Two new species of Rhyncholimnochares (Acari, Hydrachnidia) from Ecuador

South America is one of the least known areas in terms of its fauna of aquatic mites. In this work we report the presence of two new species of the water mite genus Rhyncholimnochares in the Amazonian region of Ecuador, giving the morphological description and the DNA barcode when possible.


Introduction
One of the areas still under-sampled concerning the water mite fauna (Acari, Parasitengona, Hydrachnidia) is South America. In recent work (Tuzovskij and Gerecke 2020) described several new species of the genus Rhyncholimnochares from Central and South America and provided an updated morphological delimitation of the genus along with a diagnostic key to all species. In this work, we add two new species found in the Amazonian part of Ecuador, providing morphological and -as far as possible -molecular characterization (DNA barcode).

Material and methods
Water mites were sampled by the senior author (AGV) with a 250 µm mesh size triangular net and some were kept in Koenike fluids and ethyl alcohol 100% as indicated on each species. Permanent slides were made in glycerin jelly (Cook, 1974). Total genomic DNA was isolated using the QIAGEN BioSprint 15 DNA Blood Kit (Qiagen Iberia S.L., Madrid, Spain). After digestion, water mite exoskeletons were recovered and mounted in glycerin jelly. A fragment of 658 base pairs (bp) was amplified for COI using the primer pair LCO1490 and HCO2198 (Folmer et al. 1994). Gene amplification and purification were carried out as in García-Jiménez et al. (2017).
Laser Scanning Confocal Microscopy (LSCM) images were taken with a Leica SPE microscope, with an excitation wavelength of 488 nm and an emission wavelength range of 546-670. Image stacks were processed with Fiji/ImageJ ver. 1.53f and Amira ver. 5.4.3 (see Valdecasas and Abad (2011) for details).
All measurements are given in µm, first for the holotype, followed by the data for the paratype in parenthesis (if available). We follow Cook's (1974) terminology for easy retrieval of older literature.

Diagnosis
Rhyncholimnochares munozi n. sp. can be easily distinguished from all other previously described species by the co-occurrence of, among other, the following set of characters: a) an expanded and rounded terminal tail of the ocular plate; b) comparable length of rostrum and basal part of gnathosoma; c) almost rectilinear posterior border of coxa II; d) relatively low number of genital acetabula and e) arborescent setae at the end of P-II.
The anterior end of the first pair of coxae connected by engrossed integument ( Figure 3A). A few setae along the anterior lateral edge of coxa I and on the suture line of coxa I and II. Posterior border of coxa II initially convex then straight, lateral border concave. First coxa complete lateral margin length 255 (222); medial 198 (159). Suture line between coxae I-II length, 173 (145). Second coxa lateral length, 138 (116); posterior 214 (168). Tip first coxa to end second coxa 367 (305).
A few simple setae near margins of coxae III and IV whose weak suture line is not clearly marked ( Figures 3B, 4A, B). Total length coxa III-IV 470 (415).

Etymology
The new species is named after Jesús Muñoz for his help during the stays of AGV in Ecuador.

Remarks
The bipectinate setae which, are very common on leg segments, besides the presence of simple setae, could be used as diagnostic, as they are abundant and frequently form a pattern of two rows, as already mentioned by Lundblad (1953). These setae are also common in other members of Limnocharidae (e.g. species of Limnochares) and may provide additional diagnostic value. However, they have been mentioned neither in the species described by Cook (1980) nor in the recent revision of the genus by Tuzovskij and Gerecke (2020), thus their possible diagnostic value could have been underestimated.

DNA barcoding
Not available for these specimens.

Diagnosis
Rhyncholimnochares cristinae n. sp. can be distinguished from all previously described species of Rhyncholimnochares, among others, by the following combination of characters: a) A wide incision on the anterior part of the ocular plate; b) the straight lateral border of coxa II; c) an elongated and slightly concave coxa III; P-II terminating in a palm shaped seta.

Description
Color red. Oval in shape. Integument round papillate. Dorsum without rod-shaped dorsal plate but with sparse glandularia with setae ( Figure 6F). Capitulum attached to a retractile long tubular extension of the integument. Idiosoma length up to the extreme of first coxae: 2225; maximum width 1544.
Ocular plate (OC) total length, 1022. OC fore-edge with a medial deep incision ( Figures 6A,  B and C), width 110. OC anterior part lateral margins converging; length 35; minimum width The anterior end of the first pair of coxae connected by engrossed tegument. A few ( Figure  7A) setae along the anterior lateral and medial edge of coxa I and on the suture line of coxa I and II. The anterior part of coxa I elongated. Medial border of coxa II convex, lateral straight. First coxa lateral length, 378; medial, 333; suture line coxae I-II, 266. Second coxa lateral length, 258; medial, 297. Tip first coxa to end second coxa, 582.
Legs segments with simple and bipectinate setae ( Figures 6D, E). Length of leg segments:

DNA barcoding
A 658 base pair fragment of the mitochondrial cytochrome C oxidase subunit I gene (COI) was sequenced and deposited in Genbank with accession number: MW940285 of the only specimen of R. cristinae.

Etymology
The species is named after the wife of the junior author (RGG), Cristina Quintana Colomo, for her support and encouragement to finish this work.

Remarks
As in the case of the previous species, there are plenty of bipectinate setae on legs whose pattern could be diagnostic. Lack of information in other taxa precludes their usefulness as comparative data. Only the morphology of coxae and legs and the DNA barcoding are provided for this specimen. The remaining parts of the body were lost in DNA isolation.

DNA barcoding
We could sequence a 658 base pair fragment of the mitochondrial cytochrome C oxidase subunit I gene (COI) of this specimen and is deposited in Genbank with accession number: MW940284.

Final remarks
Morphology. The bipectinate setae which are very common on leg segments (besides the presence of simple setae) frequently form two rows, a fact already mentioned by Lundblad (1953) and Rosso de Ferradas (1975). These setae are easily lost during manipulation but, due to common occurrence in other members of the Limnocharidae (e.g., species of Limnochares), they may provide additional diagnostic value.

Barcoding
At the time of writing this manuscript (23/03/2021), the sequences of the genus Rhyncholimnochares were not available in the Genbank (https://www.ncbi.nlm.nih.gov/) and only 9 sequences were present in Bold Systems (https://www.boldsystems.org/), whereas only one was public. Using BOLD Specimen Identification option, the specimen of Rhyncholimnochares cristinae has a similarity between 82.95% and 83.26% with the Rhyncholimnochares sequences retrieved from BOLD. The specimen Rhyncholimnochares sp. has a similarity between 83.1% and 83.41% with BOLD Rhyncholimnochares sequences. The similarity of R. cristinae with Rhyncholimnochares sp. is 83.89%. All these levels of similarity point to the independent species identity of examined specimens (Blattner et al. 2019).