First characterization of a taxonomically well‐resolved trophic network composed by host plants and gall midges (Diptera: Cecidomyiidae) in the Neotropical region

In the present study we described the structure of a trophic network composed by gall-midge species (Diptera: Cecidomyiidae) and their host plants in the Restinga of Barra de Maricá (Maricá, Rio de Janeiro, Brazil). Species data were retrieved from literature and different topological descriptors (links per species, connectance, and modularity of interactions) were used. All gall-midge species were monophages, with connectance of 2.8% of the 2,016 possible interactions. The network of host plants and gall midges had low number of links per species and high modularity, which indicates high specificity and specialization of plant-galling interactions in the area. This is the first characterization of a trophic network with good taxonomic resolution for the Neotropical gall midges.


INTRODUCTION
Cecidomyiidae (Diptera) is the most diverse group of gall-inducing insects in the world, with more than 6,500 described species (Gagné & Jaschhof, 2017). Nevertheless, the Neotropical fauna comprises only about 8% of the known species, a very low percentage, considering that in this region there are some megadiverse countries (Fernandes & Santos, 2014). The Brazilian fauna includes about 50% of the described Neotropical species (Gagné, 1994;Gagné & Jaschhof, 2017), most of them from the Atlantic Forest. This situation reflects the scarcity of taxonomical studies about gall midges (Araújo et al., 2019a), which makes it difficult to understand biological and ecological processes involving these insects and their host plants.
In the present study, we describe for the first time a taxonomically well-resolved network composed by host plants and gall midges at the Restinga of Barra de Maricá, municipality of Maricá, State of Rio de Janeiro, Southeastern Brazil. Restinga or coastal shrub zone is one of the most endangered vegetal physiognomy of the Brazilian Atlantic Forest due to anthropic action . For explore the network structure we used different topological descriptors (links per species, connectance, modularity, and robustness of interactions) commonly indicated to describe the architecture of binary bipartite networks (review in Dormann et al., 2009).

Data compilation
Maia studied the Restinga of Barra de Maricá for several years, from 1992 to 2011, during which this author and collaborators recorded a great amount of insect galls and their host plants, and described many gall midge species. In the present study, we compiled these data and arranged them in a database containing the gall-midge species and their host plants in order to building a list of plant-galling interactions (Table 1). All data used were previously published (Appendix 1). Only plants and gall midges identified at specific level were used in the compilation. All botanical names were updated using the database Flora do Brasil (2020) and nomenclature of gall midges was verified using Gagné & Jaschhof (2017).
The Restinga of Barra de Maricá is located in the municipality of Maricá, Rio de Janeiro, Brazil (22°52′-22°54′S and 42°48′-42°54′W). This restinga has 8 km of extension and 844.16 ha of total area . The climate of the Maricá region is classified as hot tropical, super humid, with a dry season (Alvares et al., 2013). The region has an average annual temperature of 23.2℃ and the average annual precipitation is 1,230.8 mm . It comprises several microenvironment and vegetal formations, which characterize this complex ecosystem with a very diverse flora (Oliveira & Silva, 1989).

Data analyses
The structure of interactions between host plants and gall midges was evaluated using three topological descriptors: connectance, number of links per species, modularity, and robustness. Connectance (C) is the proportion of observed interactions compared to the possible interactions in the network ranging of 0 (totally specialized) to 1 (totally connected) (Dormann et al., 2009). Number of links per species (L) is the sum of links divided by number of species. Modularity (M) is a measure of the occurrence of densely connected modules within the network (Dormann et al., 2009), ranging between 0 (when the network is not modular) and 1 (when a network is very modular). Robustness (R) is a measure of the resistance level of the network to coextinctions (Dormann et al., 2009), measured as the exponent of the curve generated by the proportion of remaining species of gall midges in function of the proportion of primary random extinctions of host plant species. The significance of observed values of each network descriptor was calculated using null models (Dormann et al., 2009). All network analyses were performed using bipartite package (Dormann et al., 2008) in the R software version 3.6.1 (R Core Team, 2020).

RESULTS
In total, we recorded 56 species of 27 genera of gall midges and 36 species of 32 genera and 24 families of host plants (Table 1). The richest gall-midge genera were Asphondylia Loew, 1850 and Dasineura Rondani, 1840, each with six species. All species of gall midges recorded are gall-inducers, except Clinodiplosis floricola Novo-Guedes & Maia, 2008 that is a free-living herbivore. All gall-midge species were monophagous, inducing galls on only one plant species (Table 1).
The host plants Guapira opposita (Vell.) Reitz (Nyctaginaceae), Eugenia astringens Cambess. and Eugenia uniflora L. sheltered more gall midges, with seven, three and three species, respectively (Table 1). The most important host family was Myrtaceae with six plant species and 13 gall-midge species. The genus Eugenia L. (Myrtaceae) stands out for having three species of host plants and eight species of gall midges.

DISCUSSION
We found a high specialization of interactions between host plants and gall midges in the present study, confirming previous studies (Carneiro et al., 2009;Araújo et al., 2019b). All species of gall midges were recorded on a single host plant species (i.e., monophagous species). The percentage of specialist species recorded in our study is higher than found by Carneiro et al. (2009) which recorded 92% of monophagous gall midges for Brazil. In other study, Araújo et al. (2019b) recorded that 79% of gall-midge species were monophages in the Slovakia. These results suggest a lower specialization of gall midges in temperate environments when compared to the Neotropical region, which have more specific gallers in their host plants, as observed for the Restinga of Barra de Maricá. It is important to note that there are differences in the duration and frequency of sampling between the present study and the others mentioned. Our network was sampled for a much longer period of time than any previously published study (almost two decades), which enhances the sampling of rarer interactions, and consequently increases the specialization of the network. However, this fact reinforces the relevance of the observed patterns, because even with such a long sampling, only species-specific plant-galling interactions were registered.
The structure of the network formed by the gall midges and their host plants proved to be highly specialized. The connectance observed in the present study (2.8%) was low as compared to other plant-phytophagous networks (review in Araújo et al., 2015). However, comparing with other networks of galling arthropods, the value observed here was higher than observed by Araújo et al. (2017) (2.3%) but lower to that observed by Araújo & Kollar (2019) (5.4%), which sampled networks in temperate forests. Our plant-galling network also showed a low number of links per species (0.608) and a high modularity (0.958), corroborating the pattern observed in other galling networks (Araújo et al., 2017;Araújo & Kollar, 2019). Our results provide evidence that supports the high specificity and specialization of plant-galling interactions (Araújo et al., 2019b). Furthermore, our results show that the network of host plants and gall midges is few robust to coextinctions. This result is due to the high specificity of the plant-galling interactions, since each species of plant lost, represents the loss of at least one species of gall midge (Araújo et al., 2017).
Main genera of gall midges recorded in our study were Dasineura and Asphondylia. The genus Dasineura is the richest in Cecidomyiidae family with 476 described species (Gagné & Jaschhof, 2017). For the Neotropical region, only 39 species in this genus are described, 10 species from Brazil (Maia & Silva, 2013). In the present study, Dasineura induced galls mainly on Myrtaceae (but also on Chrysobalanaceae, Erythroxylaceae, and Malpighiaceae). The genus Asphondylia is cosmopolitan and includes 272 gall-inducing species in the world and 100 in the Neotropical region (Gagné & Jaschhof, 2017 These two genera are characterized by highly specialized galling species, most of which are monophages (Carneiro et al., 2009), which contributed to the low connectivity of the plant-galling network.
Myrtaceae was the plant family that sheltered more gall-midge species (13 species) and presented important host plants (in terms of the number of interactions), such as Eugenia astringens, and Eugenia uniflora (three gall midge species each). Myrtaceae are one of the most diverse families of Angiospermae, with thousands of species, being important in several Neotropical ecosystems (Wilson et al., 2001). Eugenia, with about 1,000 species, is one of the most diverse genera of Myrtaceae, distributed mainly in the Central and South Americas (Merwe et al., 2004), being the genus with the greatest number of species at restingas in the State of Rio de Janeiro (Araújo & Henriques, 1984). The great number of gall midges associated to Myrtaceae plants contributed to the high modularity of the plant-galling network, since the family forms a compartment of interactions within the network. Similarly, Guapira opposita, which was the host plant species that sheltered more gall-midge species with seven species, it also contributed to the formation of a module within the network. This species is one of the most expressive plants in Quaternary coastal plains of the Atlantic Coast of Southern Brazil, very frequent at restingas, where it is widespread and one of the dominant species (Reitz, 1970).
The frequency and abundance of these plant taxa can explain the great diversity of gall-inducers associated with them. In fact, Myrtaceae have been cited in several gall inventories at restinga areas as super host family, not only in the State of Rio Janeiro, as Maricá and Carapebus (Maia, 2001), Grumari (Oliveira & Maia, 2005), Ilha da Marambaia (Rodrigues et al., 2014), Ilha Grande (Maia & Oliveira, 2010), Região dos Lagos (Carvalho-Fernandes et al., 2016), but also in Espírito-Santo (in Guarapari) (Bregonci et al., 2010), São Paulo (in Bertioga) , and Santa Catarina (in Babitonga and São Francisco do Sul) (Melo-Júnior et al., 2018;Arriola et al., 2015). The frequent presence of Myrtaceae in gall inventories at Brazilian restinga is an indication of the importance of this family for the structuring of plant-galling communities in these ecosystems. Similarly, Guapira opposita is cited as super host species in almost all inventories at restinga (review in Maia, 2013), except in Grumari and Guarapari. Recent evidence suggests that the presence of super host taxa can modify the structure of plant-galling networks in Neotropical environments, increasing the diversity and connectivity of interactions (Araújo et al., 2019c). In addition, the presence of super host species can impact the robustness of the network, because although it increases the robustness for random extinctions, the presence of closely connected species makes the network more vulnerable to directional attack (Iyer et al., 2013). These evidences suggest that Guapira opposita may have a great importance in structuring plant-galling networks in restingas, but more data are needed to measure its real role.
There is a large Linean gap in the knowledge of Neotropical cecidomyiids (Araújo et al., 2019a), which is one of the main limitations for the advancement of studies on the biology and ecology of gall-midge interactions with other species. In the present study, by using a taxonomically well-defined trophic assemblage, we elucidated for the first time the structure of a network involving host plants and gall midges in the Neotropical region. Our results show highly specialized patterns both for the interactions of gall midges with their plants. The low connectance and high modularity observed for plant-galling interactions indicates a high level of ecological and phylogenetic restrictions for the structuring of interactions within the network, which demonstrates that eventual losses of species or interactions can be hardly substituted. Thus, this high level of specificity reinforces the important of conserving this so threatened ecosystem, as each restinga area has a peculiar flora and consequently a unique assemblage of host plants and gall-midge species.

APPENDIX S1
List of published data compiled in the present study.