A New Species of Ultratenuipalpus (Acari: Tenuipalpidae) from Brazil and Re-Description of Ultratenuipalpus meekeri (De Leon), the Type Species of the Genus, with DNA Barcodes

Simple Summary The flat mite family Tenuipalpidae includes 41 genera and more than 1100 species worldwide, and is considered one of the most important families of phytophagous mites. The Ultratenuipalpus is a small genus with 25 known species present in almost all zoogeographic regions. Here, a new species Ultratenuipalpus parameekeri Castro, Ochoa & Feres sp. nov. is described from specimens collected on ferns from Brazil. It represents the first species of the genus described from the country. The type species of the genus Ultratenuipalpus meekeri (De Leon) is redescribed based on types and newly collected material from Mexico. Highly detailed low-temperature scanning electron image (LT-SEM) micrographs and DNA barcodes are provided for both species. The taxonomy of the genus Ultratenuipalpus and the ontogenetic additions of leg setae are discussed. Abstract Species of the genus Ultratenuipalpus bear a broad subquadrate propodosoma with many large, flattened, lanceolate to ovate dorsal setae. They also bear some plesiomorphic character states, such as the presence of three pairs of ventral ps setae. Here, we describe Ultratenuipalpus parameekeri Castro, Ochoa & Feres sp. nov. based on adult females, males, and immatures, collected on ferns from Brazil. We also re-describe Ultratenuipalpus meekeri (De Leon), the type species of the genus, based on types and newly collected material from Mexico, and include additional novel data (e.g., dorsal and ventral ornamentation, leg chaetotaxy, and setal measurements) in a standardized form. We include highly detailed images obtained using LT-SEM, accompanied by DNA barcodes, for both species. The ontogenetic additions of leg chaetotaxy are presented and discussed.


Materials and Methods
A portion of the samples collected of each species was maintained in 70% ethanol for subsequent use in low temperature SEM (LT-SEM) studies. Mites for LT-SEM were studied using the previously described methodology [7]. Another portion of the samples of each species was maintained in 100% alcohol for a subsequent molecular analysis.
DNA was extracted from individual mites using the QIAGEN DNeasy Blood & Tissue kits following standard protocols with the following exceptions: (1) mite specimens were carefully pierced with a sterilized minutin pin and then incubated overnight in a solution of buffer ATL and Proteinase K as per instructions and (2) a final elution was performed with 100 µL to increase the total DNA concentration. A portion of cytochrome oxidase I was amplified by PCR with previously published primers [8,9]. The amplification reactions were performed in 25 µL volumes containing 2.5 µL of manufacturer supplied buffer, 0.2 µL (5 units) of Platinum Taq polymerase (Invitrogen), 2.5 µL dNTP (0.25 mM of each base), 1 µL of each primer (10 mM), 2.5 µL of MgCl 2 (25 mM), 11.3 µL of ddH 2 O, and 4 µL of the template DNA. The samples were denatured at 94 • C for 2 min, followed by 30 cycles of 1 min denaturation at 92 • C, 1 min annealing at 50 • C, and 1.5 min extension at 72 • C, with a final elongation of 10 min after the completion of all cycles. PCR products were visualized on a 1% agarose gel saturated with GelRed (Biotium, Hayward, CA, USA). DNA was then purified with a QIAquick PCR Purification kit (Qiagen, Germantown, MD, USA). The amplified fragments were sent to Macrogen USA for sequencing. No additional primers were used for sequencing. COI sequences have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/, accessed on 15 January 2023).
All measurements are given in micrometers (µm). Measurements are presented for the holotype followed by the range for all types in parentheses. The number of leg setae is written as the total number of setae followed by the number of solenidia in parentheses. Terminology of leg and body setation is adapted from [10][11][12]. Photographs of slidemounted specimens were obtained using a Zeiss Axioscope™ microscope (Carl Zeiss Diagnosis of the genus (Based on [2]). "Body shape from elongate-ovate to broadly rounded; broad propodosoma differentiated from narrower opisthosoma (although anterior opisthosoma is broad at junction with propodosoma). Anterior margin of prodorsum usually with median forked projection forming a short notch". Prodorsum with one pair of lateral body projections anterior to setae sc2 present or absent. Posterior margin of opisthosoma with a broad rounded projection between setae h1 usually present. "Prodorsal shield divided by two oblique folds running from vicinity of the eyes angled medially to posterior margin of shield, superficially dividing the shield into three smaller plate-like regions; a small plate is indicated between setae c3-d3 on dorsal opisthosomal margin; posteroventral body margin often with band of pustulate cuticle. Dorsal opisthosoma with setae c1, c3, d1, d3, e1, e3, f3, h1, h2 present (except setae f3 absent in U. aberrans); setae f2 present or absent, when present then inserted on lateral margin aligned with lateral setae e3, f3, h1, h2; setae c2, d2, e2 absent. Setae h2 not flagellate, similar in form to h1; setae sc2, e3, f2, f3, h1, h2 flattened, lanceolate, oblanceolate, obovate to ovate, with sc2 often falcate; form of other dorsal setae variable (e.g., sc2 and f3 flagellate in U. bunyai). Three pairs of ps (pseudanal) setae present; female with ps3 positioned anteriorly on anal valves and much shorter than ps1-2, which are closely associated with each other and positioned posterolaterad anal valves; setae ps2 usually much longer than ps1; male with ps3 modified into accessory genital stylets and inserted on elongate genitoanal valves, with ps1-2 positioned as in female. Ventral, genital and anal regions membranous, without defined sclerotized plates; flap of ovipore and anus surrounded by strongly plicate and wrinkled membranous cuticle. Genital setae g2 inserted slightly anterior to g1 on reduced genital flap; g1-2 often aligned longitudinally with setae ag. Intercoxal setae (3a, 4a) not multiplied. Palps four segmented; palp tibiae with 1-2 setae; palp tarsi with 1-3 phaneres, with solenidion always present, sometimes curved, often inserted basally on palp tarsus segment at junction with palp tibia. Dorsal setae on legs inserted in lateral position. Femora of legs I-II with four setae (d, l , v , bv ); genua I-II with three setae (d, l , l ) (except some species variously described with two setae-U. acharis (genua I-II with 3-2 setae; possibly d absent), U. pterophilus (genua I-II with 2-2)); tibiae I-II usually with five setae (except two species described with four setae, U. avarua (v absent) and U. lacorpuzrarosae (possibly d absent)). Tarsal claws pad-like. Immature stages with setae c1 inserted distinctly anterior to level of setae c3. See also diagnosis of [1]. Description Diagnosis. Female: As per genus, in addition to: prodorsal setae v2, sc1 minute to short, and sc2 large, flattened obovate to ovate; dorsal opisthosoma with 10 pairs of setae (f2 present); most of the dorsal opisthosomal setae large, flattened, obovate to ovate, except setae d3 is distinctly short and c3 almost orbicular; pair lateral body projections anterior to setae sc2 and projection between opisthosomal setae h1 both present; palp four segmented, setal formula 0, 0, 2, 2. Male: Opisthosoma narrower than that of the Animals 2023, 13, 1838 4 of 30 female, with distinct transverse constriction (waist) between setae d1 and e1; many dorsal setae similar in form to those of females, except c1, d1, and e1 short to minute, d3 longer, and setae along posterior margin of opisthosoma (especially e3) narrower and more elongated than those of the female. Tarsi I-II each with two solenidia (ω paraxial and ventrolateral; ω antiaxial); tarsus III with one solenidion ω paraxial and ventrolateral. Immatures: with lateral body projection anterior to setae sc2 present (except absent in larvae); posterior projection between setae h1 absent; dorsal setae similar in general form to those of the female, except setae c1, d1, and e1 short to minute. Larvae with central prodorsum and pygidial region of posterior opisthosoma with finely colliculated integument.
Color. The body is mostly orange with the margin of prodorsum and opisthosoma with dark spots, eyes red, and legs orange. The dorsal body setae and leg setae white to translucent.
Differential diagnosis. This new species resembles Ultratenuipalpus meekeri (De Leon) (herein redescribed) as they both have dorsal setae of a similar shape and length and the same leg and palp chaetotaxy in all developmental stages. These two species also share several other characteristics, such as the pair of lateral projections anterior to setae sc2 and a single posterior projection between opisthosomal setae h1. However, the two species can be separated: the prodorsum is distinctly broader in adult females and males (measured at the widest point between setae sc1 and c1) in U. meekeri (325-345) than U. Dorsum ( Figure 18). Prodorsal region with colliculated integument anteromedially; region between setae sc2-c3 with oblique and transverse folds; pygidial region posterior to setae e1 with colliculated integument; dorsal setae similar in general form to those of females except much smaller and setae c1, d1, and e1 minute. Setal measurements: v2 2-3, Gnathosoma. Palps similar to those of female, setal formula: 0, 0, 2, 2; tibia with two setae, d 3-4, d 5, tarsus with one eupathidium 3-4 and one minute solenidion, 1 long. Setae m absent.
Etymology. The specific name parameekeri refers to the morphological similarity of this species and U. meekeri (De Leon), the type species of the genus.
Differential diagnosis. This new species resembles Ultratenuipalpus meekeri (De Leon) (herein redescribed) as they both have dorsal setae of a similar shape and length and the same leg and palp chaetotaxy in all developmental stages. These two species also share several other characteristics, such as the pair of lateral projections anterior to setae sc2 and a single posterior projection between opisthosomal setae h1. However, the two species can be separated: the prodorsum is distinctly broader in adult females and males (measured at the widest point between setae sc1 and c1) in U. meekeri (325-345) than U. parameekeri (290-315) (in females); notch in anterior forked projection is shorter in U. meekeri (8-13) than in U. parameekeri (20-27) (in females); e3 is narrower and more lanceolate on male (and to a lesser extent on females) U. meekeri than in U. parameekeri; l" on ti I on U. meekeri is thicker than on U. parameekeri; d on fe II is longer and more falcate on female U. meekeri than on U. parameekeri; c3 in larvae is narrower and more lanceolate in U. meekeri than U. parameekeri. In addition to the morphological differences, the molecular analyses confirmed that U. parameekeri and U. meekeri represent distinct species, with a 15.7% difference between their COI sequences.
Color ( Figure 20A). The body is reddish with the central region becoming darker, eyes red, and legs orange. Dorsal body setae and legs setae are white.
Color ( Figure 20A). The body is reddish with the central region becoming darker, eyes red, and legs orange. Dorsal body setae and legs setae are white.
Dorsum (Figure 32). Anterior margin of prodorsum with a short projection forming a short notch; a pair of lateral body projections anterio to setae sc2 present; posterior projection between setae h1 absent. Prodorsu region smooth; region between setae sc2-c3 with transverse folds and plic and tarsi II) and two eupathidia pζ′-pζ" (all 5). Detail of the development of leg chaetotaxy in Table 1.
Remarks. The new specimens examined in this study have similar body and setal measurements to those of the type specimens. In addition, the palp and leg chaetotaxy of those specimens match those of the type specimens.
Type material examined: Holotype: the female collected on a fern in a mangrove swamp, from San Blas, Nayarit State, Mexico, 21 March 1957, coll. D. De Leon, was deposited in the Museum of Comparative Zoology (MCZ), Harvard University. Paratypes: 2 females, 1 male, 3 deutonymphs, 1 protonymph, and 1 larva, with the same data as the holotype, were deposited in the National Insect and Mite Collection, National Museum of Natural History (NMNH), Smithsonian Institution.

Ontogeny
Studies on possible patterns of ontogenetic development of chaetotaxy provide information potentially useful for understanding mite taxonomy, phylogeny, and biology [20]. The family Tenuipalpidae has the highest number of ontogenetic studies among all Trombidiformes mites [21], with ontogenetic data available for 60 species in 20 genera [22]. However, ontogenetic development is known only for one species of Ultratenuipalpus, U. jubatus Otley, Beard & Seeman [2,22]. Here, we discuss the ontogeny of the two species, U. parameekeri and U. meekeri, which share the same pattern of additions of leg setae (Table 1).
Trochanters. Setae v are added to trochanters I, II, and III in the deutonymph and on trochanter IV in the adults. This is the standard pattern for other flat mites [22][23][24], and also for Tetranychidae [10]. Setae l are added to trochanters III in the protonymph, and this addition also occurs in U. jubatus; although the expression of setae l and v varies within the family, this pattern has been commonly reported [2,22,23,25,26].
Femora. Setae l on femora I and II are added in the deutonymph. The expression of setae l on the deutonymph also occurs in U. jubatus and in many species of Tenuipalpus [2,22,23]. Setae d and ev are present on femora III in the larva. This pattern is common in the Tenuipalpidae [22], but in U. jubatus, the addition of setae ev is delayed until the protonymph. Setae ev are added on the femora IV in the protonymph of U. parameekeri and U. meekeri (n.b., femora IV are not nude as described for U. meekeri in [17] and in the keys of [1,2]).
Genua. There is great variation in the chaetotaxy of genua I and II in the Tenuipalpidae [22,23]. Here, setae l is present on genua I and II in the larva, and setae d and l are added on genua I and II in the deutonymph. This pattern also occurs in U. jubatus and is common in the Tenuipalpus [22,23]. Setae l is present on the genu III in the larva of the new species, and the same pattern occurs in U. jubatus; while many species of Tenuipalpus add setae l or d on genu II in the deutonymph [22,23].
Tibiae. Although the number of tibial setae varies across the family, there are no post-larval additions made to the tibiae in the Tenuipalpidae [10]. Here, the number of tibial setae for both species is 5-5-3-3, as is also seen on U. jubatus, whereas setae l are suppressed on tibiae III and IV on U. avarua Xu, Fan & Zhang [1,2,22].
Tarsi. Ultratenuipalpus parameekeri and U. meekeri have a pair of tectal setae added to tarsi I-III in the protonymph, as occurs in U. jubatus. However, many species of the Tenuipalpus added these setae in the deutonymph. As is the case for many additions to leg IV, the addition of the tectal setae on tarsi IV is delayed to the deutonymph. This same pattern also occurs in U. jubatus, while many Tenuipalpus add tectal setae to tarsi IV in the adults [22,23].
The solenidion ω is added on each tarsi I-III in males of U. parameekeri and U. meekeri. Similarly, the male of U. jubatus has this solenidion added to tarsus I and II, but not to tarsus III [2]. This characteristic also occurs in other tenuipalpid genera, such as Tenuipalpus, Prolixus, and Acaricis [26][27][28], with some species of Acaricis also bearing one solenidion ω on tarsus IV.
In response to the detailed work of Lindquist [10] on the patterns of setal additions to the legs in the family Tetranychidae, ontogenetic studies regarding the family Tenuipalpidae have received increasing attention in recent years. For example, the genus Raoiella has ontogenetic data available for 13 of the 22 known species [22]. However, despite this increase in attention, there are still many tenuipalpid genera that have received little or no such focused research [22], such as the Ultratenuipalpus. Only three of the 26 known species of the Ultratenuipalpus have so far been studied ontogenetically, and it is one of the genera that should receive priority in future studies. Filling these gaps may allow an adequate comparison of ontogenetic data between species and genera of the flat mite family, and as Lindquist [10] suggests, may contribute to our further understanding of the superfamily as a whole.

Distribution, Taxonomy and Systematic
The genus Ultratenuipalpus is known from all zoogeographic regions of the world with the exception of the Nearctic and Western Palearctic regions [2][3][4]. To date, six species of the Ultratenuipalpus have been described from the Neotropical region: the new species herein described from Brazil (which represents the first record of the genus for the country), in addition to two species from Mexico (U. meekeri and U. younguisti Baker & Tuttle) and three species from Chile (U. acharis (Gonzalez), U. canelae (Gonzalez), and U. charlini (Gonzalez)) [3,29].
According to Beard et al. [2], the presence or absence of opisthosomal setae f2 may indicate a biogeographic pattern within the genus. Those species that lack f2 show a putative Gondwanan distribution, being found in Chile, Australia, New Zealand, and the Cook Islands. Those species with setae f2 present are found in China, the Philippines, Mexico, and now with the new species herein described, in Brazil. The unique exception for this pattern is U. younguisti, which lacks the setae f2 and was described from Mexico (based on specimens intercepted in the USA).
The presence of a pair of lateral body projections anterior to setae sc2 in some species of the Ultratenuipalpus (e.g., U. meekeri, U. parameekeri, and U. avarua) and the Tenuipalpus sensu stricto group could indicate a strong relationship between these two genera. Within the Ultratenuipalpus, the presence of a single posterior body projection between the setae h1 may be an important character for separating a subgrouping, since it is shared by at least six species of the genus: U. avarua, U. hainanensis (Wang), U. lacorpuzrarosae Rimando, U. meekeri, U. parameekeri, and U. umtataensis Meyer.

Conclusions
The study of body morphology, spermathecae, geographic distribution, and plant associations will allow a broader and deeper understanding of the internal relationships within the genus Ultratenuipalpus, as well its relationships with other related genera (e.g., Tenuipalpus, Extenuipalpus, Acaricis, and Prolixus). In addition, we believe that the study of possible patterns of ontogenetic additions of leg setae can provide important insights into the systematics and origin of these taxa.