 31.9% of the total species gathered in this region. In region 2, the most abundant species (28.9%) were Morellia paulistensis Pamplona and Mendes, Phaonia trispila (Bigot), and B. calopus. In this region, 44.4% of the species were singletons and doubletons. More than 52% of the total abundance of species in the CPPB was in region 3 (Taim Ecological Station). In this region, 46.8% of the specimens were S. pruna, S. argentina, and Phaonia sp. 2. We collected 26 species (36.62%) of singleton and doubletons in this region. In region 4, Phaonia sp. 1, Phaonia nigriventris (Albuquerque), and Cyrtoneuropsis incognita Malloch were the most abundant species (28.6%). Region 4 contained 48.9% of singleton and doubleton species. In region 5 (Lagoa do Peixe National Park), the most abundant species, corresponding to 55.5%, were P. nigriventris, B. calopus, B. annulata Stein and Helina sp. 6. Singleton and doubleton species accounted for 40% of the species richness (Table 3).

Differences in species composition between regions are shown by the cluster analysis (Fig. 3). The first cluster group is composed by regions 1, 2, and 3. The assemblage dissimilarity between groups from regions 1 and 2 was more than 40%. The difference between the cluster formed by regions 1 and 2 and formed by region 3 was also low, at about 45%. More significant differences between assemblage clusters were observed between group 1 (regions 1-3) and regions 4 and 5, with more than 60% dissimilarity. Among the species of muscids in Southern Brazil, including those in this study, there was high dissimilarity between clusters with a trend to increase/decrease based on geographic distance (Fig. 4).

Fig. 3
Compositional differences between Muscidae (Insecta, Diptera) assemblages of all five regions of the Coastal Plain of Pampa Biome (Rio Grande do Sul, Brazil) shown by the unweighted pair-group method using arithmetic averages (UPGMA) cluster analysis.

Fig. 4
The UPGMA cluster between Muscidae (Insecta, Diptera) assemblages of Southern Brazil. The dissimilarity analysis was performed using the Jaccard index with binary matrices. In Paraná state: green diamonds, Rodríguez-Fernández et al. (2006); pink circles, Costacurta et al. (2003). In the Rio Grande do Sul state: red stars, current study; blue squares, Krüger et al. (2010).

Diversity pattern by trophic guilds of Muscidae

The proportion of species and abundance by guild was not consistent throughout the CPPB; this difference was influenced by collection region (Tables 5 and 6). In the first three regions, the highest species richness was found in a guild of PP settings followed by OS settings; in the former, both larvae and adults are predators, and in the latter, larvae are predators and adults are saprophagous (Fig. 5A). In regions 4 and 5, this relationship was reversed, with a predominance of OS settings species followed by PP guild (Fig. 5A). In all regions, guild species with saprophagous habits in the larval and adult stages is present in the lowest proportion of the total fauna, except in region 3 (Taim), which exhibited increased richness compared with the SPS guild (Fig. 5A). The same pattern was observed for the relative abundance within each guild by region. Contrary to the relationship between the proportion of species per guild and region, the abundance of the SS guild greatly contributes to the fauna in regions 3 and 5 (Fig. 5B).

Table 5
Analysis of variance (ANOVA) and F test, testing the influence of region and ecological guilds of Muscidae on the proportional richness. Df, degrees of freedom; Sum Sq, sum of squares; Mean Sq, mean of squares.

		Df	Sum Sq	Mean Sq	F value	P
Guild		3	1.51893	0.50631	61.9787	<0.001
Region		4	0.00010	0.00002	0.0030	1.000
Guild:region		12	0.51359	0.04280	5.2392	<0.001
Residuals		120	0.98029	0.00817	–	–

Table 6
Analysis of variance (ANOVA) and F test, testing the influence of region and ecological guilds of Muscidae on the proportional abundance. Df, degrees of freedom; Sum Sq: sum of squares; Mean Sq: mean of squares.

		Df	Sum Sq	Mean Sq	F value	Pr(>F)
Guild		3	1.8376	0.61254	21.9057	<0.001
Region		4	0.0048	0.00119	0.0426	0.997
Guild:region		12	2.0160	0.16800	6.0079	<0.001
Residuals		120	3.3555	0.02796	–	–

Fig. 5
Graphical representation of proportional richness (a) and abundance (b) by the guild in the five regions of Coastal Plain of Pampa Biome (Rio Grande do Sul, Brazil). SS, saprophagous larvae and saprophagic/hematophagous adults; SPS, facultative predators/parasitic larvae and saprophagous adults; OS, predatory larvae and saprophagous adults; PP, predatory larvae and adults.

Discussion

The Muscidae in Southern Brazil, fauna similarity, and possible areas of endemism

In Southern Brazil, major taxonomic surveys of Muscidae were carried out, and a trend of greater or lesser similarity between the areas of collections was observed with increasing or decreasing distance. There was greater similarity between the data collected in CPPB and those inventoried by Krüger et al. (2010) in the Rio Grande do Sul over a 1-year collection period. We share 36 species without the morphospecies, and there was a tendency of similarity among the most abundant species in the two studies. Surveys conducted in the Paraná state (Costacurta et al., 2003; Rodríguez-Fernández et al., 2006), which is about 1000 km from the study site in the CPPB, found that the proportion of species was lower, with 17 and 23 species, and there is a high dissimilarity with CPPB (Fig. 4). Some studies (Löwenberg-Neto and Carvalho, 2009; Nihei and Carvalho, 2005) have indicated areas of endemism and diversification for Muscidae in the Paranaense Forest (Paraná state) and the Pampa. Therefore, our data indicate that the CPPB regions may represent new areas of endemism, since there is: (i) significant Muscidae richness; (ii) new records of occurrence; (iii) high rates of particular species per region; (iv) high dissimilarities with the Paraná Forest communities.

Sampling effort in the areas and conservation units in the CPPB

Differences between the samplings by Costacurta et al. (2003), Rodríguez-Fernández et al. (2006), and Krüger et al. (2010) and those in the present study include the exposure time of Malaise traps versus the number of traps installed by area. The studies by Costacurta et al. (2003), Rodríguez-Fernández et al. (2006), and Krüger et al. (2010) addressed the sampling of Muscidae assemblies on a temporal scale at the expense of a spatial scale. In contrast, we sought to directly sample on a spatial scale over time, with many replicates (traps) and a short period of exposure.

While representing a considerable sampling effort (140 Malaise traps in 35 areas), our species accumulation curve possesses no asymptote. We can argue that (i) the restricted use of Malaise traps is not problematic for the sampling of Muscidae diversity, as once compared with traps containing decomposing bait (Carvalho et al., 1984), the Malaise trap has higher efficiency in the capture of Diptera richness (Brown, 2005; Costacurta et al., 2003; Duarte et al., 2010; Krüger et al., 2010; Rodríguez-Fernández et al., 2006). Species collected with baits are often, though not always, gathered in Malaise traps; (ii) the ascending pattern of the SAC shows that more focus should be placed on traps in the large fragments than in the small fragments. It is possible that the richness has been underestimated in larger areas as the collection effort was the same in large and small areas. Therefore, small areas showed more richness in the CPPB (e.g., Pelotas and Corrientes streams, Camaquã river, Taim ecological Station, Itapuã state park). (iii) In regions 4 and 5, an increase in the number of traps per se cannot reverse the upward trend of the SAC. In such areas, it is necessary to move the collection period before January (the beginning of summer in the Southern Hemisphere) due to climatic conditions of high temperatures and low relative humidity. These conditions have probably prevented the movement of adult specimens, or else caused their death, preventing them from being collected by Malaise traps. Despite this fact, analysis with Chao2 revealed that 75% of Muscidae species were collected in some regions (Table 4), such as the areas around the Pelotas, Corrientes, and Turuçu streams. There was more richness in region 1 than at the Taim Ecological Station (Tables 3 and 4). The areas in region 1 are not protected and suffer high exploitation due to extractive practice and agricultural production practices in their surroundings.

In general, singletons and doubletons species represent 29.7% of the richness, and their proportion ranges from 31.9 to 48.9% of the species collected in different regions, which is a typical pattern for insect communities in tropical forests (Erwin, 1998; Basset and Kitching, 1991; Novotný, 1993).

Muscidae ecological guilds as indicators of priority areas for conservation

Ecological/trophic guilds of Muscidae can be used to assess environmental health as they respond to specific features. For example, one of the factors closely linked to the conservation of natural areas is the abundance of predatory species of Neodexiopsis and Limnophora. The species of these genera occur in areas where there is high availability of freshwater sources for the development of larvae, and a high abundance of prey for the larvae and adults of these species (Werner and Pont, 2006). The preys recorded in the literature include chironomids, simulids, and other small flies that do not tolerate disturbed areas (Werner and Pont, 2006). The PP guild, which includes the predators, was dominant in most areas, especially in regions 1-3 (Fig. 4). In these regions, there are many marshes, small rivers, streams, and other flooded areas, which are the surrounding physiognomy of the Patos lagoon and Mirim-Mangueira lagoon, and priority areas for conservation according to MMA (2000). Therefore, the high richness and abundance proportional to the PP guild indicate potential areas for permanent protection, such as the Pelotas streams (Pelotas, Corrientes, Turuçu) and the Camaquã river.

Other guilds may be indications of a disturbance in natural areas, specifically the SS guild, containing larvae and saprophagous adults. The species in the SS guild developed preferentially in wild and domestic animal faeces; therefore, this would be expected a priori for high diversity and abundance in Taim Ecological Station, which is a refuge for a wide variety of birds, reptiles, and mammals. Proportionality analysis showed that the SS guild contains a higher abundance in region 3. This result is not inconsistent with expectations, as the dominant species in this guild is Sarcopromusca pruna. The larvae of this species are associated with animal faeces (Pedroso-De-Paiva, 1996), while adults feed on wounds induced by bloodsucking species such as horseflies, horn flies (Haematobia irritans), and stable flies (Stomoxys calcitrans). These species are commonly associated with and recognised as vectors of many pathogens and eggs of parasites (Azevedo et al., 2007). The biology of the horn fly should be noted, whereby the adult constantly remains on the host to feed, and females move away from the host only for oviposition on newly deposited faeces, as they are dependent on high humidity in dung pats (McLintock and Depner, 1954). This peculiar behaviour should limit their capture in Malaise traps since H. irritans do not often fly, confirming the traffic of cattle within fragments of Taim. The presence of species of the SS guild, S. pruna, H. irritans, and S. calcitrans, along with the occurrence of Gymnodia species (OS guild), which also occurs in the stools of production ungulates, are an indication of disturbance.

The OS guild containing predatory larvae and saprophagous adults generally contains generalist predatory larvae in their microhabitats (Phaonia, Helina, Graphomya), and often the faeces of herbivorous vertebrates (Gymnodia) (Skidmore, 1985). The presence of species of this genus indicates a disturbance in the natural environment due to the association with cattle dung, unlike indigenous species of Phaonia, Helina, and Graphomya, where the larvae are predators in humus and mosses. The high proportion of the OS guild in Lagoa do Peixe (region 5), without the occurrence of Gymnodia species, is indicative of areas of diverse resources, consistent low anthropised area.

Closing remarks

Our results show in general that some habitats of CPPB are suffering from invasions by non-indigenous species closely associated with livestock. The invasions present a danger to the indigenous species which occupy the same space as these invaders, such as herbivorous faeces. This topic raises discussion about the implementation of buffer zones and monitoring the anthropic activities surrounding protected areas (Allan et al., 2017; Weisse and Naughton-Treves, 2016).

Conversely, the non-protected areas in Pelotas streams and Camaquã River need more legal attention by legislators to become protected areas due to the high diversity of flies and high proportionality of guilds of predators (indicators for healthy environments). Despite our sampling efforts in CPPB, the results reveal the need for additional surveys in these regions, as many more species remain to be discovered. The CPPB may be considered a hotspot for the conservation of Muscidae species, in turn, new biogeographical approaches are needed to identify new areas of endemism in the Pampa biome.

Acknowledgements

All authors have contributed substantially to the conception, design, analysis, and/or interpretation of the data in this manuscript and will take public responsibility for the content. AZS was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by the National Council of Technological and Scientific Development (CNPq) (process no. 161907/2015-6). We also thank CNPq for financing the project "Efeito da redução da área na diversidade de Muscidae (Insecta: Diptera) da planície costeira do Rio Grande do Sul" (process no. 473949/2010-5). We thank Juliano Lessa Pinto Duarte, PhD, for assistance in the genus sorting of material; Claudio José Barros de Carvalho, PhD (Universidade Federal do Paraná) for providing help with identification. We also thank the administrators of protected areas where collecting was conducted and to "Sistema de Autorização e Informação em Biodiversidade" (SISBIO) for providing the collection license (process no. 29229). RFK is supported by CNPq (process no. 308908/2016-3).

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Edited by

Associate Editor: Gustavo Graciolli

Publication Dates

• Publication in this collection
  Oct-Dec 2018

History

• Received
  9 Feb 2018
• Accepted
  10 Sept 2018
• Published
  5 Oct 2018