Camerobiid mites ( Acariformes : Raphignathina : Camerobiidae ) inhabiting epiphytic bromeliads and soil litter of tropical dry forest with analysis of setal homology in the genus Neophyllobius

A survey of the camerobiid mites living on epiphytic bromeliads and the forest floor of a Mexican tropical dry forest was carried out. We found three new species of the genus Neophyllobius, which are described in this paper; the first two, namely N. cibyci sp. nov. and N. tepoztlanensis sp. nov., were both found inhabiting bromeliads (Tillandsia spp.) and living on two tree species (Quercus obtusata and Sapium macrocarpum); the third, N. tescalicola sp. nov., was found in soil and litter under Q. obtusata. These three new species can be differentiated from other species in the genus by a combination of morphological characters in adult females, mainly those setae on femora and genua I. The idiosoma and leg setal ontogeny of a camerobiid mite is presented for the first time in this paper, illustrating chaetotaxic notations and their relative positions in N. cibyci sp. nov. larva, protonymph and adults (female and male), and establishing setal homologies among instars. Setal homology with other species in the cohort Raphignathina is briefly discussed. Additionally, a compilation and an identification key to all known species of camerobiid mites in Mexico is provided.

Neophyllobius Berlese, 1886 species have world-wide distribution, and the genus is the richest in Camerobiidae with more than 100 species.The genus was revised by Bolland (1991) and later works mainly include species descriptions (e.g., Koç & Ayyildiz 1996;Bolland 2001;Khanjani & Ueckermann 2006;Khanjani et al. 2010;Uluçay & Koç 2014).Like many other groups of mites, there is insufficient knowledge regarding their ecology and ontogeny which, in turn, restricts our knowledge of habitat specificity and the identification of homologies among species.Also, many descriptions of camerobiid mites are based on a single specimen because they are usually found singly or in low numbers (Bolland 1986), and rarely is setal nomenclature detailed.
The family Camerobiidae in Mexico comprises 13 species of Neophyllobius (Bolland 1991) (Table 1).This number is clearly low, suggesting that the group is under-represented, and shows that camerobiid mites have been reported throughout tropical and northern areas and in a variety of different habitats (inside epiphytes, tree bark or in the forest floor).
Analysis of leg chaetotaxy for different instars in rich groups, like the genus Neophyllobius, could provide systematic and phylogenetic clues for a better understanding of the groups at different hierarchical levels (Swift 2001), and may be helpful in distinguishing between lineages (Norton 1977).It is important to homologize the setal organs in the different species and among different instars because to ignore the homology and only take into account the numerical formulas in descriptions of new taxa is not phylogenetically informative.In the Camerobiidae, Grandjean's idiosomal chaetotaxy system was firstly applied by Kethley (1990), whereas the leg chaetotaxy system was implemented in adult females belonging to Acamerobia inflatus Fan & Walter, 2011(Fan & Walter 2011).However, leg chaetotaxy is frequently omitted in most recent species descriptions, with a few noteworthy exceptions (e.g., Khanjani et al. 2013Khanjani et al. , 2014)).
In this paper, we provide descriptions of three new species of Neophyllobius: two that have been found exclusively on epiphytic bromeliads and one on soil litter in Mexico.An identification key to females of all the species of Neophyllobius in Mexico is provided.The ontogeny of idiosoma and leg setation, setal notations (chaetotaxy) and their relative positions on the podomeres are presented and illustrated for one of these new species.

N. inequalis
De Leon (1958) N. equalis De Leon, 1958 NAYARIT: Navarrete Guazuma sp.De Leon (1958) According to De Leon (1958) and Bolland (1991)   Undetermined genus and species trunks or branches of two tree species: Quercus obtusata and Sapium macrocarpum.Quercus obtusata is an oak species endemic to Mexico (Arizaga et al. 2009).Additionally, soil litter samples were collected from around the base of the trunk of both Q. obtusata and S. macrocarpum.

Material and methods
Mites were extracted from soil litter samples and bromeliads with Berlese-Tullgren funnels and preserved in 80% ethanol; previously, the bromeliad leaves were cut into small pieces.Then mites were observed under a stereoscopic microscope (Olympus SZ) and were cleared in potassium hydroxide (KOH) solution.
Microscope slides were made using Hoyer's medium as a preserver for observation and illustration using a drawing tube adapted to a compound microscope (Nikon Optiphot-2).Additional observations were made with an optic microscope (Nikon Labophot-2).Line drawings were edited using the GNU Image Manipulation Program (GIMP) (The GIMP team 2014).Holotype and paratype specimens are deposited in the "Colección Nacional de Ácaros" (CNAC) kept at the "Instituto de Biología, Universidad Nacional Autónoma de México" in Mexico City, and three paratype specimens in the "Colección de Insectos de la Universidad de Morelos" (CIUM) at the "Centro de Investigación en Biodiversidad y Conservación, Universidad Autónoma del Estado de Morelos", in Cuernavaca, Morelos, Mexico.
The nomenclature for describing palp and leg chaetotaxy follows Grandjean (1944Grandjean ( , 1946) ) and Fan & Walter (2011), and the terminology of idiosomal chaetotaxy follows Kethley (1990) and Fan & Walter (2011).In descriptions of male, protonymph and larva, only specific features that were found to be different from those of females are mentioned.All measurements are given in micrometres (µm).

Etymology
The specific name incorporates the acronym of the "Centro de Investigación en Biodiversidad y Conservación" (CIByC) at the "Universidad Autónoma del Estado de Morelos" in recognition of its work on biological conservation.

Intraspecific variation
In the case of Neophyllobius cibyci sp.nov., we observed that leg setation teratologies are relatively rare, but include the following: 1) adult specimens with certain setae in different locations; for example, female right femur I with v" distal to d instead of proximal (CNAC009236), female right femur I with l' positioned before d (CNAC009234), female left femur I with l' positioned before d (CNAC009233), female tibiae I with seta dm between v' and v" (CNAC009231 and CNAC009235) instead of distal, female right tibia I with dm between v' and v" (CNAC009232) instead of distal, and male right tibia I with l'p distal to l"p instead of proximal (CNAC009238); 2) female specimens without setae on left or right legs, for example left femur I without l' (CIUM), left femur I without v" (CNAC009236), and left tibia I without v" (CNAC009236 and CIUM).

Remarks
Neophyllobius cibyci sp.nov.resembles N. farrieri De Leon, 1958 collected on "Spanish moss" (probably Tillandsia usneoides) in Oaxaca, Mexico.In both species, dorsal setae on genua I-IV are very long, extending beyond the tip of the tarsus, setae on femur I are short (about 1/6 of podomere length), and setae l' and d on femur I are nearly located at the same level.These species can be differentiated by the following characters: in N. cibyci sp.nov., dorsal setae c1 and d1 are distinctly longer than the distance between setae c1-d1 and d1-e1 respectively, setae d1 are the longest of the dorsal setae, and setae d and l' on palpal femur are heavily spinose.In N. farrieri, the lengths of dorsal setae c1 and d1 are the same as the distance between setae c1-d1 and d1-e1 respectively; setae e1 are the longest of the dorsal setae, and setae d and l' on palpal femur are weakly spinose.

Ecology
Neophyllobius cibyci sp.nov.was the most abundant camerobiid species with 17 specimens, most of which (76.5%) were collected in dry season (March and April).This species is deemed to be an inhabitant of the tree canopy because most specimens (94%) were collected on epiphytic bromeliads at 2.8 m (± 0.4) and only one female specimen (CNAC009237) was found on soil litter.Neophyllobius cibyci sp.nov.was found in low numbers, usually one specimen per sample, except for two samples which had two specimens each.The closely resembling species, N. farrieri, also inhabits epiphytic bromeliads of the genus Tillandsia.

Distribution
This species is only known from San Andrés de la Cal, Morelos, Mexico.The type locality of N. cibyci sp.nov. is about 440 km as the crow flies from the known distribution of N. farrieri (Puenta [sic.] de Nejapa, Oaxaca, Mexico).

Setal homology among Neophyllobius cibyci sp. nov. instars
The collection of different instars provides material for study and suggests the ontogenetic development pattern of gnathosoma, idiosoma and leg chaetotaxy.Although, Grandjean's idiosomal and leg chaetotaxy systems were previously applied to camerobiid mites (Kethley 1990;Fan & Walter 2011), this is the first study of complete chaetotaxy (on gnathosoma, idiosoma and legs) involving three different instars (larva, protonymph and adult) of a camerobiid mite species.Swift's (2001) study of leg chaetotaxy of Caligonellidae (Raphignathina: Raphignathoidea), included one larva and one adult female of an undetermined species of Neophyllobius.We found only one nymphal instar (protonymph); for this reason and because the presence of deutonymph is questionable in Neophyllobius, we prefer not to hypothesise the presence of setae in this particular instar (deutonymph).
There are different concepts about the leg chaetotaxy.Fundamental setae are those present when the appendages in question are first formed, that is on legs I-III in the larva or on leg IV in the protonymph.Accessory setae are any which are formed during subsequent moults (Grandjean 1941;Norton 1977;Swift 2001).Setal priority for each podomere refers to a list of setal organs that appear at the beginning of ontogeny (fundamental setae) and have greater priority, or force, than organs than develop later (accessory setae), which suggests that these setal organs are less susceptible to evolutionary regression (Norton 1977;Swift 2001).Setae of equal priority are listed together within parentheses as proposed by Norton (1977).
LeGs (Table 2).Setae v are absent on trochanters I-III in larva (Fig. 3M-O), and do not appear until the protonymphal instar (Fig. 3I-K).Seta v on trochanter IV does not appear until adulthood (Fig. 3D, H).Femora I-II of the larva with setae v" and d (Fig. 3M-N), femur III of the larva (Fig. 3O) and femur IV of the protonymph (Fig. 3L) only with seta d.Seta l' is added on femur I of the protonymph (Fig. 3I).Setae v' on femora I-III (Fig. 3A-C, E-G), and l" on femur IV (Fig. 3D, H) are added in adults.The setal priorities on femora I and II for Neophyllobius cibyci sp.nov.are: (d, v"), l', v'.Priorities on femora III and IV are: d, l", v'.Genua I-II in all instars with seta d and κ (Fig. 3A-B, E-F, I-J, M-N), and genu III only with d (Fig. 3C, G, K, O).Genu IV of the protonymph and adults only with seta d (Fig. 3D, H, L).Tibiae I-III of larva with setae dm, l', l" and φ (Fig. 3M-O).Tibia IV of the protonymph with setae dp, v'm, l" and φ (Fig. 3L); dp and l"p are added to tibiae I-III (Fig. 3I-K) of the protonymph.Seta v'm is added to tibia I (Fig. 3I) of the protonymph.Setae l'p, and d are added to tibiae I-II of adults (Fig. 3A-B,  E-F).Seta v"m appears only on tibiae I of adults (Fig. 3A, E).Setae l'p and v'm are added to tibia III of adults (Fig. 3C, G).Setae l', dp, l"p and d are added to tibia IV of adults (Fig. 3D, H).Tibia I of males with φ2 (Fig. 3E).The setal priorities for tibiae I and II in Neophyllobius cibyci sp.nov., are: (dm, l', l"), (dp, l"p, v'm), (d, l'p, v"m).Priorities on tibiae III and IV are similar except v"m is not formed.

Diagnosis
This species is characterized as follows: dorsal setae reaching setae immediately behind, seta d on femur I positioned in front of l', femur II with three setae (d, v' and v"), femur III with two setae (d and v'), setae d on genua I-IV slightly less than half as long as tibiae I-IV respectively, dorsal seta d1 less than half as long as width of body; dorsal setae c1 as long as setae d1, setae v" on femora I-II positioned distinctly in front of v'.

Etymology
The specific name makes reference to the municipality Tepoztlán, where the type locality is situated.The name of the municipality derives from two words in the náhuatl language, "Tepoztecatl" (a nahua divinity) and "tlan" (beside), so its meaning is "in the company of Tepoztécatl".

Description
Female (n = 3) (Fig. 5A-F) Holotype female (followed in parentheses by range of holotype and two paratype females).

Remarks
Neophyllobius tepoztlanensis sp.nov.resembles N. marginatus De Leon, 1958 collected from an undetermined composite plant and Quercus sp. in Nayarit, Mexico.In both species, the dorsal setae on genua I-IV are short, less than half as long as tibiae I-IV; the setae on femur I are short (about a quarter of the length of the podomere).These species can be differentiated by the following characters: in N. tepoztlanensis sp.nov., dorsal setae c1 as long as setae d1, and setae v" on femora I-II positioned distinctly in front of v'.In N. marginatus, dorsal setae c1 about half as long as seta d1, and setae v' and v" on femora I-II are positioned horizontally on nearly the same level.

Ecology
Neophyllobius tepoztlanensis sp.nov. is deemed to be an inhabitant of the tree canopy and was found on Tillandsia schiedeana and T. hubertiana at 2.7 m (± 0.4) on Sapium macrocarpum.

Diagnosis
This species is unique due to a combination of following characters: dorsal setae reaches setae immediately behind, femur II with three setae (d, v' and v"), femur III with two setae (d and v'), setae d on genua I-IV passing tibiae, long setae (about ⅓ of podomere length) on femur I; setae d on femur I positioned behind setae l', dorsal idiosomal setae pdx and c1 grouped on small and finely-striated platelet.

Etymology
The specific name refers to the parent rock of the soil (lava flow) in which soil litter, the habitat of this mite species, is deposited.The word "tescal" in the náhuatl language means covered basalt stone from ancient volcanic eruptions or volcanic lava field, and the latin suffix "cola" means inhabitant (one who inhabits).So, tescalicola means "inhabitant of volcanic lava fields".

Description
Female (n = 3) (Fig. 6A-F) Holotype female (followed in parentheses by range of holotype and two paratype females).

Remarks
Neophyllobius tescalicola sp.nov.resembles N. farrieri and N. cibyci sp.nov.In these three species, the dorsal setae on genua I-IV are very long, extending beyond the tip of the tarsus.These species can be differentiated by the following characters: in N. tescalicola sp.nov., setae on femur I are long, about ⅓ of podomere length, setae d on femur I is positioned behind setae l', dorsal idiosomal setae pdx and c1 are grouped on a small and finely-striated platelet.In N. farrieri and N. cibyci sp.nov., setae on femur I are short, about a sixth of the length of the podomere, setae l' and d on femur I are nearly positioned horizontally at same level, and dorsal idiosomal setae pdx and c1 are not joined on a platelet.

Ecology
Neophyllobius tescalicola sp.nov.inhabits soil litter of Quercus obtusata, and was collected in dry (March) and in rainy seasons (October).

Distribution
This species is only known from the type locality.
Due to the lack of setal notations, and because many descriptions of camerobiid mites are based on single specimens, it is very difficult to establish a homology hypothesis among instars and even among species.Notwithstanding this fact, our data suggest a hypothesis to how leg setae appear at different life stages on the podomeres of legs I-IV of larva, protonymph and adult.However, a detailed examination of the leg chaetotaxy of taxa in Camerobiidae will have to be conducted to establish homologies within the family and with the other raphignathoids.
The results presented here clearly show that the species richness of the family Camerobiidae in Mexico is under-represented, and that systematic surveys sampling different components of vegetation are needed.The species recorded had different habitat preferences, two of them inhabiting epiphytic bromeliads on two tree species at an average height of 2.8 m (± 0.4), and one inhabiting soil litter of one tree species.This suggests that habitat diversity determines the richness of the group but it needs to be studied in greater detail.

Table 2 .
Setal leg ontogeny of Neophyllobius cibyci sp.nov., parentheses represent a setal pair and square brackets indicate additional setae in ♂.