Preliminary dragonfly ( Odonata ) species list from the Pampa biome in Rio Grande do Sul , Brazil , with ecological notes for 19 new records for the State

An inventory of Odonata was carried out in the southern half of the state of Rio Grande do Sul, Brazil, in the Pampa biome. Originally, this biogeographical region was covered mostly by open fields and grassland, with sections of higher vegetation surrounding water bodies and rocky hills. Today the landscape is fragmented due to agricultural activities, mainly cattle farming, rice crops and forest plantations. Our survey was conducted in three municipalities from this region, between March 2015 and April 2016. Aiming at a general overview of the species composition, our sampling sites were selected on a wide basis, including lakes, bogs, temporary water bodies, small streams and river sections. Eighty two species of Odonata were collected comprising 40 genera and seven families. The dominant families were Libellulidae (56,1%), Coenagrionidae (24,5%) and Aeshnidae (7,3%). We found a diverse odonate assemblage, adding 19 new species records for the state of Rio Grande do Sul.


Introduction
One of the biggest problems towards conservation measures is the lack of knowledge on species distribution, scientifically known as the Wallacean shortfall (Cardoso et al. 2009).Jetz et al. (2012) stated that even when looking at the best known species, knowledge on their occurrence is substantially lower than the amount of available information of other important environmental variables.Insects is the least known group concerning their distribution, mostly due to their high species richness, lack of taxonomic expertise and lack of sampling (Diniz-Filho et al. 2010).In the Neotropical region, and specifically in Brazil, there are still whole biomes which are almost unknown regarding insects (Oliveira et al. 2017).An example is the Pampa biome: extending from the southern half of the state of Rio Grande do Sul (29° S), southwards through the whole Uruguayan territory and within Argentina to the temperate Patagonian steppes, and ending at 39ºS (Roig & Flores 2001).It covers only around 2% of the Brazilian territory but more than 63% of the Rio Grande do Sul State area (IBGE 2016).Although scarce, data from this region has proven high diversity and high levels of endemism, at least regarding the flora (MMA 2002, Behling et al. 2004).The Pampa is under extreme pressure from several human activities such as agriculture, cattle farming, and forestry by the conversion of grasslands into extensive plantations of Eucalyptus, Pinus and Acacia (Bencke 2009, Overbeck et al. 2009, Roesch et al. 2009).Official government data from 2008, shows that only 36% of the original vegetation remains in a fragmented mosaic (MMA 2009).In Brazil, the officially protected areas of the Pampa grasslands cover only about 0.5% of the total biome, thus, more actions are needed to recover and preserve this threatened environment (Overbeck et al. 2009).For conservation purposes, information on species diversity becomes more relevant every day, as well as knowledge on range of distribution (Oliveira et al. 2017).Knowing where species occur and their abundance is fundamental for setting up conservation priorities and red listing.Making information available to scientists and the public is also central to overcome the prevailing lack of distribution knowledge (Jetz et al. 2012).Thus, inventories can provide and improve ongoing and future management efforts (Lewis 2006).Concise information is since long needed for biologists and decision makers to prioritize specific areas for the preservation of biodiversity (Kerr et al. 2000).The Odonata fauna in Brazil is moderately known in terms of species occurrence and distribution: only 29% of the country's territory is adequately surveyed, according to De Marco & Vianna (2005), but this number is probably outdated.For the southeast and central regions several species lists are available (e.g., Costa et al. 2000, Costa & Oldrini 2005, Anjos-Santos & Costa 2006, Calvão et al. 2014, Bedê et al. 2015), while in the southern part of Brazil these studies are more scarce, the most recently published are Kittel & Engels (2014) and Renner et al. (2015Renner et al. ( , 2016aRenner et al. ( , 2016b)), which were all conducted in the Atlantic Forest biome.Thus, we were motivated to increase the knowledge of species occurrence to the Pampa biome, the southernmost part of Brazil, where no survey of Odonata has previously been made.

Study area
We sampled 63 localities distributed in four municipalities: Alegrete (AL, N = 10), Manoel Viana (MV, N = 9), Santana da Boa Vista (SB, N = 8) and São Francisco de Assis (SF, N = 36); within two different regions (1 and 2) of the Pampa biome (Figure 1).In total, the three municipality areas encompass more than 13,000,000 km 2 (IBGE 2016).The climate is Temperate (Cfb Köppen), with mean annual temperatures between 13°C and 17°C and altitudes from 50 to 200 m a.s.l.Annual precipitation mean is between 1,200 and 1,600 mm (INPE 2014).Our sites were distributed among several types of standing and running water including all types of habitats that could be considered to be used as reproduction sites for Odonata.We included small temporary pools or flooding areas, perennial waters such as bogs, swamps, ponds, lakes and rivers of various sizes.These sites included Rivers/streams (N = 27), Lakes (N = 18) and Temporary waters (N = 18), see below.

Data collection
We sampled adult dragonflies from March 2015 to May 2016, the majority of the sampling sites were visited four times during this period, excluding winter due to the lack of activity of adult Odonata in the low temperatures (below 0° C) during that season.Other sites were visited only once, e.g.temporary water and flooded areas.The sampling method followed Renner et al. (2015): hand-held insect nets by a team of two people, in sunny days, during the peak time of Odonata activity (between 09:00 h to 16:00 h).Each site was sampled during 30 minutes, by walking the edge and marginal zones, the distances walked were dependent on the size and type of waterbodies, varying from ca.50 to 250 m perimeters.We focused on adults only, since the majority of the larvae are still unknown (e.g., Garrison et al. 2006).Adults are often dispersing long distances (Corbet 1999), and we expect that some of our specimens might therefore derive from other regions.Our aim, however, was not to discern vagrants from reproducing species, but to get an account of species present in the area.
To quantify our sampling effort, we choose to present a rarefaction curve (Mao tau) and the Jakkinfe 1 estimator.It is a general statistical technique for reducing the bias of an estimator by removing subsets of the data and recalculating the estimator with the reduced sample.Specifically, Jackknife1 depends only on the uniques (species found in only one sample) because the richness estimated is changed only when a sample that contains one of these species is deleted from a subset of samples (Gotelli & Colwell, 2011).We also quantified our sampling effort by using a Jackknife estimation of total species richness according to Smith & van Belle (1984).

Sampling sites
We sampled many different environments ranging from temporary water bodies (small sites) to big river sections.Some of these places were in good environmental conditions, only marginally affected by human disturbances, and notable by the presence of species considered rare by us.These were small erosion sites with temporary water flow, small streamlets/swamps where cold and clear water was flowing from the underground (Figure 2a), temporary flooding zones at a major river (Figure 2b) and also swampy areas with varied and well vegetated edge zones (Figure 2c).Most of the present threats to such unique environments are related to the expansion of forestry practices, which are growing notably and quickly in the regions where the studies took place.

Species List
Eighty-two species belonging to 40 genera and seven families were collected in the three municipalities (Table 1).Seven specimens, newly emerged males and/or females, were impossible to determine to species level; so these were classified as "sp.".Among these we found two specimens of an undescribed species belonging to the family Coenagrionidae.This species is currently under description by Dr. J. Muzón (La Plata, Argentina, personal communication), and no further information is therefore given here.Libellulidae was the dominant family (56,1%, N = 46) followed by Coenagrionidae (24,5%, 20) and, Aeshnidae (7,3%, 6), as shown in Table 2.The richest genus was Erythrodiplax, represented by eight species, this genus was also the most abundant in all seasons, occurring in virtually all sampling sites along with Pantala flavescens Fabricius, 1798, a known migratory and widely distributed disperser (Troast et al. 2016).Some species occurred only once (uniques) in this study and were considered regionally rare, for example: Minagrion waltheri Selys, 1876, Brechmorhoga nubecula Rambur, 1842, Macrothemis lutea Calvert, 1909 and Dasythemis venosa Burmeister, 1839.The rarefaction curve, which was based on the sampling events data, gave us a view of the sampling effectiveness of this study (Figure 3), tending to reach its asymptote.According to Smith & van Belle (1984) calculations using Jackknife as estimator, we expect 101 species to occur in the region, meaning we reached 81% of the total estimated diversity.

New records
Excluding the single undescribed species, we found 19 new records for the Rio Grande do Sul State; some of these species we considered rare and some seem to be well distributed in South America.Here we present some habitat preferences and ecological aspects of these species, based on the literature found and on our field observations.3.1 -Mnesarete lencionii Garrison, 2006 (Calopterygidae) Rare, at least for the latitude where our survey took place; this species is thought to be distributed all over Southeastern Brazil (Garrison 2006).The species resembles the congeneric M. pruinosa (Hagen in Selys, 1853), differing from it by the presence of a dark pseudopterostigma.Only one male was captured, it was found in a small fast flowing rivulet used for drainage of rice fields.At the same place we also found other calopterygids such as Hetaerina rosea Selys, 1853, which at the time of sampling, was abundant in the area.This species of Mnesarete was recorded from Argentina and Paraguay (Garrison 2006), and from Brazil from MG (Vilela et al. 2016) and SP (Garrison 2006).

-Argia lilacina Selys, 1865 (Coenagrionidae)
An inconspicuous coenagrionid due to its diminutive size (less than 30 mm), whose females shows pale brownish to green coloration and males are a bit more colored, varying from grayish blue to black.This species was quite common and we collected more than 80 specimens.It was mostly found in small streams flowing in open/field areas.According to Takiya et al. (2016), this species was previously recorded for Bolivia, Paraguay, and Argentina, and in Brazil for CE, TO, MT, GO, MG, MS, ES, SP and RJ.

-Minagrion waltheri Selys, 1876 (Coenagrionidae)
This Minagrion species was considered a rare species in our dataset, it occurred in small numbers along the margins of lentic waters, such as swamps and marshes (Garrison et al. 2010).It occurs in areas dominated by cattails (Typha), preferring slow moving clear water streams (Santos 1956(Santos , 1965)).Our specimens occurred at only one sampling site, which had the same environment as that provided by Santos (1956Santos ( , 1965)).The clean water seemed to pertain to an underground flow giving it zero or almost zero turbidity.The previously known records for this species are from MG (Bedê et al. 2015) and SP (Selys 1876; Santos 1965).We thus expand its distribution more than 1,200 km southwards.The species may well occur in between these two regions, but there are no records, probably due to lack of sampling.

-Oxyagrion rubidum (Rambur, 1842) (Coenagrionidae)
This species was locally common, alongside with O. terminale Selys, 1876, and difficult to identify in the field due to the resemblance of the two species.The environment where it was found was characterized by dense marginal vegetation on rivers, streams and ponds.This species is known from Uruguay (von Ellenrieder et al. 2009), Paraguay (Heckman 2010), Argentina and Chile (Muzón et al. 2014).From Brazil there is only one record in SP (Costa et al. 2000).Belle, 1982 (Gomphidae) This is a small gomphid, pale colored which makes it inconspicuous in the forest and fields.It is known by its agile and rapid flight that makes it difficult to collect (Belle 1982).This genus is easily identified by its unique characters: two latero-dorsal hooks on the male S10 (Garrison et al. 2006).We consider this species as poorly known since the only records found are the original description from Argentina and MG by Belle (1982).Our unique specimen (male) was caught in a rocky fast flowing stream with sandy margins and covered by low vegetation.

-Brechmorhoga nubecula Rambur, 1842 (Libellulidae)
This species was considered rare to our sampling efforts, since it was found on only one occasion in a fast flowing stream, with a shaded marginal zone.It could be easily confused with some species belonging to the Macrothemis genus.According to Kompier (2015) it can be overlooked due to its secretive habitats, which was proven true by our sampling experience.According to Heckman (2006) this species occurs in almost all southern American countries, and in Brazil there are records from CE (Takyia et al. 2016), RJ (Assis et al. 2004, Kompier 2015), MG (Souza et al. 2013, Bedê et al. 2015) and SP (Costa et al. 2000).

-Dasythemis venosa Burmeister, 1839 (Libellulidae)
This forest species was found only in well preserved environments, such as small forest fragments, in shaded areas or natural clearings (at fallen trees), perching on dry twigs above the water.Records of this species are from Argentina and Paraguay (Heckman 2006) and from Brazil there are records from SP (Costa et al. 2000) and MG (Souza et al. 2013).

-Diastatops obscura (Fabricius, 1775) (Libellulidae)
A conspicuous species that has dark colored wings and butterfly-like flying style.Our specimens were found in rich marginal vegetation of streams and lakes.The species can be easily confused with its congener Diastatops intensa Montgomery, 1940, which also occurred at the same localities.This species is known from many countries in South America, including Paraguay and Argentina (Heckman 2006); from Brazil there are records from, MG (Bedê et al. 2015, Vilela et al. 2016), MS (Dalzochio et al. 2011), MT (Calvão et al. 2014), RJ (Kompier 2015) and SP (Costa et al. 2000).The species probably occurs in all Brazilian states, but was not previously detected in RS due to lack of sampling.

-Dythemis nigra Martin, 1897 (Libellulidae)
At our sampling sites this species seemed to be rare, found on only one location, a small slow-flowing clear water stream.Our specimen was caught perching on twigs in a shaded marginal zone close to the water.The distribution records for this species, according to Takiya et al. (2016), are from Mexico, Panama, Trinindad and Tobago, Colombia, Venezuela, Guyana, Suriname, French Guiana, Ecuador, Peru, Paraguay, Argentina and Brazil: AM, BA, CE, ES, GO, MG, MS, MT, PA, PE, RJ, SC and SP.

-Erythemis vesiculosa Fabricius, 1775 (Libellulidae)
A very conspicuous species, characterized by its flying habits when it is hovering at low height above the water surface.Numerous specimens were observed, mostly at the lakes, and male-to-male territorial disputes and fight behavior was observed on several occasions.This species also has a wide distribution since there are records from many countries in South America.In Brazil the records are from several states: MG (Bedê et al. 2015), MS (Dalzochio et al. 2011), MT (Calvão et al. 2014), RJ (Assis et al. 2004, Kompier 2015) and SP (Costa et al. 2000).Ris, 1911 (Libellulidae) This is a tiny libellulid species whose males have bright yellowish colors that make the identification easy in the field.In our survey it occurred only on two locations, which were slow flowing water areas fed by small streams, corroborating the information provided by Costa et al. (2001) describing the larvae of the species.It is known to occur in Brazil (Pirassununga, SP), Paraguay and Argentina (Jurzitza 1981, Costa et al. 2001, Garrison et al. 2006).

-Gynothemis venipunctata Calvert, 1909 (Libellulidae)
A species which is easily identifiable in the field by the yellowish or amber spots on the male wing bases.Our six specimens were caught flying in a similar motion to that of Macrothemis, at about 1.5 m height, above open field areas, corroborating the observations made by Garrison (1983).Also, some were seen at a distance, flying in swarm-like formations consisting of some 5 to 10 specimens close to tree tops of about 10 m height.The species is known from Venezuela (De Marmels 1983) and in Brazil from MS (Costa et al. 1998), RJ (Kompier 2015) and SP (Costa et al. 2000).

-Idiataphe longipes Hagen, 1861 (Libellulidae)
We considered this species as common only in the peak of the summer season (Dec -Feb), since it was found in large numbers, usually perching on dry twigs along the water's edge.It seems to prefer lakes with diverse marginal or aquatic vegetation.There are records from several countries of South America: Colombia, Peru, Venezuela, Paraguay and Guyana.From Brazil the records are from the following states: SP (Costa et al. 2000), RJ (Kompier 2015), ES (Heckman 2006) and MG (Bedê et al. 2015).Ris, 1909 (Libellulidae) Most of our 14 specimens were caught at temporary waters or small creeks with sandy and rocky bottom.Its flight behavior resembles in some ways that of Gynothemis venipunctata; a slow and fragile style, very different form the great majority of dragonflies.Also, opposite to most of its congeners, M. heteronycha was found mostly in open areas instead of forest with closed canopy.According to the literature, it occurs in Paraguay, Argentina (Ris 1913, Garrison & von Ellenrieder 2006) and Brazil: SP, RJ, MG, MS, ES and SC (Costa et al. 2000, Dalzochio et al. 2011).Calvert, 1909 (Libellulidae) A very interesting and unexpected finding.Our specimens were caught in tandem along a flooding area near to a river sand bank (Figure 2b).At first sight they were thought to be gomphids, due to the long abdomen (big overall size for a Macrothemis species) and general appearance.It is a rare species described from Brazil, known to occur in the state of SE which Biota Neotrop., 17(4): e20170374, 2017 http://dx.doi.org/10.1590/1676-0611-BN-2017-0374http://www.scielo.br/bn is the species type locality (Calvert 1909) and recently registered in CE (Nobre & Carvalho 2014).Here the distribution of the species is expanded southwards about 4,000 km.

-Micrathyria spuria Selys, 1900 (Libellulidae)
A common species, easily identified by the abdominal markings and the dorsally whitish cerci.We found this species mostly in lakes and swamps with abundant marginal vegetation.It showed the typical Micrathyria behavior of perching on tips of emergent vegetation or dry twigs.The females we caught were found away from the water bodies, perching and foraging around trees and bushes.The species is known to occur in Venezuela (De Marmels 1983), Paraguay and Argentina (von Ellenrieder 2009) and Brazil: MG, MS, PR, RJ and SP (Costa et al. 2000(Costa et al. , 2002)).Calvert, 1909 (Libellulidae) Most of our specimens were found in the same localities as M. heteronycha; temporary waters or small perennial rocky creeks.Some of them were found away from the water.This species has records from Panama, Colombia, Peru, Venezuela, French Guiana, Guyana, Surinam, Bolivia, Paraguay, and from Brazil it has been recorded in BA, ES, MG and RJ (Costa et al. 2000) and AM, CE, MS, PA and PI (Takiya et al. 2016).

-Orthemis attenuata Erichson, 1848 (Libellulidae)
The males resemble a bit the darker species of the genus Erythemis, while the females have a typical and unique color scheme: dark brown with yellowish stripes.Most of our specimens were caught in a temporary river flood pool, which was muddy at the time of our sampling efforts, in the spring season.Strong male-to-female harassment was observed, as well as male-to-male territorial disputes.There are records from several countries in South America (von Ellenrieder 2012); in Brazil it is known to occur in PA (Pinto & Carvalho 2009), RJ (Kompier 2015), BA, ES and MT (von Ellenrieder 2012).Hagen, 1867 (Libellulidae) This species is known by its crepuscular habits and erratic flight, usually found over marshes hunting mosquitoes (Paulson 2001).Our specimens were caught during the first minutes of our sampling efforts early in the morning (09:00 am), flying at irregular intervals in shaded areas which made them difficult to see and to capture with insect nets.This species was only found in the flooded areas, near to the Ibicuí River.In Brazil the records are known from the following states: MS (Costa et al. 1998), MT (Juen et al. 2014), RJ (Costa et al. 2002) and SP (Costa et al. 2000).

Discussion
Conservation strategies depend basically from information about diversity distribution, biogeography, population and community ecology.Therefore, inventories play a key role for the development of such measures.In this survey, we increased the knowledge on the Odonata fauna of Brazil's Pampa biome.We found 82 species, reflecting the diversity of our sampling sites; including several types of aquatic systems, most of them located in man-influenced areas, mostly by agriculture.Of these species, no less than 19 were new to the state and one new species not described in this paper.Naturally the number of species in the area will increase by including more sample sites and resampling ours, but never-the-less we already can see a highly diverse fauna in the region.We registered a large number (46) of Libellulidae species, which could be the result of many widespread generalists occurring in the mosaic of open fields and agriculture areas with little riparian fragmented forest, a landscape which is known to favor the fast and agile flying dragonflies, supporting the findings of Machado (2001).As suggested by Corbet (1999), there are different responses from Anisoptera and Zygoptera to environmental conditions, since the latter are known as low range dispersers (Vieira & Cordero-Rivera 2015).In general, there are clear relations between environmental factors (biotic and abiotic) and species composition, these factors acting as determinants of presence and absence of some species due to ecological and physical restrictions (e.g., Paulson 2006, Juen et al. 2007).This is naturally an oversimplified division as Zygoptera is a taxonomical and not an ecological unit and contains both good (Flenner & Sahlén 2008) as well as weak (Lorenzo-Carballa et al. 2015) dispersers, but as an average assumption it is valid also when analysing large scale species patterns (Heiser & Schmitt 2013).We found many rare species in the study (singletons n = 20), all having a restricted occurrence.We consider the following possible explanations: first, we found a high number of species of Coenagrionidae (20), some of which can mirror good ecological conditions even in a highly fragmented region, acting then as bioindicators (Clausnitzer 2003, Suhling et al. 2006, Samways & Sharrat 2010, Renner et al. 2016a).Following this idea, in the open grassland with longer dispersal distances in the open, more exposed surroundings between suitable habitats (Juen et al. 2007), fewer of the smaller bodied species have the possibility to occur.This is due to some genera having ecological restrictions related to aquatic vegetation (i.e.plant diversity) and water quality (e.g., Argia, Homeoura, Oxyagrion) as stated by Garrison et al. (2010).Second, in well preserved areas or large forested areas, higher number of Zygopterans such as Heteragrionidae, Calopterygidae and again coenagrionids are expected to be found, many of which have specific environmental restrictions and ecological needs, mostly regarding to diversity of plants and vegetal structures in the riparian areas (Juen et al. 2014, Carvalho et al. 2013).This fact can explain the occurrence of Minagrion waltheri (Coenagrionidae) in only one locality in SA, which is a well-preserved area that keeps its natural features, as well crystal clear water and high diversity of aquatic/riparian vegetation (Figure 2a).These specialized species can function as powerful tools when distinguishing priority areas for preservation, as many of such restricted species have been proposed to be good indicators of environmental quality (Clausnitzer 2003, Sahlén 2006, Koch et al. 2014).
When compared to other studies from the Neotropics (De Marco et al. 2014, Monteiro et al. 2013) our collection efficiency captured only 81% of the expected diversity, tending to reach its asymptote, implying that the actual number of species in the region could be as high as 101 (cf., Figure 3).We have a relatively low number of sampling sites and, hence, cannot see the full picture of the odonate diversity in the Pampa biome, given its dimensions.However, we tried to include every kind of aquatic environment which could shelter any species of Odonata, from small puddles of temporary water to big rivers, lakes and perennial bogs; more than one of each.To our surprise, we found several interesting habitats in which we recorded most of the species that we considered rare.These habitats could be starting point for future conservation actions to be taken for diversity maintenance in the area.Special focus should therefore be given to the environments whose original features are still preserved, such as small rivulets flowing in ravines, swampy and well vegetated areas supplied by underground waters and forest remnants with waterbodies (Figures 2a, b, c).
Future studies should, if possible, include a bigger selection of environments from more municipalities to obtain a more complete sample of the communities.In addition, more frequent sampling during the seasons might also contribute to the inventory, given that some species are related to specific conditions to be active, e.g.crepuscular flight, which occur among many Aeshnidae that are active only in the twilight.There are also species that are active during rainy conditions (Garrison 1989, Wasscher 1990, Corbet 1999), some of which might occur in the Pampa region.
The knowledge achieved in other biomes of Brazil is much deeper than that from the Pampa.But this biome is considered a highly biodiverse Biota Neotrop., 17(4): e20170374, 2017 http://www.scielo.br/bnhttp://dx.doi.org/10.1590/1676-0611-BN-2017-0374system (Overbeck et al. 2009), and in this context, species surveys can supply valuable initial information for the actions needed to preserve and restore these environments.With this survey we tried to add further information and improve the knowledge of the group Odonata from the Pampa biome of southern Brazil, showing that even under such fragmented and altered conditions diversity is still high; this fact acknowledges that future measurements of conservation and restoration are needed.

FIGURE 1 :
FIGURE 1: Map of Brazil and the Pampa biome domain in Rio Grande do Sul, rectangular insets from the two sampling regions (1 and 2), red dots marking the sampling sites.

FIGURE 2 :
FIGURE 2: Some of the sampling sites which were remarkable by good environmental conditions: (a) Swampy area, with slowly flowing clear water in SF; (b) Flooding area near to Ibicuí River, MV; (c) Swamp close to a forested edge zone in SB.

FIGURE 3 :
FIGURE 3: Rarefaction curve showing the efficiency of our sampling, stabilization expected to be reached if we continue our efforts.S(est): estimated species number; 95% CI: confidence interval, upper and lower.

TABLE 2 :
Number of Odonata species collected per family in Rio Grande do Sul within the Pampa biome.