Insect galls from Amazon rainforest areas in Rondônia (Brazil)

: This study aimed at survey insect galls of an Amazon rainforest area in Rondônia, Brazil. We found 152 gall morphotypes in 103 plant species. Fabaceae were the host with the greatest gall richness. Leaves were the most galled organ. Globose and glabrous galls were the most frequent. Cecidomyiidae were responsible for most of the galls. This is the first record of 110 galls morphotypes and 23 host plants species in this biome. Ten gallers are endemic in Brazil. Five genera of Cecidomyiidae were first recorded in Rondônia as well as Schismatodiplosis lantanae Rübsaamen, 1908.


INTRODUCTION
The insect galls are abnormal growths of plant tissue induced by insects, within which they develop, finding shelter against adverse environmental conditions, protection against predators and parasitoids, and food source (Mani 1964).The galling insects present high specificity in relation to their host plant, being usually monophagous.In addition, each galling species produces a morphologically and structurally unique gall in its host plant, so that the gall is considered as the extended phenotype of the galler (Weis et al. 1988, Stone & Schonrogge 2003, Carneiro et al. 2009).
The Neotropical Region presents a high diversity of galling insects, but its taxonomic knowledge is still very scarce (Gonçalves-Alvim & Fernandes 2001).In Brazil, some ecological studies indicate that the greatest diversity of Cecidomyiidae species is found in Amazon rainforest, the largest Brazilian phytogeographic domain, but only 2% of these species are known for this biome (Julião G.R., unpublished data).
Most of the species known in the Brazilian territory were collected in Atlantic Forest and Cerrado areas, with about 80% and 14.5% of the total of the species registered to Brazil, respectively (Proença B. & Maia V.C., unpublished data).
So far, only five inventories with host plants data, characterization of the galls' morphotypes, records of inducers and associated fauna have been carried out in areas of the Amazon rainforest.One of them was performed in the state of Amazonas (Almada & Fernandes 2011) and the other four in Pará (Maia 2011, Silva et al. 2011, Araújo et al. 2012, Carvalho & Mota 2018).Data on galling insects and their host plants remain unknown for the other Brazilian states which include this phytogeographic domain, as the state of Rondônia.This gap impairs the knowledge of the diversity of these insects, as well as studies on the biogeography and geographic distribution of the same.
In this context of lacking information, the main objective of this work is to survey and characterize the insect galls of an Amazon rainforest area in Rondônia, contributing to the knowledge of the galls in this phytogeographic domain.The specific objectives were: i) record the galling inducers and associated fauna; ii) verify the average of gall morphotypes per host plant species; iii) indicate the new records of host plantas and gall morphotypes in the Amazon rainforest area; iv) evauate the gall richness; v) analyze the gall mige (Cecidomyiidae) endemism.
Along each trail, aerial organs of herbaceous, shrubby and arboreous plants were surveyed for insect galls up to two meters in height.All galls were photographed in the field and characterized by plant organ of occurrence, shape, color, presence or absence of trichomes, and number of internal chamber.Each morphotype was individually conditioned in plastic bags and labeled with informations about the collection site, and number of the host plant.Branches (whenever possible with flowers and fruits) were removed from each host plant, and pressed in the field for preparation of exsiccates.Each host plant received an identical label to its corresponding gall.The terminology of gall shapes followed Isaías et al. (2013).
Both galls and host plants were transported to the Laboratório de Diptera in Museu Nacional/UFRJ, Rio de Janeiro State, Brazil.For each gall morphotype, part of the samples was dissected to observe the number of internal chambers and remove immature insects, and the remaining part was conditioned in closed transparent plastic pots for rearing the adults, including inducers, parasitoids, predators and inquilines.The rearing pots were checked every day for newly emerged adults.All insects obtained were transferred to microvials with 70% ethanol.The gall midges (Cecidomyiidae) were mounted in microscopy slides, following the methodology outlined by Gagné (1994).The excedent specimens remained in 70% ethanol.The Cecidomyiidae were identified to genus level using the keys of Gagné (1994) and to species based on original descriptions.Adults of microhymenoptera and Coleoptera were identified by Dr. Maria Antonieta P. Azevedo and Dr. Sérgio Antônio Vanin, respectively.The Coleoptera specimens were deposited in the Entomological Collection of the Museu de Zoologia da Universidade de São Paulo (MZSP) and the microhymenoptera in the Entomological collection of Museu Nacional/UFRJ (MNRJ).

Analysis and data comparison
The average of gall morphotypes per host plant species was calculated using simple arithmetic mean.Data obtained about the host plant families, genera and species, as well as the total and average of morphotypes found were compared to other inventories in the Amazon rainforest area.We searched for previous records of galls in each host plant genus and species verified in this study.We only compared the gall morphotypes for the host plants identified at species level.It was considered as new records to the Amazon rainforest the host plant species and gall morphotypes that was not recorded before in the other inventories in this phytogeographic domain.
All botanical species names, including the genera, were conferred at Flora and Funga do Brasil website (2019) to verify possible synonyms that could cause the data misinterpretation, such as the indication of first record of some of the galls to this phytogeographic domain.

Endemism analysis and galling species richness
The host plant endemism was based on data of Flora and Funga do Brasil website (2019).The galling species were considered endemic when associated exclusively with endemic host plants.
The relationship between galling species richness and plant family size was evaluated.According to plant size hypothesis, gallers are richer in species on plants from taxa also richer in species (Fernandes 1992).To model the relationships between galling species richness and plants size, we use zero-altered poisson models and zero-altered negative binomial models following the routine of the package "msne" and the "gamlss" (Hilbe 2014).The response variable (galling species richness on plant galled) is a count and cannot obtain the value zero.We refer to the variable as being zero truncated (Hilbe 2014).But the analyses were not able to deal with the variance heterogeneity.Because the residues did not behave well we use the non-parametric Spearman correlation test (Sprent & Smeeton 2007) to determine relantionships between galling species richness and plant family size.Statistical analysis was conducted using the R statistical package (R Core Team 2017).

RESULTS
We found 103 host plant species, distributed in 69 genera and 35 families, hosting 152 insect galls morphotypes.The number of host plant families, genera and species, as well as the number of the gall morphotypes are shown in Table I.Fabaceae (n=43), Bignoniaceae (n=33) and Sapindaceae (n=7) presented the highest richness of galls.Among the plant genera, Adenocalymma Mart.ex Meisn.(Bignoniaceae), Bauhinia L., Machaerium Pers., and Inga Mill.(Fabaceae) presented the highest number of galled species with eight, five, five and four host species, respectively.Bauhinia ungulata L. (Fabaceae) and Vismia guianensis (Aubl.)Choisy (Hypericaceae) were the superhost species presenting nine and five distinct morphotypes, respectively.
The number of glabrous galls (n=116) was much higher than that of hairy (n=36).Most galls presented only one internal chamber (83.5%).
We recorded 110 new gall morphotypes.The predominant color of the galls was green (n=108), but we also found brown (n=58), yellow (n=8) and reddish (n=2) morphotypes.The majority of the galls were induced in only one organ of the host plant, except three morphotypes induced by gall midge (Cecidomyiidae): one on stem, petiole and tendril of Tanaecium pyramidatum (Rich.)L. Lohmann (Bignoniaceae); other on stem and leaf of Callichlamys latifolia (Rich.)K. Schum (Bignoniaceae); and the third on stem and leaf of Vismia guianensis (Hypecariaceae).
The inducers of 86 morphotypes could not be determined.
The characterization of the galls, regarding the organ of occurrence, shape, color, indumentum, number of internal chamber and inducing insect is shown in Table III.
In addition to the gallers, other arthropods were found in 16 gall morphotypes, acting as parasitoids, successors and inquilines.Among these, the parasitoids were the most frequent group, occurring in 11 gall morphotypes, and represented exclusively by microhymenoptera.The successors, however, made up the most diversified guild, composed of Thysanoptera, Formicidae (Hymenoptera) and Hemiptera each occurring in one morphotype.The inquilines were the less frequent, occurring only in one morphotype and being represented only by Cecidomyiidae (Diptera).
The In the first field work, in 2011, we collected a higher number of galls (n=106) than in the second one, in 2012, (n=70).Monte Negro was the locality with the highest number of galls (n= 91), followed by Campo Novo de Rondônia (n= 52) and Cacaulândia (n= 37).Gall (Fig. 8): on leaf, conical, light green, with small white trichomes, with concavity on the apex and one-chambered.Galler: Cecidomyiidae.Exuviae and adults obtained.Associated fauna: Hymenoptera (parasitoid).Larvae obtained.Localities: Monte Negro (Cachoeira) and Campo Novo de Rondônia (Marimbondo's trail).Collected in October 2011 and May 2012.
Comments: First record of Adenocalymma coriaceum hosting an insect gall in Amazon rainforest areas.
Comment: First record of Tanaecium hosting insect galls in Amazon rainforest areas.
Comments: First record of Celtis hosting insect galls in Amazon rainforest areas.
Comments: First record of Hippocratea hosting insect galls in Amazon rainforest areas.
Comments: First record of Prionostemma hosting insect galls in Amazon rainforest areas.
Inga cayennensis Sagot ex Benth (native species) (n=1) Gall (Fig. 85): on leaf vein, globoid, reddish brown, covered with small trichomes, with a central hole and one-chambered.Galler: indeterminate.Locality: Monte Negro (Cachoeira).Collected in October 2011.Comments: Some of the galls are gregarious.Inga cayennensis was recorded hosting a brown, glabrous and parenchymatical leaf gall in Amazon rainforest by Maia (2011).First record of this gall morphotype in Amazon rainforest areas.
Comments: First record of Uncaria hosting insect galls in Amazon rainforest area.
Comments: First record of Metrodorea hosting insect galls in Amazon rainforest area.

DISCUSSION
According to Araújo et al. (2019), the Atlantic Forest and Cerrado are the most studied Brazilian areas, with 23 and 22 gall inventories, respectively.Areas of Amazon rainforest remain neglected in this aspect, although this study and several other surveys point out high gall richness in this area (Almada & Fernandes 2011, Araújo & Espírito-santo Filho 2012, Maia 2011, Julião et al. 2014a, b, 2005).The first insect gall survey in Rondônia was done by Price et al. (1998), but this author did not identify the host plants neither characterized the gall morphotypes.Araújo & Espírito-santo Filho (2012), Julião et al. (2005, 2014b) and Yukawa et al. (2001) found 194, 1,150, 236 and 84 gall morphotypes, respectively, in Brazilian Amazon rainforest areas.However, due to lack of botanical identification we cannot compare their results to our data.
More recently, five inventories, with host plants identification and gall characterization, have been published for areas of Amazon rainforest by Araújo et al. (2012), Almada & Fernandes (2011), Maia (2011), Silva et al. (2011), and Carvalho & Mota (2018) in Pará and Amazonas states.These authors found 112, 309, 77, 27 and 27 morphotypes, respectively, but none of them applied the survey methodology used in the present study.This methodological difference may influence the results obtained, since they imply differences in the sampling effort.The average of galls per host plant species (n=1.4) in this study was the third highest when compared to other studies, where Araújo et al. (2012) and Maia (2011) reported averages of 1.7 and 2.0, respectively.
Fabaceae were recorded as the host plant family with the greatest gall richness in this study and also by Maia (2011) and Araújo et al. (2012) in Oriximiná municipality, by Carvalho & Mota (2018) in Santarém municipality, all localities situated in Pará state (in Amazon rainforest area).Some studies indicate that the greatest gall richness is related to the family of host plant with greatest diversity of species, in different architectural groups, in a certain area (Gonçalves-Alvim & Fernandes 2001).Fabaceae are known to be one of the main hosts of insect galls in different areas of the Neotropical region (Maia 2011).This family stands out as the third largest family of plants in the world (Lewis et al. 2005).Fabaceae species can be found in almost all Brazilian biomes and ecosystems, presenting a cosmopolitan distribution (Queiroz 2009).These plants are well represented in areas of Amazon rainforest, especially those with tree habit (Ducke 1949).Bignoniaceae and Sapotaceae also presented high gall richness in the study of Araújo et al. (2012).According to Julião et al. (2014a), the families with the highest gall richness in Amazon rainforest are Fabaceae, Sapotaceae, Burseraceae, Chrysobalanaceae, and Lecythidaceae.
In the present study Bauhinia L. (Fabaceae) and Adenocalymma Mart.ex Meisn.(Bignoniaceae) were the genera with the highest number of galls, results also found by Araújo et al. (2012).However, Maia (2011) and Almada & Fernandes (2011), verified the highest gall richness in Protium (Burseraceae) and Inga (Fabaceae).Species of Bauhinia were previously recorded in Amazon rainforest by Araújo et al. (2012) and Carvalho & Mota (2018), and in Cerrado areas by Coelho et al. (2009), hosting two and one gall morphotypes.Due to the low gall richness observed, these authors did not consider Bauhinia as a superhost genus.Therefore, we considered this the first record of Bauhinia as superhost in Amazon rainforest.
V i s m i a g u i a n e n s i s ( A u b l .) Ch o i sy (Hypericaceae) is considered, for the first time, a superhost species in this phytophysiognomy.Other species of Vismia were previously recorded as superhosts by Almada & Fernandes (2011).
Other studies also recorded the leaves as the most galled organ plant (Maia 2011, Almada & Fernandes 2011, Silva et al. 2011, Araújo et al. 2012, and Carvalho & Mota 2018).Generally, leaves are the most attacked plant organ.According to Isaías et al. (2014), among all registered galls in Brazil, 52.6% are induced on leaves and 35.2% on stems.It is believed that this pattern is related to the fact that the leaves are a constant and abundant resource for the gallers (Maia 2001).We did not found galls in flowers or fruits, as well as Maia (2011), Silva et al. (2011), Almada & Fernandes (2011), and Carvalho & Mota (2018).Galls on buds and tendrils were less frequent, but they were also observed by Araújo et al. (2012).
The globoid morphotype was the most abundant in our study.According to Isaías et al. (2014), this is the most common shape in the Neotropical region.Most galls were glabrous, as also found by Silva et al. (2011a), Maia (2011), Araújo et al. (2012) and Carvalho & Mota (2018) in the Amazon rainforest.
Cecidomyiidae were the most frequent gallers, reinforcing the pattern of this family as the main taxonomic group among the gallers in the Neotropical region, responsible for more than 90% of all recorded galls (Mani 1964, Carneiro et al. 2009).The inducers of 86 gall morphotypes (56.5% of the total) could not be determined, because of the presence of parasitoids, occurrence of insects with diverse feeding habits in the same gall, and collecting of empty galls.Additionally, we can also mention that about 78% of the galls were found in only one or two trails, resulting in insufficient material for identify the inducers.
In spite of comprising more than 6,500 species in the world (Gagné & Jaschhof 2021), only 29 species of Cecidomyiidae have been reported in Amazonian Forest (Maia 2021).This is a very low value, especially if we consider the high number of gall morphotypes induced by gall midges in Amazonian forest inventories.
Although there are no studies on endemisms of Cecidomyiidae, Maia & Mascarenhas (2017) proposed some species as endemic when they are exclusively associated with endemic host plant species.Based on this same argument, we suggest the endemism of the gallers which are associated with Adenocalymma coriaceum A.DC. Benth.(Fabaceae).Unfortunatly, none of these gall midge species have been described so far.
Regarding the associated fauna, the presence of parasitoids, inquilines, predators and successors have been reported in several surveys such as in Cerrado by Maia & Fernandes (2004); in Amazon rainforest by Maia (2011); and in Atlantic Forest (Restinga) by Rodrigues et al. (2014).According to Maia (2001), parasitoids are usually the most frequent natural enemies of galling species in Atlantic Forest (Restinga) and Cerrado areas, as well as throughout the world (Gagné 1994).However, Maia (2011) observed that the inquilines were more frequent than parasitoid and predators in an Amazon rainforest area.In the present study, parasitoids were more frequent than inquilines, being recorded in about 7.2% of the gall morphotypes, whereas the later were found in 0.6%.
Several insects are pointed out as inquilines, such as Diptera (Sciaridae and Cecidomyiidae), Thysanoptera, Coleoptera, Lepidoptera, Hemiptera and Hymenoptera.However, in our study inquilinous and predator guilds were less diverse and little frequent, being composed only by Cecidomyiidae.In these studies, the term "inquiline" is used in a broad sense, including cecidophages, true inquilines and kleptoparasites.We observed the same to the successors, which included only Hemiptera, Thysanoptera and Hymenoptera (Formicidae), differing from other studies where mites (Acari), springtails (Collembola) and booklice (Psocoptera) were found (Bregonci et al. 2010, Maia et al. 2008).But it is important to mention that our data resulted from two collecting periods, while in the other studies the period of investigation was longer than our.
Larger plant taxa potentially offer more hosts and consequently bear a greater number of galling insects (Fernandes 1992).Galling insect richness was positively correlated with plant family size as larger plant families hosted more galling species than smaller plant families.Host plants from related or closer lineages contain similar chemical compounds and plants belonging to the same taxon in a sympatric condition would result in the speciation of galling insects via host replacement (Jermy 1984, Futuyma et al. 1995, Joy & Crespi 2007).These results corroborate previous studies carried out at the Neotropical region such as Carneiro et al. 2014.The taxon of the host plant appears to be the best predictor of the number of insect species at one site.

CONCLUSIONS
Our data confirm the high richness of insect galls in Amazon rainforest areas of Rondônia.Fabaceae presented the highest gall richness and Bauhinia ungulata was considered superhost.Leaves were the most galled plant organ and no galls were found in flowers and fruits.The average of morphotypes per host plant species was 1.4 and the most frequent morphotype was globoid.Most galls were glabrous and one-chambered.Cecidomyidae were the most frequent galling inducers.All results are similar to the previously known patterns to Brazil.
The plant size hypothesis was corroborated, reinforcing the importance of the plant composition for gall richness.
Twenty-three botanical species were recorded as host plants in Amazon rainforest for the first time, as well as 34 botanical genera and 100 gall morphotypes.We suggest the endemism of ten morphospecies of Cecidomyiidae.
The associated entomofauna was few abundant and less diverse when compared with other inventories in Cerrado and Atlantic Forest.

Table I .
Distribution of insect gall morphotypes per host plant families in Rondônia, Brazil, with number and percentage of galled plant genera and species.

Table II .
Comparison between the results of this study and others in Amazon rainforest areas.Data on number of gall morphotypes, host plant species and families, average of gall; botanic families with the highest richness of gall; and botanic genera super-hosts of galls are showed.

Number of gall morphotypes Host families Host species Average of gall morphotypes/ Host plants Botanic families with the highest richness of gall Super-hots botanic genera References
Locality: Cacaulândia (Fazenda do cabeça).Collected in May 2012.Adenocalymma sp.3 (n= 2)

Table III .
Gall characterization (shape, color, number of internal chamber, trichomes, galled plant organ, and inducing insects) in Amazon rainforest areas, in three municipalities of Rondônia (Brazil).
Larvae and adults obtained.