New species of Daidalotarsonemus and Excelsotarsonemus (Acari, Tarsonemidae) from the Brazilian rainforest

Abstract Three new species of Tarsonemidae, Daidalotarsonemus oliveirai Rezende, Lofego & Ochoa, sp. n., Excelsotarsonemus caravelis Rezende, Lofego & Ochoa, sp. n. and Excelsotarsonemus tupi Rezende, Lofego & Ochoa, sp. n. are described and illustrated. Measurements for these species are provided, as well as drawings, phase contrast (PC), differential interference contrast (DIC) and low temperature scanning electron microscopy (LT-SEM) micrographs. Some characters, which have not been used or clearly understood, are described herein. Biological, ecological and agricultural aspects about the role of these species in the rainforest and its surrounding environment are briefly discussed.


Introduction
Currently, Daidalotarsonemus De Leon (Acari, Prostigmata, Tarsonemidae) consists of 26 described species (Lin and Zhang 2002, Lofego et al. 2005, Sousa et al. 2014. It is one of the few genera of Tarsonemidae which have been documented on all continents, except Antarctica (Lindquist 1986, Lin andZhang 2002). The known geographical distribution of Excelsotarsonemus Ochoa & Naskręcki is much more restricted, previously recorded only from Costa Rica. The two genera are closely related and considered to be sister genera (Ochoa et al. 1995, Ochoa andOConnor 1998). Both are considered to be plant inhabiting taxa, apparently with a preference for plants located in humid places, where there is an abundance of fungi, bacteria and lichens. In Brazil, the Amazon and Atlantic Rainforests are biomes which fit these requirements, because of their high temperature and rainfall index.
In recent years, significant advances in microscopy have expanded our knowledge of the morphological characters of organisms, which has led to a better understanding of the taxonomy and ecology of species (Fisher and Dowling 2010). One of most effective techniques that has been integrated to study mite morphology and biology is Low Temperature Scanning Electron Microscopy (LT-SEM), in which a sample is instantly frozen with liquid nitrogen, making a frozen snap-shot of the specimen as it occurs in nature available for microscopic study (Bolton et al. 2014). This procedure is critical for understanding not only external morphology, but also ecological and behavior characteristics, not accessible using light microscopy.
The objective here is to describe new species of Daidalotarsonemus and Excelsotarsonemus found in a rainforest in Brazil using phase contrast (PC), differential interference contrast (DIC) light microscopy and LT-SEM microscopy techniques. The LT-SEM study led to a better understanding of the morphology of these species and their respective genera and is discussed herein.

Material and methods
Several leaves of Annona muricata L. (Annonaceae), Theobroma cacao L. (Malvaceae) and Spondias purpurea L. (Anacardiaceae) were collected in and the area surrounding a section of the rainforest near Santa Cruz State University campus (UESC), 14°47'45"S; 39°10'18"W, Ilhéus, Bahia State, Brazil. The region is characterized by having high relative humidity (75-90%) and high precipitation (100-330 mm/ month) indexes throughout the year. Mites collected in the study were prepared and analysed using three different microscopy techniques: phase contrast (PC), differential interference contrast (DIC) and low temperature scanning electron microscopy (LT-SEM). The terminology used herein follows that of Lindquist (1986), except fot the gnathosomal setae dgs and vgs (Magowski and Di Palma 2000). For each structure, all the measurements are provided in micrometers (µm), followed by the range of all specimens measured in parentheses, including the holotype. The following abbreviations are used for institutions where the types were deposited: Acari Collection of the Departamento de Zoologia e Botânica (DZSJRP), São Paulo State University, São José do Rio Preto, São Paulo, Brazil; United States National Museum of Natural History (USNM), Smithsonian Institution, housed in Beltsville, Maryland 20705, USA.
Etymology. The species name oliveirai is in honor of Dr. Anibal Ramadan Oliveira (UESC -Universidade Estadual Santa Cruz from Ilhéus-BA) for his contribution to study of mites and for all his support during the samplings in the region. Diagnosis. Females of this species resemble those of Excelsotarsonemus kimhansenae Ochoa & OConnor because of the shape of dorsal setae v1, sc2, c1 and c2, and the ornamentation pattern on the prodorsum; but they are distinguished by the asymmetric shape of setae e and the U-shaped cerotegument accumulation between prodorsum and tergite C in Excelsotarsonemus caravelis sp. n., whereas setae e are orbicular and smooth and tergite C surface is smoother in E. kimhansenae. The accumulation of the cerotegument between the tergites was easily noticed in all microscopy techniques used (Fig. 16), and it is being considered a taxonomic feature, useful for distinguishing these species.
Etymology. The region where this mite was found is the same place as the first Portuguese explorers arrived in Brazil, at the end of 15 th century. On their trip, they used caravels, which had big sails. The name caravelis is used because several dorsal setae of this mite species are held in the upright position resembling those sails. Note. Setae f has a unique modification as it is oblanceolate dorsal view, with four faces attached by the main vein, giving a deep concavity at either site, with a central furrow dorsally shoe-like; all margins serrate (Fig. 21H). Similar setal complex modification has been observed in E. mariposa (setae d, f and e) and other Excelsotarsonemus and Daidalotarsonemus species under DIC. However, it is under the LT-SEM that we can understand their complexity. Diagnosis. Females of this species resemble those of Excelsotarsonemus kaliszewskii Ochoa & Naskręcki (Ochoa et al. 1995) because of the similar shape of setae sc2, c1 and d. However, setae c2 and e of Excelsotarsonemus tupi sp. n. are setiform-like, while in E. kaliszewskii these setae are falcate and elongate. In addition, the humps on the prodorsum and the muscle attachments of tergite D are very different in shape between these two species, being more ornate and prominent in E. kaliszewskii.  smooth; Palps moderately short 6-8 (7), with 2 small subterminal setae and terminal projections. Pharynx fusiform, 15 (14-16) long and 8 (7-9) wide at maximum width region. Gnathosoma, idiosoma and legs covered with tiny dimples, each around 0.3 (0.2-0.5) in diameter.    -f 11 (9-15), e-f 13 (11-16) and h-h 10 (9-14). Seta sc2 located lateral to sc1. Dorsal cupules not easily seen.
Etymology. The species name tupi is in honor of a Tupi people, one of the most important native indigenous tribes in Brazil which used to live in all coastal region where this mite species was found.

Discussion
Some characters of Daidalotarsonemus, Excelsotarsonemus and other tarsonemids in general have been misunderstood or have not been clearly interpreted, certainly because of the reliance on only light microscopy technology. This becomes clear by comparing LT-SEM micrographs with the drawings of species described previously. The use of LT-SEM and other SEM techniques by acarologists is useful to truly understanding morphological details of the mites, and contributing to more accurate and reliable taxonomic and systematic studies.
The extension of the prodorsum over the gnathosoma in the genera Daidalotarsonemus and Excelsotarsonemus is a feature mentioned by Lindquist (1986) and Ochoa et al. (1995), respectively. Using the LT-SEM, it was observed the gnathosoma has the ability to protract and retract, being covered by the prodorsum and the coxisternal plates I (Figs 4, 18 and 25).This is a difficult character to discern using light microscopy, mainly because slide mounting distorts it by the flattening of the specimen between the slide and the coverslip, often pushing the gnathosoma forward. In the Daidalotarsonemus species studied, it was observed these mites are able to partially retract the gnathosoma under the propodosoma, leaving the distal part, including the palps exposed. The two species of Excelsotarsonemus are able to retract the entire gnathosoma, similar to turtles, under the propodosoma and over the apodemes 1.
Both genera studied, especially Excelsotarsonemus, have some dorsal setae (especially sc2, c1, d, e and f) with very broad and intricate folding patterns. It is not clear the function of these setae yet. Each one has strong veins that probably help it raise up and maintain itself perpendicular to the body. These sail-like setae might allow them to become airborne, gliding within the canopies and colonizing new trees (Ochoa and OConnor 1998). Setae e and f, because of their position and the way they lay above tergite H, seem to have different functions, perhaps related to protection, entrapping fungal spores and/or improving the aerodynamic characteristics of the mites. Some setae have even more complicated patterns, e.g. the setae e of Excelsotarsonemus caravelis (clearly asymmetric) and setae f of Excelsotarsonemus tupi (asymmetrical and with internal cells). Furthermore, setae d in both species apparently sits on the modified setae e or f like a lid (Fig. 21G, 21H, 28F, 28H). Tergite EF and its setae are supported by plate H, which is concave; both plates are partially covered by the posterior projection of tergite D (Fig. 28I).
It was noticed the production of cerotegument (Krantz 2009) over the body of both genera. Using LT-SEM, the cerotegument was captured extending over the body with fungi, lichens and bacteria accumulating on it. The cerotegument along with its attached material are shed at the edge of the tergites, especially on the propodosoma and tergites C and D (Figs 2C, 4C, 9C, 16C, 18C, 23C and 25C), indicating a way these mites might disseminate microorganisms and even plant pathogens. Although these mites were preserved in 70% alcohol for about eight months, the cerotegument still contained fungi and bacteria. The primary purpose of this substance appears to be water retention, but it also may allow the mite to cover itself in another layer of particles if the substance is sticky (Walter and Proctor 2013). This fact has important biological and agricultural implications. First, this substance allows them to carry debris over their body when they disperse between the canopies. Also, the cerotegument could protect the mite against harmful fungi, being a barrier between them and the soft cuticle. Lastly, by carrying fungi and bacteria, they may act as reservoirs of microorganisms (including plant pathogens) to their host plants, and spreading them throughout the forests and surrounding crops. More studies on the biology and feeding parameters of these genera are necessary to better understand their role and impact.
The discovery of three new mite species in such a small sampling area is remarkable. Although South America has five of the biodiversity hotspots biomes of the world (Myers et al. 2000), just 10 tarsonemid species have been described based on specimens found in this region (Lofego et al. 2005, Lofego and Gondim Jr. 2006, Lofego and Feres 2006, Lofego et al. 2007, Moraes et al. 2002. In addition, two species of Daidalotarsonemus and three of Excelsotarsonemus were found in very similar rainforest areas in Costa Rica (Ochoa et al. 1991;Ochoa et al. 1995, Ochoa andOConnor 1998). Most of these mite species in Costa Rica and Brazil were collected on cocoa trees. This tropical crop has broad leaves which are often covered with fungi and lichens, making it a perfect collecting trap of falling insects and mites from the surrounding tree canopy. Undoubtedly, there is much more to be learned of the species composition, biology and ecology of tarsonemid species present in this rainforest. It is also alarming to think about how much biological information is probably being lost even before it becomes known to the scientific community due to deforestation. For this reason, it is imperative to conduct more surveys to increase the knowledge of the fauna of Tarsonemidae and other mite families in forest canopies around the world. bourn (DPI-FDACS) and Dr. Evert Lindquist (BRCAC) for their comments and suggestions. To the Smithsonian Natural History Museum and National Agricultural Library (NAL-USDA), SEL-USDA for support and assistance with references for this study. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; USDA is an equal opportunity provider and employer.