Insects galls of Pantanal areas in the State of Mato Grosso do Sul , Brazil : characterization and occurrence

Two Pantanal areas were investigated in Corumbá, Mato Grosso do Sul. The field works were carried out in five trails, in August and December/2011 and April/2012. Seventy-six gall morphotypes were found in 52 host plants, 22 of them were identified at species level and 30 at genus. Fabaceae, Myrtaceae and Sapindaceae were the plant families with the greatest richness of gall. Leaves were the plant organ with the greatest gall richness. Six gall shapes were found – globoid, lenticular, fusiform, cylindrical, conical, and marginal roll. The three first were the most common. Five colors were observed – green, brown, red, yellow, and black. Green and brown predominated. Most were glabrous and one-chambered. These features have been indicated in several Brazilian biomes. Diptera (Cecidomyiidae) were the most frequent gallers. The associated fauna comprised parasitoids (Eulophidae, Braconidae, Eupelmidae, Eurytomidae, Platygastridae, and Pteromalidae), inquilines (Cecidomyiidae: Trotteria Kieffer, 1902, Hymenoptera: Tanaostigmoides Ashmead, 1896, Hemiptera, Lepidoptera and Thysanoptera) and predators (Pseudoscorpionida: Olpiolum medium Beier, 1931, Cecidomyiidae: Lestodiplosini and Hymenoptera: Formicidae). Parasitoids were the most frequent. Multiparatism was observed. Our data represent the first contribution to the knowledge of the associated fauna in the Pantanal.


INTRODUCTION
Galls are atypical plant developments that provide shelter, food and protection to the inducing organism or its offspring (Shorthouse et al. 2005).They are induced by mites and insects, each species being responsible for the development of a morphologically and structurally unique gall in the host plant (Dreger-Jauffret and Shorthouse 1992).The galls can occur in any plant organ: roots, fruits, stems, flowers, leaves and others.They are classified according to their structural complexity as simple (without reorganization of the vegetal tissue) and complex (with reorganization of the vegetal tissue), to their morphology as clavate, conical, cylindrical, fusiform, globoid, lenticular, rosette, marginal roll, etc., to the number of internal chambers as one-chambered (with a single chamber) or multichambered (with two or more chambers) and in relation to the presence or absence of trichomes as glabrous or pubescent.Insect galls are induced by six orders: Coleoptera,

1544
SHARLENE ASCENDINO and VALÉRIA C. MAIA Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Thysanoptera (Mani 1964).A rich and frequent fauna of arthropods can be found in association with the galls, acting as parasitoids, predators, inquilines and/or successors.
Gall inventories in Brazil refer mainly to the Atlantic Forest and Cerrado in the Southeast Region (Maia 2013, Coelho et al. 2009).For the other Brazilian biomes and regions there is a lack of inventories and taxonomic studies.Julião et al. (2002) published the first insect gall inventory of the Pantanal (focus of the present study), where 133 gall morphotypes in 75 plant species were recorded.More recently, Urso-Guimarães et al. ( 2017) published a wide inventory of insect galls of the Mato Grosso do Sul, where 32 gall morphotypes in 23 plant species were recorded in Pantanal areas.These two studies surveyed the same Pantanal sites, the "Base de Estudos do Pantanal" (BEP) and the São Bento farm, both situated in the municipality of Corumbá, State of Mato Grosso do Sul (MS), central-western Brazil.
The Pantanal biome is the largest floodplain in the world, flooded most of the time and with periodic flooding (Oliveira 2007).With an area of about 150,000 km², which occupies 1.8% of the Brazilian territory (IBGE 2004), it is considered the smallest Brazilian biome, influenced by three others: Amazonia, Cerrado, and Atlantic forest (MMA 2015).In Brazil, the Pantanal spreads over two States: Mato Grosso (MT) and Mato Grosso do Sul (MS).Although MS is smaller in territorial extent than MT, it comprises most of the Pantanal, 65.0% (IBGE 2004).
The present study aimed at increasing the knowledge of the diversity of insect galls in areas of the Pantanal, contributing to the taxonomic knowledge of galling insects and host plants in this biome and in Brazil, and adding new data on the associated fauna.
The vegetation was examined in search of galling insects and all plant organs were investigated, except subterranean roots.Fertile branches were collected from each host plant for the preparation of exsiccates.They were examined by botanists of Universidade Federal Rural da the use of stereoscopic microscope to obtain the immature stages and determine the number of internal chambers.
All insects were determined preliminarily in order and/or family and preserved in 70% alcohol.Cecidomyiidae (Diptera) were later mounted on microscope slides following the methodology outlined in Gagné 1994, the genera were identified using the keys of Gagné 1994 and the species based on their original descriptions.They were deposited in the Entomology collection of the Museu Nacional of Rio de Janeiro (MNRJ)/UFRJ.The other arthropods were identified in the lowest possible taxonomic level by specialists: Pseudoscorpionida by Dr. Mark Harvey (Western Australian Museum), Coleoptera by Dr. Paulo Magno (Museu Nacional, Amazônia (Pará) for species identification and the exsiccates were deposited in this Institution.Each gall morphotype was photographed in the field.Vegetable branches with galls were obtained and separated by host species and gall morphotype (to avoid mixing of material), labeled, numbered (the same number of the corresponding exsiccates) and were conditioned and transported in plastic bags to laboratory of Diptera of the Department of Entomology (Museu Nacional do Rio de Janeiro/ MNRJ-UFRJ).Part of the material was conditioned in closed transparent plastic pots, labeled and lined with humid paper.These pots were examined daily for emergence of adult insects (inducers, parasitoids, inquilines, predators and successors).The other part of the sample was dissected with  UFRJ) and Hymenoptera by Dra.Maria Antonieta Pereira de Azevedo (Museu Nacional, UFRJ) and the specimens were deposited in the scientific collections of the institutions of these specialists.
The other insects (Hemiptera and Lepidoptera) were identified only at order level.
The botanical names were updated using the Flora do Brasil website (Flora do Brasil 2020).The geographic distribution of the host plant species in Brazil, as well as data on endemism were retrieved from this same website.

RESULTS
Seventy-six gall morphotypes were found in 52 host plants, 22 of them were identified at species level and 30 at genus (Table I).All host plant species are native, among them, three are endemic in Brazil: Andira inermis (W.Wright) Kunth ex DC, Blanchetiodendron blanchetii (Benth.)Barneby  The mean number of gall morphotypes per plant morphospecies was 1.46.Fabaceae, Myrtaceae and Sapindaceae were the plant families with the greatest richness of gall, with 15, 13 and nine gall morphotypes, distributed in ten, eight and three plant species, respectively.
Concerning the gall external morphology, six shapes were found -globoid, lenticular, fusiform, cylindrical, conical, and marginal roll.The three first were the most common, with 38.1% (36.0% in the BEP and 43.7% in the SBF), 25.0% (26.2% in the BEP and 25.0% in the SBF), and 17.1% (19.6% in the BEP and 6.2%, respectively.All other shapes presented lower percentages (less than 10.0%): cylindrical (9.8% in the BEP, not found in the SBF), conical (8.1% in the BEP and 6.25% in the SBF), and marginal roll (not found in the BEP and 6.2% in the SBF).Five colors were observed -green, brown, reddish, yellow, and black.The green color predominated, with 43.4% (44.2% in the BEP and 37.5% in the SBF), followed by brown, with 35.5% (36.0%inthe BEP and 37.5% in the BEP, with one morphotype found in both localities).The other together represented less than 30.0% of the total: yellow (14.7% in the BEP and 25.0% in the SBF), reddish and black, both colors found only in the   The inducers of 40 gall morphotypes (52.6%) were determined (52.5% in the BEP and 56.3% in the SBF).They belong to three insect orders: Diptera (Cecidomyiidae) responsible for 35 gall morphotypes, Hemiptera (four) and Coleoptera (one).Among the determined gallers, most were Cecidomyiidae (44.2% in the BEP and 56.2% in the SBF).Hemipteran and coleopteran galls were found only in the BEP.Cecidomyiidae were represented by two supertribes: Lasiopteridi and Cecidomyiidi.Among the former, the genus Neolasioptera Felt, 1908 was identified (with five morphospecies in the BEP and a single one in the SBF).Among the latter, three tribes were recorded: Asphondyliini (Schizomyiina), Clinodiplosini, Lopesiini, all obtained in the BEP and represented by a single morphospecies.The other gall midges were identified at family level.
The associated fauna comprised parasitoids, inquilines, and predators (Table SII -Supplementary Material).The parasitoids were the most frequent.They were found in 22 gall morphotypes (28.9%) and included 21 morphospecies distributed in five families of Hymenoptera: Eulophidae (eight morphospecies), Braconidae (three morphospecies), Eupelmidae (three morphospecies), Eurytomidae (four morphospecies + a single pupa which could not be morphospecified), Platygastridae (one morphospecies), and Pteromalidae (two morphospecies).Among them, Eulophidae were the most frequent.Besides these records, other seven parasitized gall morphotypes were found, but as the Hymenoptera were obtained only as larva, they could not be separated in morphospecies.
The parasitoids were obtained mainly from leaf (86.9%), but also from stem galls (13.0%).If we consider the total number of leaf galls, 34.5% of them were parasitized.Similarly, if we consider the total number of stem galls, 20.0% were parasitized.Most parasitoids occurred in glabrous galls (82.6%), but some of them were obtained from pubescent galls (17.4%).The parasitized glabrous galls represented 30.1% of the total of the glabrous galls, and the parasitized pubescent galls represented 30.8% of the total of the pubescent galls.The parasitoids were found in four gall shapes, globoid, lenticular, fusiform, conical, being most frequent in globoid galls (43.5%).Ten plant families hosted parasitoids, among them, Fabaceae, Myrtaceae, Sapindaceae and Polygonaceae comprised together 65.2% of the records.
Comparing the two studied physiognomies, we can realize that the BEP comprised greater gall richness than the SBF.In the former, we found 61 gall morphotypes in 41 host plants, 20 of them were identified at species level and 21 at genus level; and the medium number of gall morphotypes per plant morphospecies was 1.4.In the latter, we found 16 morphotypes in 12 host plants, only two identified at species level and 10 at genus; and a similar medium number of gall morphotypes per plant morphospecies (1.3).A single gall morphotype occurred in both physiognomies on Calyptranthes sp.(Myrtaceae).In the BEP, Myrtaceae and Fabaceae were the superhost families, the former with eight galled species each.Myrtaceae hosted 12 gall morphotypes, whereas Fabaceae 11.In the SBF no plant family comprised more than four galls.Because of it, no superhost family was pointed out.
Data on galls morphology, inducers, associated fauna, sites and dates of collection are presented below.The data were organized in alphabetical order by family plant, genus and species.The number of gall morphotypes for each botanical taxon is given in brackets.The previous gall records in each plant species or genus in Brazil were indicated, as well as the new records.Tabebuia sp. ( n=1) Gall (Fig. 9): on leaf (abaxial surface), globoid, yellow, glabrous, solid.Galler: Hemiptera.Other    2002) reported 133 gall morphotypes on 75 plant species and pointed out Bignoniaceae, Fabaceae, Sapindaceae and Hippocrateaceae as the plant families with the greatest number of galled species and gall morphotypes.Urso-Guimarães et al. ( 2017) recorded 32 gall morphotypes on 23 plant species and pointed out Sapindaceae as the superhost plant family.In our survey, we found 76 gall morphotypes on 52 plant species and we indicated Fabaceae, Myrtaceae and Sapindaceae as the superhosts.These differences can be attributed to differences in the sample efforts and collection methodology.Julião et al. (2002) adopted the methodology of randomly walking and surveyed 27 sites, totaling 54 hours of collection.Urso-Guimarães et al. ( 2017) sampled two sites, where a 10m wide transect was staked out.Each site was sampled once, totaling two hours of sampling effort.
In our survey, we adopted the same methodology of Julião et al. (2002) and investigated three sites, totaling 72 hours.So our sample efforts were done for a longer period of time than that of Julião et al. ( 2002), but we investigated a smaller extension of area.These data reinforce the impact of the sample efforts in the gall richness.Nevertheless, Fabaceae and Sapindaceae were pointed out previously as an important host plant family.In fact, several Brazilian inventories in different biomes confirm the superhost status of Fabaceae (as for example: Santos et al. 2011-Caatinga, Luz et al. 2012transition area of Caatinga-Cerrado, Coelho et al. 2009-Cerrado, Maia et al. 2014 -Atlantic Forest).The high gall richness of Fabaceae can be explained by the plant richness hypothesis (Southwood 1960(Southwood , 1961)), which predicts that the richest plant taxa comprise the greatest insect gall richness.
Three endemic species were found as host plants and they comprised together six gall morphotypes.Considering that almost all insect gallers are monophagous and each species induces a gall that is anatomically and physiologically different from those induced by other related species (Shorthouse et al. 2005), there is a great probability that these gallers are endemic too.
The mean number of gall morphotypes per plant morphospecies in our study was 1.  Santos et al. 2011 -Caatinga, andCarneiro et al. 2009 -Cerrado), but the presence of superhost species increases this mean.In our inventory, Paullinia pinnata L. presented the highest number of gall morphotypes, five, and can be indicated as superhost species.This plant together with two others (Paullinia ingifolia Rich.ex Juss.and Andira inermis) helped to increase this mean (without them, the mean decreases to 1.31).
About 76.3% of the gall morphotypes were observed on leaves.The highest frequency of leaf galls is an already known world pattern.Most galls were globoid, green or brown, glabrous and onechambered.These features have been indicated in several Brazilian biomes as the most common, suggesting a widespread pattern.Nevertheless, Julião et al. (2002) pointed out the predominance of fusiform galls.Diptera (Cecidomyiidae) were the most frequent gallers.The same result was found by Julião et al. (2002), but differing from these authors, we did not find Hymenopteran and Lepidopteran galls.As Urso-Guimarães et al. 2017 identified the inducer of a single gall morphotype, we cannot compare the results.Cecidomyiidae are in the entire world the most frequent galling insects (Gagné and Jaschhof 2014).Among the cecidomyiids, we recorded the occurrence of Neolasioptera Felt 1908 and Trotteria.The former is known from the Pantanal by a single newly described species, Neolasioptera pantaneira Maia, 2017(Maia et al. 2017), the latter is reported for the first time in this biome.
In our study, the parasitoids were the major component of the associated fauna, as in other Brazilian inventories (Maia 2001, Maia and Fernandes 2004, Maia et al. 2008) (Fernandes et al. 1997, Maia andFernandes 2004), but not in the Pantanal.
The percentage of parasitized leaf galls was higher than that of stem galls.This result suggests that the galling larvae are more protected against parasitoids in the stem than in leaf galls.The tissue hardness can possibly explain it (Levin 1973, Southwood 1986), as stem galls are harder than leaf galls.According to Craig et al. 1990, the hardening of gall wal1s prevents penetration of the ovipositor of parasitoid wasps that would reach the larva and inhibits feeding on the gal1 tissue by herbivores.Another gall feature that can affect the rate of parasitism is the presence of trichomes.According to Richardson (1943), plant trichomes can act as structural defenses as they impair feeding and mobility.In the present work, we observed similar percentages of parasitized glabrous galls (30.1%) and parasitized pubescent galls (30.8%), so our results cannot confirm the trichomes' protective function.
Data on gall inquilines and predators are scarce in Brazil, as few studies with this focus have been published.Nevertheless, the taxonomic taxa observed in our study have already been recorded (Maia 1995, 2001, Maia et al. 2008), except Tanaostigmoides (Hymenoptera) which is recorded for the first time here.It is interesting to emphasize that records of pseudoscorpions in galls are known only in Brazil and Africa (Maia 2001, Maia and Souza 2013, Rodrigues et al. 2014, Maia 2013, Carvalho-Fernandes et al. 2012, Judson and Heurtault 1996).Although their association with galls on Fabaceae is previously known, this is the first record of pseudoscorpions in galls on Andira inermis (W.Wright) Kunth ex DC (Fabaceae).Besides, Olpiolum medium Beier, 1931 is reported for the first time in insect galls.
Callimicra sp.(Coleoptera, Buprestidae) was found in a lenticular gall on Ficus sp.(Moraceae).In the Neotropical Region, records of Buprestidae in galls are scarce, as well as in the entire world (Maia 2012, Bellamy andScholtz 1986).According to Hespenheide 2013, larvae of most species of Callimicra Deyrolle, 1864 are phytophagous and live on their host plants.Hanson (2009) recorded species of Callimicra in association with Ficus hirsuta Schott (Moraceae) and Ficus sp.We cannot affirm if this beetle is galler, but as it was the single dweller obtained from a gall without evidences of invasion, we believe that this can be the first record of Buprestidae galler in Brazil.
As in Julião et al. (2002) and Urso-Guimarães et al. ( 2017) there is no record of the associated fauna, our data represent the first contribution to the its knowledge.
The BEP comprised higher gall richness than the SBF, but the sample effort was greater in the former than in the latter.Nevertheless, some patterns were observed in both localities: leaves were the most galled plant organ; there was a predominance of globoid galls, brown and green colors, glabrous and one-chambered galls; and cecidomyiids were SHARLENE ASCENDINO and VALÉRIA C. MAIA the most frequent galling insects.Parasitoids were found in both areas, but they were represented by different species.
In spite of being the third study about insect galls of the Pantanal, several new records were pointed out, showing that the gall diversity of this biome can be greater than expected.Although the harsh environment hypothesis which predicts that insect galls are especially abundant in plant species found in hot and dry habitats (Fernandes andPrice 1988, 1992) has not been tested, the present inventory does not add data in favor of it.Studies with this approach are necessary to confirm or reject this hypothesis.
The taxonomy of the inducers is other important challenge as most part is still superficially identified or is unknown.It requires a long-term research with frequent collections to follow the entire lifecycle of the gallers and obtain all stages necessary to their identification.

Figure 1 -
Figure 1 -Municipality of Corumbá in the State of Mato Grosso do Sul.

Figure 2 -
Figure 2 -Study sites in BEP (Base de Estudos do Pantanal): (a) banks of the Miranda River; (b) riparian forest of the banks of the River Miranda.
46.The values recorded by Julião et al. (2002) and Urso-Guimarães et al. (2017) were 1.77 and 1.39, respectively.Previous studies on insect galls in Brazilian biomes have reported values ranging from 2.1 to 1.1 (Maia et al. 2014).So our result fits into the known range.Most host plant species comprise a single gall morphotype (see Maia 2011 -Amazonian forest, Maia 2001 -Atlantic forest,

TABLE I (continuation)
Total Verbenaceae J.St.-Hil Lippia alba (Mill.)N.E.Br.ex Britton and P.Wilson BEP (RP) . They were represented by Eulophidae, Braconidae, Eupelmidae, Eurytomidae, Platygastridae and Pteromalidae, families already recorded as parasitoids of galling insects in Brazil (Maia and Azevedo 2009).Eulophidae were the most frequent one, as previously pointed out by Maia and Azevedo 2009 at restinga areas (Atlantic forest).Multiparatism has already been recorded in galls of the Atlantic forest (Maia 1999, Maia and Monteiro 1999, Maia and Tavares 2000, Maia and Azevedo 2009), and Cerrado