A new genus and new species of macronyssid mite (Mesostigmata: Gamasina: Macronyssidae) from Brazilian caves including molecular data and key for genera occurring in Brazil

A new genus and a new species of macronyssid mites is described based on material collected from caves in Minas Gerais state, Southeastern Brazil, and additional occurrences from caves in Rio de Janeiro (Southeast) and Pará (Eastern Amazon region). The new genus may be distinguished from other Macronyssidae genera by the combination of sternal shield with a distinct X-shaped area; sternal shield bearing three pairs of setae and two pairs of pores; sternal gland absent; anterior spur on coxa II lacking; adults with dorsal shield entire and j and J series complete; adults and protonymphs with setae J5 minute; males with strong-pores on dorsal shield and peritrema short extending from posterior margin of coxae II to the anterior margin of coxae IV. A key for genera occurring in Brazil is provided, as well sequences from the mitochondrial large subunit of ribosome (16S).


Introduction
The family Macronyssidae Oudemans, 1936 includes mites that can be associated with a variety of vertebrates, such as birds, reptiles and mammals, including humans (Radovsky 2010; Orlova et al. 2017; Bassini-Silva et al. 2019. The life cycle of these mites comprises five stages of development: egg, larva, protonymph, deutonymph and adults (male or female) (Radovsky  Sequencing and editing chromatogram -PCR products found positive in 1% agarose gel electrophoresis were purified using the Ampure® (Agencourt) kit and sequenced using a 3730 DNA Analyzer. Sequencing primers as for PCRs. Chromatograms were resolved in ChromasPro 1.41 (Technelysium Pty Ltd).
Sampling, alignment and 16S secondary structure -Newly obtained sequences and those downloaded from GenBank are reported in Table 2, along voucher material collection numbers. This fragment was chosen because it led to the largest sample of terminals identified to species and with vouchering data. It is beyond the objectives of this article perform a throughout phylogenetic analyses of Macronyssidae, something that would require more markers and terminals. The aim of including this fragment is just to verify if it supports the inclusion of the new genus in the relatively well sampled Ornithonyssus. Preliminary automatic alignment was performed using MUSCLE (Edgar 2004) and then manually aligned with aid of sequence editor BioEdit 7.2.1 (Hall 1999) considering the 16S secondary structure as reported for other arthropods (Smith and Bond 2003; Gillespie et al. 2006. A full annotated alignment is provided in the supplementary material. Therein, matching parentheses and dots were used to indicate stem and loop regions, respectively; structural helix numbering is given after Gillespie et al. (2006). Length variable regions inferred to be ambiguously aligned were classified into regions of expansion and contraction (REC), non-pairing regions of ambiguous alignment (RAA) and excluded from downstream phylogenetic analyses. Access to the Brazilian genetic heritage was registered in SisGen under register number A68A31E.
The model HKY+G was chosen using jModeltest and used in Maximum likelihood and Bayesian analyses. They agree to each other and similar to Nieri-Bastos et al. (2011) recovers L. pererai and O. wernecki in a basal position relative to remaining Ornithonyssus species ( Figure 1C). Chiasmanyssus cavernicola n. gen. n. sp. branches basal in the topology obtained, being recovered in an isolated position relative to Ornithonyssus and Lepronyssoides species from which 16S sequences are available.

Taxonomy
Family Macronyssidae Oudemans, 1936 Diagnosis according to Radovsky (2010). Diagnosis -Female large, its sternal shield square, distinctly sclerotized, with a smoother area on central sternal shield X-shaped; sternal shield bearing three pairs of setae and two pairs of pores; without sternal glands; anterior median protusion in epigynal shield extends under the sternal shield; anterior spur on coxa II lacking. Adults with dorsal shield entire, reticulated Table 3 Pairwise mean interspecific p-distances values between sequences of 16S from species of macronyssid mites deposited in GenBank and newly sequenced from the new genus. Values with cells shadowed in gray are intra-specific mean distances in case of more than one sequence available for that species. and with j and J series complete. Adults and protonymphs with setae J5 minute; all setae on idiosoma, legs and gnathosoma slender and smooth, except palp with disto-ventral seta on genu spatulate. Females and protonymphs with chelae simple and slender, without spines or other processes. Male with four pairs of conspicuous pores near setae j3, z4, J3 and one laterally between J4 and J5 setae; peritrema short, extending from posterior margin of coxae II to the anterior margin of coxae IV and peritrematic shield with nodule fused to the middle of the shield.
Etymology -The generic name comes from Greek word "chiasma" referring to the distinct X-shaped area on sternal shield and "nyssus" , to prick, referring to the parasitic life.
Diagnosis -Female: Elongated dorsal shield, holotrichous, bearing 27 pairs of setae, all setae long and subequal, except j2, z2, s1-s2 small (and J5 minute). Unarmed dorsum with approximately 84 pairs of long setae and ventral opisthosoma with approximately 48 pairs of setae; peritrema long, extending from level of anterior margin of coxae II to the middle of coxae IV; palpal trochanter with a small blade-like ventral process. Male: anterior margin of the dorsal shield not discernible from the soft cuticle; with neotrichous condition on opisthonotal region; a pair of additional minute setae, laterally to J4 and J5, on neotrichous area of opisthonotal region. Four pairs of caudal setae on unarmed dorsum and four pairs of setae on unarmed venter. Protonymph: Subrectangular podonotal shield with lateral borders biconcave at level of z2 and z4, posterior region almost square; with j2 very close to j1. Trapezoidal pygidial shield with J4 and Z4 small than others (except J5 minute). Unarmed dorsum with 12 pairs of long setae. Unarmed venter with four pairs of median setae between ventral shields and a pair of caudal setae, without setae flanking the anus.

Description. Male -Measurements summarized in table 4 (Figures 5-7)
Idiosoma -( Figure 5A-B). As in female but smaller. Dorsum ( Figure 5A). Dorsal shield entire, occupying most of the dorsal idiosoma; anterior margin of the dorsal shield not discernible from the soft cuticle; posterior narrow to setae J5; reticulate sculpture; dorsal shield with j and J series complete, j1, j2 and j3 on dorsal shield and J5 minute, bears approximately 56 pairs of setae; neotrichous area on opistonothal region; a pair of additional minute setae,  laterally to J4 and J5, on neotrichous area of opistonothal region; one pair of lyrifissures near on margin lateral to setae j1 and six pairs of pores visible on dorsal shield, four pairs of conspicuous pores near setae j3, z4, J3, one laterally to J4 and J5 setae, and two supplementary pairs positioned close together, in the dorsal shield margin, laterally the setae J2; four pairs of caudal setae on unarmed dorsum. Venter ( Figure 5B). Tritosternum as in female. Venter has a holoventral shield; reticulated sculptured; bears two pairs of lyrifissures and three pairs of setae on sternal region; third pair of pores near the metasternal and genital setae on shield; ventral region broadened posteriorly, with 10-11 setae on each side. Anal region with three post-anal setae, cribrum wide and anal ring present. Endopodal plates small and triangular, between coxae III and IV. Unarmed ventre with four pairs of setae. Peritrema much shorter than in female, extending from posterior margin of coxa II to anterior margin of coxa IV. Peritrematic shield anteriorly fused to dorsal shield, with nodule fused to the middle of the shield. Peritrematic shield posterior to stigma as in females.
Legs ( Figure 6A-C and Figure 7A-C). Legs as in female. Chaetotaxy of legs as in female, including tarsus I sensory field ( Figure 7B-C).
Description. Protonymph -Measurements summarized in table 4. (Figures 8-9). Idiosoma -( Figure 8A-B). Smaller than adults. Dorsum ( Figure 8A): Subrectangular podonotal shield with lateral borders biconcave at level of z2 and z4, posterior region almost square; some line sculpture on dorsal shield surface; 11 pairs of setae, with j2 very close to j1; two pairs of pores near setae j3 and s5 on podonotal shield. Dorsum with two pairs of medial setae between the shields (J1-J2) and a lateral pair (J3). Mesonotal shield (or median diastema or platelets) comprising three pairs of platelets. Pygidial shield trapezoidal, as long as wide, with pairs of setae: J4-J5, Z3-Z5 and S5; J4 and Z4 much smaller than other setae (except J5, minute) on the pygidial shield; three pairs of pores near setae Z3, Z4 and J5 on pygidial shield. Unarmed dorsum has three pairs of free "nodules", two pairs large near to lateral edges and one pair very small on anterior region. Unarmed dorsum with 12 pairs of long setae, dorsal and ventral caudal setae may be present 1 or 2 minute barbs (clearly observed just under immersion). Venter ( Figure 8B). Tritosternum as in adults. Sternal shield resembles a pentagon; three pairs of slender and long sternal setae; two pairs of lyrifissures posterior to st1 and st2. Unarmed venter with five pairs of setae, four pairs of median setae between ventral shields and a pair of caudal setae (without seta flanking the anus); setae pair between coxae IV are slender than other ventral setae. Anal shield subtriangular with postanal setae long and subequal; Peritreme short, extending from middle of coxae III to middle of coxae IV.
Host -Unknown Etymology -The specific name "cavernicola" refers to its habitat.   Domrow 1958, Glauconyssus Uchikawa 1991, Ichoronyssus Kolenati 1858 and Parichoronyssus by bearing less than three pairs of pores on the sternal shield. These genera have the third pair of sternal pores on the shield or as in Synasponyssus on the complex sternal-median shield, included in the so-called group "A" by Radovsky (2010).

Discussion -The new genus differs from Synasponyssus, Bewsiella
Chiasmanyssus cavernicola n. gen. n. sp. resembles all other genera of Macronyssidae by sharing the peritrematic shield posterior to the stigma fused to the parapodal shield around the posterior margin of coxa IV in adults, a character regarded as apomorphic in the family, but absent in several genera (Bewsiella, Glauconyssus, Ichoronyssus, Synasponyssus and Parichoronyssus) probably close to the laelapine stem (Radovsky 2010), and by the presence of an extra seta in the tibia III, genu III and IV.
Those setae are most likely lost in some derived genera: i) all extra seta absent from Chiroecetes; ii) only extra setae on genu III in Megistonyssus Radovsky 1966; iii) tibia III and genu IV both with one extra seta in Mitonyssoides and Acanthonyssus; iv) only genu IV with one extra seta in Macronyssus and Radfordiella (one species R. oudemansi Fonseca 1948); v) Pellonyssus, Atrichonyssus Radovsky 2010 and Ophionyssus have two extra setae in genu IV. Radovsky (2010) suggests that absence or presence of one or more extra setae on any or all tibia III, genu III and IV is useful for setting apart closely related genera within the family.
Chiasmanyssus n. gen. differs from all other genus described by the morphology of the sternal shield in females with its distinct "X" shaped area. Some genera have the sternal shield with some distinct sclerotization but not comparable to this: Chelanyssus Zumpt and Till 1953 and Steatonyssus have a sclerotized band in posterior margin (Till and Evans 1964; Till and Evans 1966; Radovsky 1966); Chiroptonyssus has a heavily sclerotized posterior band extending anteriad on the sides of the shield and Lagidonyssus has the shield divided into two parts, which anterior part shortest and a posterior part (from first pair of pores) more strongly sclerotized and with setae st2 and st3 (Fonseca 1935b; Radovsky 1967; 2010.
The presence of the seta J5 minute is similar to Macronyssus and Megistonyssus, among the most derived genera. However, it is distinguished from these genera by the absence of anterior spur of coxa II. In Macronyssus, the protonymphs have only 10 setae in shield podonotal, with j1 setae are anterior to the podonotal shield and in Megistonyssus the pygidial shield has only four setae pairs and a setae pair flanking the anal shield in the unarmed venter (Radovsky 1966; 1967; 2010.
Chiasmanyssus n. gen. is closely related to genus Cryptonyssus Radovsky 1966. It can be set apart, however, by only five pairs of setae on venter, four between the shields and one caudal (without setae flanking the anus); shape of pygidial and podonotal shield and presence of only 12 setae pairs on the dorsal integument. Furthermore, females lack marginal setae on dorsal shield, while males lack stout or spinose anterior setae on coxae II and III (Radovsky 1966; 1967.
Despite the scanty molecular data that could be retrieved from GenBank, the 16S fragment sequenced apparently is a good bar-coding marker, since it has a considerable gap between intra-and inter-specific distances. This fragment also supported Chiasmanyssus n. gen. as an independent lineage, highly supported in a clade recovered as sister-group of Ornithonyssus and Lepronyssoides.
Finally, it remains as a mystery the host of the new species. As the large number of individuals dwelling on caves floor, vertebrates that use them as nesting sites or shelter are candidates. However, authors have been surveying bats for several months, the most abundant vertebrates found in those caves and, despite finding most of mesostigmata lineages usually associated to bats in neotropics, could not find Chiasmanyssus n. gen. on them thus far. 3. Podonotal shield with 10 setae pairs; setae j3 present; J5 present and minute; pygidial shield with 6-7 setae pairs; 2 median setae between dorsal shield; normally 4 setae pairs between ventral shields but may be more than 10 setae pairs when neotrichous . . . . . . . . . . Macronyssus -Podonotal shield with 8-9 setal pairs; setae j3 absent; J5 absent; pygidial shield with 1-3 setae pairs; 3 median setae between dorsal shield; 2-3 setae pairs between ventral shields, never neotrichous . .  13) 13. Venter neotrichous with about 5 or 6 pairs of setae between shields (except C. venezolanus); some caudal setae on the pygidial shield peg-like and/or with short barbs; leg II much stouter than other legs; claws II much larger than other claws and claws I as claws III and IV . .