Diversity of Tephritidae and Agromyzidae (Diptera: Brachycera) in Flower Heads of Asteraceae in the Chaco

The Chaco is an international biome, connecting four countries: Paraguay (230,000 km 2 ), Bolivia (90,000 km 2 ), Argentina (520,000 km 2 ), and Brazil (Mato Grosso do Sul state (MS), with around 9,000 km 2 and in the middle of South America. Brazilian Chaco is restricted to Porto Murtinho region, MS. The daisies (Asteraceae) with near 24,000 species worldwide is characterized by herbs and shrubs that coevolved with several taxa of endophagous insects: dipterans Agromyzidae, Ceciidomyidae and Tephritidae; Coleoptera (Apionidae), Hemiptera (Miridae), Lepidoptera (Blastobasidae, Gelechiidae, Pterophoridae, Pyralidae, and Tortricidae) and the parasitoids of this endophagous insects, which found in the daisies’s flower heads ideal conditions for food, breeding site and shelter. The Neotropical florivorous flies are the Agromyzinae (Agromyzidae), and Tephritinae (Tephritidae), which in their larval stage feed on Asteraceae inflorescences. To report the species of florivore flies, their host plants and parasitoids in flower heads of Asteraceae from the Brazilian Chaco, we sampled inflorescences of 25 species (± 500 flower heads/species) that were kept in containers to the emergence of the florivorous flies or their parasitoids sampled in the three phytophysiognomies. The adult insects after 48 hours of their emergence were fixed in 80% ethanol for later identification. A total 25 species of Asteraceae were evaluated in the Brazilian Chaco, being collected 17,000 flower heads. Nine tribes of two Asteraceae subfamilies were sampled, from which 15 species of florivorous flies were recovered. We found 5 genera with 9 of Tephritinae (Tephritidae), 6 species of Melanagromyza (Agromyzinae, Agromyzidae), and 104 parasitoids (Hymenoptera) of the florivorous flies.


Introduction
The florivorous flies are the most importante guild of insects feeding on daisis's flowers in Brazil. They belong to the subfamilies Agromyzinae (Agromyzidae), and Tephritinae (Tephritidae). Those flies, in their larval stage, feeds in Asteraceae inflorescences (flower heads).

The mainly aims of this research
1. To Report the species of florivore flies (FF), their host plants, and their parasitoids, in flower heads of Asteraceae from the Brazilian Chaco. 2. To quantify abundance, species richness and diversity of the FF in three phytophysiognomies in the Brazilian Chaco.

Materials and methods
Sampling was carried out in the municipality of Porto Murtinho-MS, Brazil, in three different phytophysiognomies of the Brazilian Chaco. Flower heads of Asteraceae were collected at: Eldorado Farm (21 ° 42′20.9 "S 57 ° 47'45. 6 The flower heads of all Asteraceae species found in the field were collected, preferably 200 in pre-anthesis per species. The flower heads were inserted in plastic bags, identifying the species of plant and the area. The collection carried out monthly and during from May 2017 to April 2018. Exsiccates from all Asteraceae evaluated were prepared.
The climate of the region in the Brazilian Chaco is considered tropical Aw by the Köppen classification, with hot and rainy summer and dry and milder winter. The rainfall varies between 1,100 mm and 1,800 mm. The vegetation in the Chaco is made up of shrubs, deciduous, microphiles and spinach. The soil is highly saline, with little water drainage, which is why in rainy season it generates temporary floods characteristic of the Pantanal.
The Asteraceae inflorescences collected were taken to the Laboratory of Systematic and Taxonomy of Tephritidae (LabTaxon), Universidade Federal da Grande Dourados (UFGD), where the flower heads were counted and placed in 500 ml plastic cups, with the juxtaposed openings forming a cage. The cups were attached with adhesive tape, forming a closed container that made it possible to contain and obtain species of florivorous flies and their parasitoids. After emergence of florivorous flies and/or their parasitoids, they remained alive for a period of 48 hours to acquire the chromatic pattern of the body and wings. They were subsequently conserved in 92% ethyl ethanol. After 20 days without any fly's emergence, inspections on the containers were stopped, and the remaining material discarded.
The Wonders of Diptera -Characteristics, Diversity, and Significance for the World's Ecosystems 4 The exsiccates of the sampled Asteraceae were sent for identification of the species by Professor Dr. Jimi Naoki Nakajima (Universidade Federal de Uberlândia), specialist in Asteraceae. The duplicates of the identified Asteraceae were deposited at the CGMS Herbarium of the Biodiversity Museum-FCBA/UFGD in Dourados-MS, as the specimens voucher.
Statistical analyzes: Infestation rates were calculated using the equation: Number of FF/Number Flower heads x 100, being N the number FF (= number of Florivorous flies) divided by the Number of Flower heads (total of Flower heads/Asteraceae species). Were analysed the diversity in each phytohysiognomy. The diversity index (Shannon-Weaner) (H), Species Richness Index (Margalef), (Alpha), and Uniformity or Equitability Index (E) were calculated.
A total 472 adults of florivorous flies from two families and 15 species (9 of Tephritidae and 6 of Agromyzidae) were recovered, being 163 agromyzids (Melanagromyza), and 309 tephritids from nine species were recovered from 15 species Asteraceae species. The Tephritid species belong to five genera: Cecidochares Bezzi 1910; Dictyotrypeta Hendel 1914; Tomoplagia Coquillett 1910; Trupanea Schrank 1795, and Xanthaciura Hendel 1914. We found nine morphospecies of Tephritinae. All specimens of Agromyzidae are grouped in a single genus: Melanagromyza Hendel 1920. The recovered parasitoids are (n = 104) Hymenoptera await identification by specialists. Herein, trophic interactions are reported between florivorous flies with 15 species of Asteraceae in the Brazilian Chaco ( Table 2).
Herein, for the first time we present the several associations of florivorous fly species with their Asteraceae (flower heads) host species in the Brazilian Chaco. The trophic interactions established here were the following: Chromolaena margaritensis was colonized by Xanthaciura sp.1; Aspilia elata by Dictyotrypeta sp.1, and Dictyotrypeta sp.  Table 2).
The highest rates of infestation by florivorous flies in Asteraceae in the Brazilian Chaco were reported in the following species: Acmella grisea (34%); Aspilia elata   (6) with Asteraceae. Trupanea was also the only Tephritidae genus associated with more than one tribe (three). The Asteraceae species from the Tageteae and Vernonieae tribes, each presented trophic associations with only one tephritid genus: Trupanea and Tomoplagia, respectively ( Table 2).
Praxelis clematidea was the host in which the higher abundance of parasitoids (38), and presented trophic interaction with only one specimen of Xanthaciura sp.1. In the species of the tribes Eupatorieae and Heliantheae, ocurred higher florivore fly species richness, being obtained three species from each host plant. In the Heliantheae species were registered greatest abundance of Agromyzidae (136 adults), from which four of the six registered species of all the reared agromizids. In the species of Eupatorieae there was obtained higher abundance of tephritines (185), and 64 parasitoids ( Table 3).
Characteristics of trophic interactions between florivorous flies and Asteraceae as a function of the Chaquean phytophysiognomies: Only the species Trupanea sp.1   Table 3.  None of the species of tephritids reported for WSS was specific to this phytophysiognomy ( Table 4).

Grassy-woody Steppe Savanna
The phytophysiognomy with highest species richness (S = 6), and abundance of Tephritinae (268) was GWSS. The highest abundance of parasitoids (50) was found in the phytophysionomy Park Steppe Savanna (PSS). PSS presented the highest diversity by the Shannon (H) index, despite it haven't the greatest species richness (S). Probably this is due to the fact that this index takes into account the homogeneity of species in the environment. In the Grassy-Wood Steppe Savana (GWSS) the highest value of diversity was registered for the Margalef index. This index consider the sample size, which can be explained by the high abundance of the florivore fly Cecidochares sp.1 in this phytophysiognomy (Tables 4 and 5).
Applying the rarefaction method in the samples ("curve of collector"), it was possible to obtain estimates of the species richness of Tephritinae from the Chaquenha community. It indicated that the samples were insufficient to reach the asymptote of curve of collector. This means that the sampling effort was not enough to detect all species of florivorous flies present in the Brazilian Chaco. Due to the mosaic of phytophysiognomies in this biome, the few sampling points proved to be insufficient, to estimate the total species richness of florivorous flies in the Brazilian Chaco, even doing repetitions in the four year seasons (Figure 1).  Table 5.

Discussion
The Brazilian Chaco presented a lower florivorous species richness than other neighboring Neotropical biomes already evaluated. Eighteen genera of Tephritinae (Tephritidae) and 80 species occurring in Brazil were reported by Prado [9]. In the Cerrado Biome, 12 genera are listed [10], and 10 genera have already been cataloged in the Atlantic Forest [11].
This research represents the first inventory of florivorous flies (Tephritidae and Agromyzidae) feeding in Asteraceae flower heads in the Chaco. Associations of Melanagromyza species (Agromyzidae) with Asteraceae species are reported for the Cerrado Biome [10], but there was yet no data for the Brazilian Chaco.
Due to the lack of keys for several taxa of Neotropical Tephritinae, many publications were unable to perform specific identification of florivorous flies. Species of Cecidochares, Dicyotrypeta, Trupanea and Xanthaciura are constantly only morphospecified.
In the Brazilian Chaco, the abundance of tephritids was higher than that of agromyzids, as well as the species richness (9 Tephritinea spp. against 6 probable species of Melanagromyza spp. (Agromyzinae, Agromyzidae). This pattern was also repeated in the Cerrado Biome [10]. According to several authors in the Neotropical Region, the most frequent and abundant species of florivore Diptera are the tephritines (Tephritidae). Herein, tephritines and agromyzines presented similar frequencies. Therefore, this pattern is not well understood and needs further studies in the Chaco biome to clarify its patterns of co-occurrence.
The Brazilian Chaco presented 16 trophic interactions between 15 species of florivorous flies and their Asteraceae species. In the Cerrado Biome, 49 species of Asteraceae are reported as hosts of florivore dipterans [10]. Herein, Chromolaena margaritensis hosted Xanthaciura sp.1 and Melanagromyza sp. 6. These are the first records for such associations worldwide. The flower heads of C. margaritensis has already been reported as a host for Cecidochares species in South Brazil [12].
Tomoplagia species were specific to the Vernonieae tribe. This relationship is already known [9]. In this research, Trupanea jonesi was the most generalist species, infesting two distinct asterace tribes: Heliantheae and Tageteae. The polyphagy of T. jonesi is well known, more than 100 associations with their host plants have been established [13].
The low diversity of florivorous flies recorded in the Brazilian Chaco can be explained by the low diversity of Asteraceae species there or by insufficient sampling effort. Probably, the species richness of florivorous flies is positively correlated with the species richness of sampled Asteraceae in the Chaco. Biomes richer in Asteraceae show a greater diversity of these trophic interactions. The Cerrado presents a high diversity of Asteraceae and associated species of Tephritinae, as has pointed out by other inventories [9,11,12].
Tomoplagia matzenbacheri and Tomoplagia minattai found in this research are new records for the state of Mato Groso do Sul (MS). The only previous work developed in the state of MS with florivorous flies was carried out by Uchoa, Wachter-Serapião & Roque [14] in the Cerrado, a fragment of the Atlantic Forest and an agroecosystem (orchard).
The Brazilian Chaco presented 25 species of Asteraceae, which apparently represents 92% of the species cataloged for this Biome [15]. However, many of the species recorded in this study do not appear in the floristic inventories of the Brazilian Chaco and vice-versa. New floristic studies must answer if there is a subsampling or if it is the result of the divergent interpretation among different authors about the characterization of phytophysiognomies truly Chaqueans.
Herein, Dimerostemma grazielae was host to florivorous flies in the Brazilian Chaco. This species of Asteraceae is considered rare in Brazil [16]. Calea Diversity of Tephritidae and Agromyzidae (Diptera: Brachycera) in Flower Heads of Asteraceae… DOI: http://dx.doi.org /10.5772/intechopen.96352 rupicola that was recorded on the APA trail (GWS) (=APA Waterfall Municipal Park = Parque Municipal Cachoeira do Apa) is considered endemic to the state of Mato Grosso do Sul, also reported in the Pantanal region [15]. C. rupicola has not been associated with florivorous flies in this research. In the state of Mato Grosso do Sul there are, at least, 32 endemic species of Asteraceae.
The Wood Steppe Savanna (WSS) presented pioneer plants such as Conyza bonariensis and Erechtites hieracifolius, indicating that environment as the most degraded. A conjecture of elements is related to the low diversity in this place: deconfiguration of the flora by human action, reducing the species richness of Asteraceae, and as well as, some punctual flooding during the summer period, caused by anthropic action.
In this study, the occurrence of Pectis odorata was reported in the Brazilian Chaco. This plant was common in flooded areas, mainly in the WSS. Pectis gardneri, presents adaptations for the common water deficit in the Brazilian Chaco, as pointed out by Antunes [17]. New floristic studies can answer if there are also adaptations to constant flooding, in species of Asteraceae and other taxa in the Brazilian Chaco.
Herein, we added new data about biodiversity of Tephritinae (Tephritidae) and Agromyzinae (Agromyzidae), presenting essential information to fight for environmental preservation, as well as contribute to the catalog of flora and fauna from Chaco. Human-caused phenomena, such as global warming and habitat destruction, have increasingly threatened the planet's biodiversity. Endemic species are the most susceptible to disappear, due to their ecological sensitivity. As there is a dependence on florivorous flies for their host Asteraceae, a relationship extremely species-specific, both taxa have a greater chance of co-extinction. The savannas are at high risk of species extinction, due to the loss of habitat being much greater than the conservation efforts by human community and political authorities.
Finally, in the Brazilian Chaco: Cecidochares sp.1 was the most abundant species of florivorous fly upon the Asteraceae flower heads. A species of this same genus, Cecidochares connexa (Tephritinae) has been successful employed for the biological control of an exotic Asteraceae in Ghana, West Africa (Chromolaena odorata). This plant species was introduced on Africa by decade of 1930 and to humid regions of tropical Asia, with negative impacts on agriculture and regional biodiversity [4].
Trupanea species were the most polyphagous in the Chaco. From the Heliantheae tribe of Asteraceae we recovered the highest abundance of Melanagromyza spp. (Agromyzinae, Agromyzidae). From the species Eupatorieae tribe were reared highest abundances of tephritids and their parasitoids (Hymenoptera). Park Steppe Savanna (PSS) was the phytophysiognomy among the three evaluated that presented the highest diversity (H′) of Tephritinae, probably, due to the heterogeneity of this phytophysiognomies in the Brazilian Chaco.

Conclusions and perspectives
1. In the Brazilian Chaco do occur at least 15 florivore fly species, nine of Tephritinae (Tephritidae), from five different genera, and six species of Melanagromyza (Agromyzinae, Agromyzidae); 2. The Grassy-Woody Savanna is the Chaquean phytophysiognomy that harbored higher species richness; 3. Some florivore fly (Tephritinae and Agromyzinae) species needs to better studied to employ in programs of biological control for invasive Asteraceae in the Neotropical Region.