Diversity of Ophioniformes wasps (Hymenoptera: Ichneumonidae) in a Central-West Brazilian Savanna area

This study aimed to assess the diversity, abundance and seasonal dynamics of Ophioniformes wasps collected throughout a year, as recorded in Malaise traps set at the Reserva Ecológica da Universidade Estadual do Mato Grosso do Sul, a Brazilian Savanna area in Aquidauana city, MS. A total of 621 specimens of Ophioniformes belonging to 11 subfamilies, 30 genera and 106 species were sampled. Despite the impressive number of species found, in a pattern of a few relatively abundant species and many rare ones, our data estimate even higher richness, probably between 125 to 165 species in the studied area. The genera Diadegma, Nonnus, Diradops, Meniscomorpha, Syzeuctus, Ophiopterus and Thyreodon are recorded for the first time in Mato Grosso do Sul State. Among the climatic variables evaluated, mean temperature and relative humidity were positively and significantly correlated with richness and abundance. Our data highlights the huge unknown species richness of these parasitoids in Brazil and the need of taxonomic studies efforts in the future.

Members of this group for which the host biology is known are all koionbionts and all except Tryphoninae (the sister group to all other subfamilies) are endoparasitoids . They parasitize larvae and pupae of a broad array of holometabolous insect orders, most commonly Lepidoptera, but also Hymenoptera, Coleoptera (Quicke, 2015 and references therein) and less commonly Raphidioptera (Spöck, 2002).
In Brazil, there are 368 described species of recorded Ophioniformes (Yu et al., 2016;Fernandes et al., 2020a) from almost all 18 subfamilies, except for Hybrizontinae, Sisyrostolinae, Stilbopinae and Tatogastrinae. Surveys of ichneumonid fauna including this clade, with identification of at least subfamily level, were conducted predominantly in the South (Kumagai & Graf, 2000;Kumagai & Graf, 2002) and Southeastern regions of Brazil (Guerra & Penteado-Dias, 2002;Kumagai, 2002;Sandonato et al., 2010;Comério et al., 2012;Tanque et al., 2010). Over the last years, Darwin wasps faunistic surveys have been conducted in the Brazilian Central-West and Northeast regions (Aranda & Graciolli, 2015;Fernandes et al., 2019Fernandes et al., , 2020b. The Brazilian Savanna (known as Cerrado) is recognized as a crucial area for conservation due to its relatively high diversity and presence of ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 endemic species (Mittermeier et al., 2011). It occupies 1,983,017 km² of the Brazilian territory, extending over thirteen federation states, including most of Mato Grosso do Sul state (MS) (IBGE, 2019). Despite its significance for species conservation, deforestation rates in this biome were 2.5 times higher than in the Amazon rainforest between the years 2002 and 2011 (Strassburg et al., 2017). Fast increase of agribusiness and infrastructure development combined with badly enforced environmental laws are leading to a major biodiversity collapse of this biome (Colli et al., 2020). Improvement on the knowledge of Ichneumonidae fauna is helpful to enable taxonomic studies and reviews (Klopfstein et al., 2019), to use this information as a tool for environmental assessment (Mazón, 2016), and even to discover possible biological control agents (Townes, 1971;Gupta, 1991;Neto et al., 2004). So, this study aimed to assess the diversity, abundance and seasonal dynamics of Ophioniformes wasps collected throughout a year using Malaise traps in the Reserva Ecológica da Universidade Estadual do Mato Grosso do Sul, from a Brazilian Savanna area.

Study area
All Ichneumonid specimens here recorded were gathered by the project "Diptera from Mato Grosso, Mato Grosso do Sul and Rondônia states: diversity, systematics and distributional limits", which employed mass collecting using Malaise traps, from September/2011 to August/2012, completing a full year of continuously (day and night, approximately 26,300 hours) operated three Malaise traps. Each of these traps had 3.4 m² of catch area and contained flasks filled with ethanol 70% which were emptied every fortnight periods. The Malaise traps were placed at the Reserva Ecológica da Universidade Estadual do Mato Grosso do Sul (Aquidauana city, MS; Fig. 1A-C), each one in a collecting sites, with the following characteristics: Seasonal Deciduous Forest with closed vegetation (20°26′03.7″S, 55°39′20.8″W); Seasonal Deciduous Forest with open vegetation (20°25′59.0″S, 55°39′20.8″W); Riparian Forest (20°26′07.2″S, 55°39′32.8″W). The study area climate is classified according to the Köppen criteria as tropical Savanna climate (Aw): with mean monthly temperatures above 18℃, and a relatively dry winter, with less than 60 mm of precipitation in the driest month (Alvares et al., 2013).

Specimens treatment and data analysis
Specimens were sorted, dry mounted, labelled and deposited at the Museu de Zoologia da Universidade de São Paulo (MZSP) Hymenoptera collection. Individuals were identified in subfamilies sensu Bennett et al. (2019) and those belonging to the Ophioniformes were sepa-rated into genera. Further identification was restricted to morphologically similar individuals based on characters in Gauld (1991Gauld ( , 2000, Gauld et al. (1997Gauld et al. ( , 2002 and authors' experience within the group. Naming of described species was not possible because of limited knowledge of the Brazilian fauna. Henceforward, morphospecies are referred here to as species. To characterize the assemblage, we estimated richness (S), total (At) and relative (Ar) abundances, numbers of species represented by one and two individuals (singletons and doubletons) and number found in one and two samples (uniques and duplicates). The species accumulation curve was constructed using the Mao Tau method and the estimated richness was evaluated through the estimators Chao 1, Chao 2 (classic formula option for both), Jackknife 1, Jackknife 2, and Bootstrap with 500 randomizations. These analyses were performed using EstimateS 9.1.0 software (Colwell, 2013).
A rank-abundance curve on a logarithmic scale (log₁₀) was generated to evaluate dominance in the assemblage based on the abundance of species in all samples. The dominance indices of Simpson and Berger-Parker were calculated. The monthly number of collected species and individuals of Ophioniformes was correlated with meteorological data through the Coefficient of Pearson to search for possible relationships between the abiotic variables and the temporal distribution of species. The environmental data of mean temperature, relative humidity and precipitation were obtained from Instituto Nacional de Meteorologia (INMET). The data normality was assessed through the Shapiro-Wilk test and the abundance data were log transformed. These analyses were performed using PAST 4.02 software (Hammer et al., 2001).
High-resolution photographs were taken using a Leica M205C stereomicroscope attached to a Leica DFC 295 camera. Multiple photographs of the subject were taken at different focal stages and combined using multiple stack methods with Leica LAS (Leica Application Suite V3.6.0) or Helicon Focus 6.7.1 software.
The performance of species richness estimators varied, with Jackknife 2 estimating the greatest number of species (165.56) and Bootstrap being the most conservative, suggesting about 84% of the inventory of species completeness (Fig. 3, Table 3).    Among the climatic variables evaluated, relative humidity was the only variable negatively correlated with Ophioniformes abundance and richness (Table 4). The greatest richness also coincided with the period of higher abundance (Table 1 and Fig. 4) and the Pearson correlation test found a positive relation between richness and abundance variables (RP = 0.91269, p < 0.05).   2002; Guerra & Penteado-Dias, 2002;Kumagai & Graf, 2002;Tanque et al., 2010;Comerio et al., 2012;Fernandes et al., 2019). A relatively high number of Ophioniformes species were found in the present survey. Despite our efforts, rarefaction curves indicate an even greater actual richness, estimated between 125 to 165 species. The comparatively high number of singletons and uniques samples suggests the necessity of greater sampling efforts, as observed by Sääksjärvi et al. (2004) who recorded a relatively high local diversity of Pimplinae (Pimpliformes) in Amazonian Peru, but also represented by a relatively high number of singletons. Fernandes et al. (2017) pointed out the necessity of long-term sampling for the collection of rare specimens of the small family Sclerogibbidae. An interesting example can be observed in Supeleto et al. (2020): even after extensive collection trips, only six specimens of Acrosnemus Supeleto, Aguiar & Santos, 2020 (Ichneumonidae: Cryptinae) were sampled showing the importance of recurrent sampling.

DISCUSSION
Among the Ophioniformes subfamilies collected, Nesomesochorinae is for the first time recorded in a Savanna area in Center-West Brazil. Although Lycorininae has not collected in our study, Shimbori et al. (2017) recorded the subfamily in the municipality of Campo Grande, only 140 km distant from Aquidauana.
It is well known that temperature and humidity are important factors regulating parasitoid life (e.g., Gauld, 1987;Ouedraogo et al., 1996;Emana, 2007). Our figures support Aranda & Gracioli (2015) results that peak abundance in Hymenoptera in the Brazilian Savanna is from August to October (Table 2).
Anomaloninae is a subfamily currently with 25 species of nine genera recorded in Brazil (Fernandes et al., 2020a). Shimbori et al. (2017) recorded the presence of seven of the recorded Brazilian genera of this subfamily (except for Castrosion Bradshaw, 1997 andOphiopterus Brullé, 1846) in the state of Mato Grosso do Sul. The genus Ophiopterus (Fig. 6B) Fernandes et al., 2020a). O. coarctatus has been recorded in the Amazon (Morley, 1912), Guaratuba in its south region (Brullé, 1846) and in the state of Espírito Santo (Azevedo et al., 2015). The genus is recorded here for the first time in Central-West Brazil.
Five species of the cosmopolitan genus Anomalon Panzer, 1804 are recorded in Brazil (Yu et al., 2016;Fernandes et al., 2020a). Apart from the savanna this genus is also known to occur in the Amazon (Townes & Townes, 1966) and Caatinga biomes (Fernandes et al., 2019(Fernandes et al., , 2020b and in coconut palm and coffee crops Comério et al., 2012). Ophionellus Westwood, 1874 is a small new world genus (21 species) with three Brazilian species (Yu et al., 2016;Fernandes et al., 2020a). Besides the records for Mato Grosso do Sul state, this genus has been recorded in the south by Brullé (1846) and in soy crops attacking the noctuid moths Chrysodeixis includens (Walker, 1858) and Heliothis virescens (Fabricius, 1777) (Massarolli et al., 2018).
The cosmopolitan Banchinae subfamily has 28 described species in 13 genera reported in Brazil (Yu et al., 2016;Fernandes et al., 2020a). All the genera found in this study are first records for the Central-West region of Brazil. Thirty-five species of Diradops Townes, 1946 (Fig. 5C) are restricted to the New World, two of them recorded in Brazil: Diradops castanea (Brullé, 1846) and Diradops aculate (Brullé, 1846) both occurring in the South (Brullé, 1846). The genus was well represented in our survey, with four species most frequently collected in August. Little biological information can be found in the literature, but they are known to attack Notodontidae and Noctuidae moths (Surgeoner & Wallner, 1975;Kasparyan & Ruíz, 2007;Fernandes et al., 2010).
The subfamily Campopleginae stands-out by the relatively high abundance and richness recorded. The incidence of this subfamily may be associated with the agricultural areas surrounding the study site. Campopleginae play an important role as a biocontrol agent against crop pests (e.g., Moraes et al., 1991;Azidah et al., 2000; Bortolotto et al., 2014). All genera identified in this study were also found occurring in organic crops in the State of São Paulo by Sandonato et al. (2010).
The genus Charops Holmgren, 1859 represented here by two species was recently reviewed by  for Brazil, with the description of seven species. Marconato et al. (2008) found this genus attacking geometrid moths in Erythroxylum microphyllum St.-Hilaire, a plant species typical of the Brazilian Savanna (Mendonça et al., 1998). As rare as Charops, Cryptophion Viereck, 1913 was represented by only one specimen collected in September. The works of Onody et al. (2013) and Onody & Penteado-Dias (2016) were important contributions for the taxonomy and biogeography of this genus, showing its presence in the Atlantic forest and Savanna in the Brazilian southeast. Gauld & Janzen (1994) observed specimens reared from the sphingid moth Erinnyis ello (Linnaeus, 1758) in Costa Rica. Besides Sphingidae, this genus is also known to attack saturniid moths (Gauld & Janzen, 1994).
A relatively high richness and abundance were observed for Dusona Cameron, 1901 in our survey. Kumagai & Graf (2000) observed four Dusona species occurring in rural and urban area in the South of Brazil. This genus has more than 400 described species worldwide and only four of them have been recorded in Brazil. The last Brazilian species was described in 1926 (Hancock, 1926).
Eiphosoma was the most common genus of this subfamily in our survey. Onody et al. (2012) reported fourteen Eiphosoma species occurring in organic crops in the state of São Paulo. Seven species of this genus were reported in a Savanna fragment situated in the same state (Melo & Penteado-Dias, 2009). This is a medium-sized genus with 56 valid nominal species restricted to the New World, with 23 species recorded in Brazil (Yu et al., 2016;Fernandes et al., 2020a). Shimbori et al. (2017) re-corded five species for the Mato Grosso do Sul state. Species of this genus are known to be endoparasitoids of Lepidoptera with host records of the families Crambidae, Noctuidae and Pyralidae in Brazil (Sauer, 1946;Lima, 1953).
Little is known about the genus Dimophora Förster, 1869 in Brazil. This small genus includes 19 described species, but only one from the Neotropical region: Dimophora daschi Gauld, 2000 described from Costa Rica (Yu et al., 2016). The presence of this genus was reported by Azevedo et al. (2015) in Espírito Santo state. We record this genus for the first time in the Brazilian Central-West region.
Pristomerus Curtis, 1836 is a cosmopolitan, relatively large genus comprising 140 valid nominal species (Yu et al., 2016). Pristomerus spinator (Fabricius, 1804) is the only species occurring in Brazil. Patel & Habib (1984) recorded this species attacking Spodoptera frugiperda (Smith, 1797) in corn crops in the Southeast region. Temelucha Förster, 1869 is also a cosmopolitan genus comprising 235 species with fourteen of them are known to occur in the Neotropics (Yu et al., 2016). Although no described species have been previously recorded in Brazil, the genus has been documented in different regions including the Northeast (Fernandes et al., 2019), Southeast (Azevedo et al., 2015) and for the first time hereby in Central-West region.
The subfamily Ctenopelmatinae was represented in our survey by only one specimen of the New World genus Physotarsus Townes, 1966 collected only in September. Six species are known to occur in Brazil (Zhaurova & Wharton, 2009). This genus has been recorded as parasitoid of Trochophora lobata (Erichson, 1848), a sawfly of the family Argidae in Costa Rica (Gauld et al., 1997).
Mesochorinae are currently classified in 10 genera Araujo, 2018) with 871 described species (Yu et al., 2016). From these, 700 species belong to the cosmopolitan genus Mesochorus (Fig. 5F) with 49 species recorded in Brazil (Araujo et al., 2019). Most of these species were described by Dasch in 1974 and are endemic to the Atlantic Forest, while only one is described for the Savanna biome (Araujo et al., 2019). Members of this genus are hyperparasitoids mostly of Ichneumonoidea (occasionally Diptera: Tachinidae) within Lepidoptera and sawfly larvae and also in immature Hemiptera (Quicke, 2015 and references therein). The six species we found are likely undescribed since the number of Mesochorus species is probably underestimated, as already pointed out by Araujo et al. (2019).  Santos, A.D. et al.: Ophioniformes wasps in a Brazilian Savanna Pap. Avulsos Zool., 2021;v.61: e20216145 9/13 Metopiinae is a medium-sized cosmopolitan subfamily with 862 described species worldwide (Yu et al., 2016). Fernandes et al. (2020a) registered nine genera and 29 species occurring in all regions of Brazil (apart from the Northeast) of Brazil. The genus Colpotrochia Holmgren, 1856 comprises 62 species with five Brazilian species. This genus was recorded parasitizing Spodoptera frugiperda (Smith, 1797) in Brazilian corn fields (Silva et al., 2012).
Exochus Gravenhorst, 1829 is a large genus comprising 281 cosmopolitan species (Yu et al., 2016). This genus was studied recently in Brazil by Melo et al. (2015), who described E. atlanticus from the Atlantic rainforest and provide new geographical records of E. ablatus Sithole, 2002 andE. izbus Gauld &Sithole, 2002 in Savanna and semideciduous forest in southeast region.
Wasps belonging to the New World genus Nonnus Cresson, 1874 (Fig. 5E) were the only representatives of the subfamily Nesomesochorinae in our survey. Its members are relatively large and conspicuous wasps with 20 described species (Wahl & Bennett, 2020). Nine species are recorded in the Brazilian states of Amazonas, Espírito Santo, Goiás, Mato Grosso, Rio de Janeiro, Rio Grande do Sul and São Paulo (Townes & Townes, 1966;Comério et al., 2012;Azevedo et al., 2015;Fernandes et al., 2020a) and for the first time here for Mato Grosso do Sul state. Host records are unknown for Nonnus, thus the biology of this group remains a mystery (Wahl & Bennett, 2020).
Ophioninae wasps are commonly collected in Brazil due to their large size and attraction to light (Burbutis & James, 1979). This cosmopolitan subfamily comprises 32 genera and more than one thousand species (Yu et al., 2016). Some species of this subfamily are parasitoids of important agricultural pests (like S. frugiperda), but they are not suitable for biocontrol release programmes (Quicke, 2015). Most species of this subfamily belong to Enicospilus Stephens, 1835 which comprises around 700 species (Yu et al., 2016) with 61 of them recorded in all Brazilian regions (Fernandes et al., 2020a). We record here for the first time the genus Thyreodon Brullé, 1846 (Fig. 5B) in the Central-West Brazilian region. This genus comprises 45 species, nine of them with Brazilian occurrence (Yu et al., 2016;Fernandes et al., 2020a).
Tersilochinae is a cosmopolitan medium-sized subfamily with more than 560 described species in 27 genera (Khalaim & Ruíz-Cancino, 2020). Fernandes et al. (2020a) registered three genera and 13 described species in the south and southeast regions of Brazil. Stethantyx Townes, 1971 is a Neartic and Neotropical genus with 47 species (Yu et al., 2016). Recently taxonomic contributions to this genus were made by Khalaim (2017) with the description of Stethantyx durrelli Khalaim, 2017, increasing the recorded species in Brazil to 11 (Fernandes et al., 2020a). Biological information regarding this genus is scarce but it seems to parasitize weevils (Parker et al., 1950).
The subfamily Tryphoninae is a cosmopolitan group with 54 genera and about 1,300 species (Bennett, 2015). They are koinobiont ectoparasitoids of Lepidoptera larvae and pre-pupae (Konishi, 2014). In our survey this subfamily is represented by several species of the genus Netelia Gray, 1860, a large genus comprising more than 330 species, (Bennett, 2015), of which only nine are known to occur in Brazil (Yu et al., 2016); the most recent description being N. oeceticola (Blanchard, 1941), but the total number of species present might be much higher (Bennett, pers. comm.) This genus was recorded as a parasitoid of the noctuid moth Mocis latipes (Guenée, 1852) in the Southeast region (Lourenção et al., 1982).
In summary, this study recorded for the first time the presence of the genera Diadegma (Campopleginae) and Nonnus (Nesomesochorinae) for the Mato Grosso do Sul state and the genera Diradops, Meniscomorpha, Syzeuctus (all Banchinae), Ophiopterus (Anomaloninae) and Thyreodon (Ophioninae) for the Central-West region. Many genera recorded here (e.g., Dusona, Diradops, Enicospilus, Netelia) are relatively little-studied in the Neotropical region and the number of undescribed species in these genera highlights the huge unstudied species richness of Ichneumonidae Ophioninae wasps in Brazil and the need to study and conserve this critically important part of the world's biodiversity.