Morphometrical Identification and Phylogenetic Analysis of Rhinonyssidae (Acari: Mesostigmata) Parasitizing Avian Hosts: New Molecular Data

Citation: Sánchez-Carrión, S.A.; Dimov, I.; Márquez Jiménez, F.J.; de Rojas Álvarez, M. Morphometrical Identification and Phylogenetic Analysis of Rhinonyssidae (Acari: Mesostigmata) Parasitizing Avian Hosts: New Molecular Data. Microorganisms 2023,11, 1783. https://doi.org/10.3390/ microorganisms11071783 Academic Editor: Kirill Sharshov Received: 2 June 2023 Revised: 5 July 2023 Accepted: 6 July 2023 Published: 10 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Article Morphometrical Identification and Phylogenetic Analysis of Rhinonyssidae (Acari: Mesostigmata) Parasitizing Avian Hosts: New Molecular Data Susana A. Sánchez-Carrión1, Ivan Dimov 2, Francisco J. Márquez Jiménez 3and Manuel de Rojas Álvarez 1,* 1Departament of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Profesor García González 2, 41012 Sevilla, Spain; [email protected] 2Department of Clinical Anatomy and Operative Surgery Named after Professor M.G. Prives, Pavlov First Saint Petersburg State Medical University, L’va Tolstogo Str. 6-8, Saint Petersburg 197022, Russia; [email protected]u 3Department of Animal Biology, Vegetal Biology and Ecology, Faculty of Experimental Sciences, Universidad de Jaén, 23071 Jaén, Spain; [email protected] *Correspondence: der[email protected]; Tel.: +34-954-55-64-50 Abstract: Members of the family Rhinonyssidae are tiny hematophagous endoparasitic mites that inhabit the nasal cavities of birds and can cause trauma to their hosts. Traditionally, identifying species in this group has relied on observing their morphometrical characteristics. Nevertheless, determining species within this particular group has become more challenging due to the rising number of newly discovered species. Moreover, the morphometrical traits vary depending on the specific genus or group of species being studied. In this study, the complete internal transcribed spacer ITS1, 5.8S rDNA, and ITS2 regions of the ribosomal DNA from eighteen species of rhinonyssid mites belonging to four genera were sequenced to assess the utility of this genomic region in resolving taxonomic questions in this group and to estimate the phylogenetic relationships among the species. Mites were collected by dissecting the nasal cavities of birds under a stereomicroscope. Specimens used for morphometrical analyses were cleared in 85% lactic acid for 1–48 h and mounted in Hoyer’s medium. Other specimens were preserved at − 20 ◦ C for molecular studies. From the data obtained in this study, it can be concluded that a thorough review and an accurate morphometrical identification and determination of the discriminatory traits are needed in this group of mites. Moreover, although the ITS1–5.8S–ITS2 fragment solves different taxonomic and phylogenetic problems at the species level, it would be necessary to test new molecular markers, or even a combination of nuclear and mitochondrial markers or different domains of the nuclear 28S rDNA, to discover a reliable taxonomic situation for rhinonyssids. Keywords: mites; molecular systematics; Rhinonyssidae; morphometrical; phylogenetic 1. Introduction Rhinonyssid mites are permanent hematophagous endoparasites that move slowly and inhabit the nasal cavities of birds, taking advantage of the most vascularized areas to feed [ 1 ]. Usually, these mites do not cause significant pathological problems in their hosts, although their way of feeding could cause direct damage to birds (Rhinonyssidosis avium disease) [ 2 ] and can cause trauma and rupture of the nasal tissues. The family Rhinonyssidae includes around 600 species described worldwide, which are currently grouped into 11 genera [ 3 ]: Larinyssus [ 4 ], Locustellonyssus [ 5 ], Mesonyssus [ 6 ], Ptilonyssoides [ 7 ], Ptilonyssus [ 8 ], Rallinyssus [ 9 ], Rhinoecius [ 10 ], Rhinonyssus [ 11 ], Sternostoma [ 8 ], Tinaminyssus [12], and Vitznyssus [13]. The first studies on these parasites date back to the late 19th century, during which several European researchers mentioned the presence of nasal parasites in birds [ 14 ]. Microorganisms 2023,11, 1783. https://doi.org/10.3390/microorganisms11071783 https://www.mdpi.com/journal/microorganisms Microorganisms 2023,11, 1783 2 of 14 Berlesse and Trouessart defined the genus Ptilonyssus and the subfamily Rhinonyssinae in 1889 [ 8 ]. It was not until 1935 that new species were included, and, years later, the works of Castro and Pereira pioneered a larger and more complete compendium [ 13 ]. Strandtmann, in the middle of the 20th century, increased the morphological information on this family of mites [ 15 ]. Fain began his studies on rhinonyssids from Europe and Africa in 1957, followed by a huge scientific work on rhinonyssids [ 16 ]. Several authors continued to make important contributions to our knowledge of the family Rhinonyssidae in the second half of the last century. Furthermore, the classification of the family Rhinonyssidae has always been based on the morphometry of the mites found. However, many of the morphological characteristics that have been used for discriminatory purposes are based on characteristics that gradually change among closely related species, with great variability observed in groups of species that are taxonomically and ecologically close. This eventually leads to ambiguous identifications, as well as groupings of species that may be closely related, such as the “sairae” group of Ptilonyssus [ 17 ], the “melloi” group among the Tinaminyssus [ 18 ], or the “coniventris” group in the genus Rhinonyssus [19] The new techniques of molecular biology, through the study of different molecular markers, have allowed us to establish relationships between the genetics of populations and distinct aspects of morphology [ 20 ], providing a complementary approach to the accurate identification of species in different groups of parasites and establishing phylogenetic relationships among them [21,22]. Despite these facts, few molecular studies on rhinonyssid mites have been carried out. The small size, the great difficulty of identifying the huge number of morphologically similar species, the lack of adequate descriptions and illustration of the species [ 23 ], and the disagreement among acarologists regarding the discriminatory morphological features for identifying genera and species may explain the high number of synonymies published by different authors. Thus, in the case of the family Rhinonyssidae, there have been few molecular studies and few molecular markers have been tested as nuclear markers, such as the ITS1–5.8S–ITS2 fragment and the 28S region, or as mitochondrial rDNA (e.g., cytochrome oxidase I (COI) and 16S). This means that molecular data in the GenBank database regarding different markers and/or the number of rhinonyssid species are scarce [17,24,25]. The aim of the present study was to determine the level of variation among the ITS1–5.8S–ITS2 sequences of several different species and populations, and to analyze the phylogenetic relationships among rhinonyssid mites. For this purpose, our dataset was aligned with other ITS1–5.8S–ITS2 sequences that were obtained previously [25,26] The resulting phylogenetic tree was analyzed according to the current taxonomy of the family Rhinonyssidae. Our data addressed some taxonomic questions, such as the status of the taxa included in species complexes that comprise very closely related species, and the usefulness of cheliceral morphology for identifying genera. 2. Materials and Methods Eighteen species of rhinonyssid mites belonging to four genera were analyzed using new samples collected for this study in Spain and Russia. Three different populations belonging to the same species and two genera were included, alongside some sequences retrieved from GenBank (Table S1). Mites were collected by dissecting the nasal cavities of birds under a stereomicroscope. The specimens used for morphometrical analyses were cleared in 85% lactic acid for 1–48 h (depending on the original opacity) and mounted in Hoyer’s medium. Other specimens were preserved at −20 ◦C for the molecular studies (Table S2). Hosts were obtained in different ways. Hunting birds like Streptopelia turtur and Columba palumbus were provided to us by hunters. The “Centro Zoosanitario de Sevilla”, which is in charge of the bird population control program developed in the city of Seville, captured the individual birds and provided them to us frozen for parasite examination Microorganisms 2023,11, 1783 3 of 14 (e.g., Columba livia,Streptopelia decaocto,Passer domesticus). Most of the remaining surveyed bird species were obtained dead under various circumstances: on the roads, because of high-voltage transmission lines, or even due to adverse weather (elevated temperature and lack of water during the summer, etc.) (Table S2). 2.1. Morphometrical Study and Identification For an accurate identification of each of the species and populations which was considered, illustrations from the dorsal and the ventral views were made on paper using a Nikon Eclipse Microscope with a camera lucida. All specimens from each population were analyzed using the discriminatory morphological features considered by different authors. In addition, a brief description of each genus is reported here. For Tinaminyssus spp., the most important morphometrical features for discriminating the species are the shape of the chelicerae, a length of 450–630 µ m, the presence and shape of the poststigmatal shields, chaetotaxy and the shape of the dorsal podosomal shield, the arrangement of the z6 setae, the length of the Jv1 and Jv2 setae, the gnathosomal formula, the number of ventral setae and convexitas coxae, and the presence of spina coxae [ 27 – 29 ]. Ptilonyssus spp. are mites with oblong bodies 380–760 µ m long. The dorsal side of the podosoma has only a podosomal shield, the dorsal side of the opisthosoma has only one shield (either opisthosomal or pygidial), and the stigma has oblong peritremes and is located dorsolaterally. There can be presence or absence of the mesosomal shield, an absent poststigmal shield, terminally located gnathosoma, presence or absence of the deutosternal denticles, absence of the tritosternum, presence or absence of the sternal shield, presence of genital and anal shields, an anus with the anal shield located ventrally, the presence of asperos, and absence of the convexitas and spina coxae [3,7,13,16,27,28,30,31]. The Rhinonyssus species consists of mites with generally rounded idiosomes, a length of 430–970 µ m, the presence of a podosomal shield, the absence of opisthosomal and pygidial shields, dorsolaterally located stigmata without peritrems, presence or absence of mesosomal shields, the absence of a poststigmal shield, terminally located gnathosoma, the absence of deutosternal denticles, the absence of a tritosternum, presence or absence of the primary scutum, the presence of a genital shield, an anal shield that is present or strongly reduced, an anus located on the ventral or dorsal side of the opisthosoma, the presence or absence of asperos, and an absence of convexities and spines on the coxae [7,15,16,27,28,30,32,33]. The Sternostoma species is characterized by an oval body and a length of 330– 630 µm . On the idiosoma, the two dorsal shields are podosomal and opisthosomal. Pygidial shields are absent. The stigmata are located dorsolaterally, and the peritremes are absent. Mesosomal shields are present or absent. Poststigmal shields are absent. The gnathosoma are located ventrally, and only the most distal part can extend beyond the frontal edge of the idiosoma. Deutosternal denticles are present or absent. The tritosternum is absent. Sternal, genital, and anal shields are present. The anus has an anal shield situated ventrally or terminally. Asperos can be present or absent [3,7,16,27,28,31,32,34]. Moreover, twelve morphometrical measures and two ratios between the measures (LOS/WOS and LPS/WPS) were retrieved. Morphometrical measurements of the dorsal and ventral sides of Tinaminyssus spp., Ptilonyssus spp., Rhinonyssus spp., and Sternostoma spp. were made, namely, the length of the body (including palps) (LB), the width of the idiosoma (WI), the length of the podosomal shield (LPS), the width of the podosomal shield (WPS), the length of the opisthosomal shield (LOS), the width of the opisthosomal shield (WOS), the length of the anal shield (LAS), the width of the anal shield (WAS), the length of the ventral view of the gnathosoma (including palps) (LG), and the width of the gnathosoma (WG) [26]. Furthermore, in 2016, Dimov [ 35 ] introduced abbreviations and Latin terms for the setae and some other structures, as follows: MS, mesosomal shield (mesosomal scutulum); LMSL, length of the left mesosomal shield; LMSR, length of the right mesosomal shield; Microorganisms 2023,11, 1783 4 of 14 WMSL, width of the left mesosomal shield; WMSR, width of the right mesosomal shield; PSS, poststigmal shield; PgS, pygidial shield; Dd, deutosternal denticles; Cc, convexity on the coxa (convexitas coxae); Sc, spines on the coxa (spina coxae); α 1– α 4, microphytes of the opistosomal shield; dch, chelicera finger; bch, base of the chelicera; An, anus; LPgS, length of the pygidial shield; WPgS, width of the pygidial shield. Some of these features have been considered for morphological identification. 2.2. Molecular Study DNA was isolated from single mites using the NucleoSpin ® Tissue XS DNA extraction kit manufactured by Macherey-Nagel (GmbH & Co. KG, Düren, Germany). Briefly, the mites were coarsely crushed with a plastic pestle in a 1.5 mL microcentrifuge tube containing 20 µ L of an extraction buffer according to the manufacturer’s protocol. Each sample was checked on SYBR Safe stained with 2% Tris-borate-ethylenediaminetetraacetic acid and 0.8% agarose gel. The PCR mix for amplifying the ITS region consisted of 10 µ L of 10 × PCR buffer, 1 µ L of 10 mM dNTP, 6 µ L and 50 mM MgCl 2 , 1.5 µ L of 10 µ M forward primer, 1.5 µ L of 10 µ M reverse primer, 10 µ L of template DNA, and 0.5 µ L of Taq DNA polymerase (2.5 units). The samples were autoclaved with double-distilled water for a final volume of 50 µL. For amplification of the ITS region, the initial denaturation step was carried out at 94 ◦ C for 2 min, followed by 35 cycles of 20 s at 94 ◦ C for denaturation, 30 s at 53 ◦C for annealing the primers, and 45 s at 72 ◦ C for primer extension. A final extension step was carried out for 7 min at 72 ◦ C. The primers were as follows: forward, 5AGAGGAAGTAAAAGTCGTAACAAG-3 0 ; reverse, 5-ATATGCTTAAATTCAGGGGG3 [36]. The PCR products were sequenced in two directions using the Sanger method by the commercial company Allgenetics (A Coruña, Spain). GenBank accession numbers for the sequenced samples are provided in Supplementary Table S1. Sequence Alignments and Phylogenetic Analyses The sequences of the ITS region were aligned using MEGA v.5.2 [ 37 ] and adjusted manually, whereas DAMBE v.5.0 [ 38 ] was used to optimize the alignments. Pairwise distance matrices were generated using the Kimura two-parameter model (K2P) [39]. Phylogenetic trees were inferred on the basis of the nucleotide data and built using two methods. Maximum likelihood (ML) trees were generated using PHYML v.3.0 [ 40 ], and Bayesian inference (BI) trees were built using Mr. Bayes v.3.2.6 [ 41 ]. JMODELTEST 2.1.10 [ 42 ] was used to select the best-fitting model of nucleotide substitution for the alignment of the ITS region. Models of sequence evolution were chosen for the subsequent analyses according to the Akaike Information Criterion [ 43 , 44 ]. The best-fitting nucleotide substitution models selected by JMODELTEST included a general time-reversible model with a gamma-distributed rate variation of GTR + G for the ITS region. These nucleotide substitution models were used to analyze the phylogenetic relationships using PHYML. Support for the topology was examined by bootstrapping the original dataset 1000 times (the heuristic option) [ 45 ]. The best-fitting nucleotide models selected by JMODELTEST and available for BI in MrBayes were GTR + G (nst = 6 with gamma rates) For BI, the standard deviation of the splitting frequency was used to assess convergence; the chains were sampled every 500 generations and each dataset was run for 10 million generations. Burn-in was determined empirically by examining the log-likelihood values of the chains. Phylogenetic trees based on the ITS region were rooted with a species of the superfamily Dermanyssoidea, namely, Dermanyssus gallinae. 3. Results 3.1. Morphometrical Identification All mites were identified strictly on the basis of the descriptions, measurements, and the best illustrations and redescriptions of females that different authors have carried out, Microorganisms 2023,11, 1783 5 of 14 with which the specimens considered in this study were compared. Thus, a summary of the most important discriminatory traits is reported in Supplementary Table S3. Previous works were used to identify the species [3,6,7,13,15,16,18,19,27,27,31,32,46–63]. The specific publications are presented in Supplementary Table S3 within the descriptions of each identified species. Molecular Results There was little variability in the lengths of the different sequences, which ranged between 449 and 536 bp, with an average of 506 bp. The nucleotide composition (Supplementary Table S4) showed a higher content of adenine and thymine (29.6% and 31.7%, respectively), while cytosine had the lowest concentration (14.5%), followed by guanine (24.2%). In the collection of these data, the values of the external elements were not considered. Table 1shows the fragment lengths (in base pairs) of the ITS1, 5.8S, and ITS2 fragments. The boundaries were determined by using the conserved areas flanking the 18S, 5.8S, and 28S sequences according to Navajas et al. [36] and de Rojas et al. [64]. Table 1. Lengths of transcribed internal spacers 1 and 2 (ITS1, ITS2) and the rDNA gene 5.8 (in base pairs). Species ITS1 5.8S ITS2 Tinaminyssus melloi (Zaragoza) Castro, 1948 218 184 - Tinaminyssus melloi (Utrera) Castro, 1948 218 182 90 Tinaminyssus columbae (Utrera) Castro, 1948 200 182 90 Tinaminyssus columbae (Zaragoza) Castro, 1948 200 182 90 Tinaminyssus minisetosum Butenko, 1984 201 181 90 Tinaminyssus streptopelioides Butenko, 1984 218 182 90 Tinaminyssus streptopeliae Fain, 1962 218 182 94 Tinaminyssus bubulci Zumpt & Till, 1955 198 181 - Ptilonyssus hirsti (Montellano) Castro, 1948 214 189 - Ptilonyssus hirsti (Utrera) Castro, 1948 214 189 87 Ptilonyssus motacillae Fain, 1966 214 191 82 Ptilonyssus muscicapae Bregetova, 1970 213 189 82 Ptilonyssus motacillae phoenicuri Fain, 1966 213 189 82 Ptilonyssus fringillae (Montellano) Fain & Sixl, 1971 226 188 87 Ptilonyssus pari (Russia) Fain & Hyland, 1963 226 188 89 Rhinonyssus echinipes Hirst, 1921 206 177 89 Rhinonyssus himantopus Strandtmann, 1951 197 181 90 Rhinonyssus neglectus Hirst, 1921 210 177 89 Microorganisms 2023,11, 1783 6 of 14 Table 1. Cont. Species ITS1 5.8S ITS2 Rhinonyssus tringae Fain, 1963 193 177 89 Sternostoma boydi Strandtmann, 1951 236 184 90 Sternostoma fulicae Guevara & Úbeda, 1975 232 184 90 Thus, when the fragments of the ITS1–5.8S–TS2 region of the species considered in the present study were aligned, an alignment was obtained with 576 positions, including gaps, with 197 conserved and 353 variables. The pairwise distance matrix produced by a pairwise comparison of all considered taxa is given in Supplementary Table S5, which also includes the percentages of similarity between the taxa. Table 2shows a summary of the intergeneric, interspecific, and intrapopulation distances, as well as the percentage of similarity. Table 2. Variation in the genetic distances using Kimura’s two-parameter model and the percentage of intergeneric, interspecific, and intrapopulation similarity (*, genetic distance between P. fringillae– P. pari). Level Genetic Distance (K2P) % Genera Tinaminyssus–Sternostoma 0.26–0.34 77.6–81.1 Tinaminyssus–Rhinonyssus 0.33–0.54 70.5–79.5 Tinaminyssus–Ptilonyssus 0.22–0.39 73.8–82.8 Sternostoma–Rhinonyssus 0.32–0.42 72.7–77.8 Sternostoma–Ptilonyssus 0.23–0.32 76.4–82.5 Ptilonyssus–Rhinonyssus 0.31–0.48 71.7–79.3 Species Tinaminyssus 0.05–0.27 82.5–96 Sternostoma 0.1 91.5 Ptilonyssus 0.08–0.30 (0.01 *) 72.6–94.1 Rhinonyssus 0.08–0.48 78.3–93.2 Populations T. melloi (Zaragoza–Utrera) 0 100 T. columbae (Utrera–Zaragoza) 0 100 P. hirsti (Montellano–Utrera) 0.04 96.7 3.2. Phylogenetic Study of the Species Included in the Alignment In the analysis of the sequences aligned with Mega version 11.01 and Clustal W version 2.0, an alignment was obtained for the ITS1–5.8S–ITS2 fragment of 576 base pairs, finding 197 preserved positions and 353 variables. Phylogenetic trees were constructed as explained in the Materials and Methods section. The topology of the Bayesian tree, including all the considered taxa with Dermanyssus gallinae as an outgroup, is shown in Figure 1. Each node in this tree shows the Bayesian posterior probabilities (PP) followed by the ML bootstrap values (BS). Microorganisms 2023,11, 1783 7 of 14 Microorganisms 2023, 11, x FOR PEER REVIEW 7 of 14 gallinae as an outgroup, is shown in Figure 1. Each node in this tree shows the Bayesian posterior probabilities (PP) followed by the ML bootstrap values (BS). Figure 1. Phylogenetic tree (BI topology) of rhinonyssid mites of different species and geographical origins based on the ITS1-5.8S-ITS2 fragment. Values next to the nodes indicate the Bayesian posterior probabilities (PP), followed by the ML bootstrap values (BS). Support is only indicated on nodes with PP > 0.95 and BS > 75%. Each species shows the GB access number and the length of the amplified fragment. (*) Sequences were retrieved from GenBank. 4. Discussion The question of delimiting species in molecular genetics remains a problem to be solved because there is no objective limit which indicates that two species are distinct [24]. However, Dobler and Farrell [65] observed that, in the case of parasitic organisms, “gene flow between parasite populations in different host species may be limited, even if the parasites are not 100% specific”. This structure can favor the formation of races or speciation. The family Rhinonyssidae is believed to have descended from ectoparasitic ancestors related to the Macronyssidae [4]. Rhinonyssid genera vary in their degrees of host specificity, with some genera being restricted to one host family and others found in hosts from Figure 1. Phylogenetic tree (BI topology) of rhinonyssid mites of different species and geographical origins based on the ITS1-5.8S-ITS2 fragment. Values next to the nodes indicate the Bayesian posterior probabilities (PP), followed by the ML bootstrap values (BS). Support is only indicated on nodes with PP > 0.95 and BS > 75%. Each species shows the GB access number and the length of the amplified fragment. (*) Sequences were retrieved from GenBank. 4. Discussion The question of delimiting species in molecular genetics remains a problem to be solved because there is no objective limit which indicates that two species are distinct [ 24 ]. However, Dobler and Farrell [ 65 ] observed that, in the case of parasitic organisms, “gene flow between parasite populations in different host species may be limited, even if the parasites are not 100% specific”. This structure can favor the formation of races or speciation. The family Rhinonyssidae is believed to have descended from ectoparasitic ancestors related to the Macronyssidae [ 4 ]. Rhinonyssid genera vary in their degrees of host specificity, with some genera being restricted to one host family and others found in hosts from different orders [ 48 ]. In North America, genera with broad host ranges are Ptilonyssus (passeriform, caprimulgiform, falconiform, and apodiform hosts), Rhinonyssus (anseriform, podicipediform, and charadriiform hosts), Sternostoma (passeriform, piciform, and charadriiform hosts), and Tinaminyssus (ciconiiform and columbiform hosts). Among the Microorganisms 2023,11, 1783 8 of 14 more host-specific rhinonyssids, Rhinoecius species parasitize owls (Strigiformes), Rallinyssus species parasitize rails (Rallidae), and Larinyssus species parasitize gulls and terns (Laridae) [3]. 4.1. Morphological Analysis Throughout the course of their study, the principles governing the construction of the natural rhinonyssid system have undergone significant changes. These changes have often required a complete review of the supraspecific classification, with the most challenging aspect being the development of criteria for determining the genera and subfamily ranks, such as the morphology of the chelicerae (Figure 2). Thus, firstly, an accurate identification of the studied species is needed. In the Supplementary Materials, Table S3, a brief description including all discriminatory characteristics of the species identified in this study is shown. Microorganisms 2023, 11, x FOR PEER REVIEW 8 of 14 different orders [48]. In North America, genera with broad host ranges are Ptilonyssus (passeriform, caprimulgiform, falconiform, and apodiform hosts), Rhinonyssus (anseriform, podicipediform, and charadriiform hosts), Sternostoma (passeriform, piciform, and charadriiform hosts), and Tinaminyssus (ciconiiform and columbiform hosts). Among the more host-specific rhinonyssids, Rhinoecius species parasitize owls (Strigiformes), Rallinyssus species parasitize rails (Rallidae), and Larinyssus species parasitize gulls and terns (Laridae) [3]. 4.1. Morphological Analysis Throughout the course of their study, the principles governing the construction of the natural rhinonyssid system have undergone significant changes. These changes have often required a complete review of the supraspecific classification, with the most challenging aspect being the development of criteria for determining the genera and subfamily ranks, such as the morphology of the chelicerae (Figure 2). Thus, firstly, an accurate identification of the studied species is needed. In the Supplementary Materials, Table S3, a brief description including all discriminatory characteristics of the species identified in this study is shown. Figure 2. Morphology of the chelicerae of the species identified in this study. Scales are shown in µm. It is noteworthy that, with regard to the genus Tinaminyssus, in 1948, Castro [13] described Neonyssus melloi as a new species from some specimens found in the nasal cavities of Columba livia domestica L. According to this author, the new species was characterized by its very strong legs with highly developed nails, a gnathosome that was small in size in relation to the body, chelicerae of uniform diameter, and peritremal scutes surrounding the stigmata. In 1960, Fain [6] created the genus Mesonyssus to include the Neonyssus species endowed with long cheliceral fingers. Therefore, Neonyssus melloi became part of the new genus Mesonyssus. However, in 1958, Strandtmann and Wharton [9] had defined the genus Figure 2. Morphology of the chelicerae of the species identified in this study. Scales are shown in µ m. It is noteworthy that, with regard to the genus Tinaminyssus, in 1948, Castro [ 13 ] described Neonyssus melloi as a new species from some specimens found in the nasal cavities of Columba livia domestica L. According to this author, the new species was characterized by its very strong legs with highly developed nails, a gnathosome that was small in size in relation to the body, chelicerae of uniform diameter, and peritremal scutes surrounding the stigmata. In 1960, Fain [ 6 ] created the genus Mesonyssus to include the Neonyssus species endowed with long cheliceral fingers. Therefore, Neonyssus melloi became part of the new genus Mesonyssus. However, in 1958, Strandtmann and Wharton [ 9 ] had defined the genus Tinaminyssus on the basis of a series of characteristics, including all those assigned by Fain to the genus Mesonyssus. Therefore, the generic name proposed by Strandtmann and Wharton had priority, as indicated by Domrow in 1969 [ 27 ], who considered that the correct name for the genus should be Tinaminyssus. Microorganisms 2023,11, 1783 9 of 14 4.2. Molecular Analysis As shown in the Supplementary Materials, Table S4, the length of ITS1 varied between 232 bp in Sternostoma fulicae and 193 in Rhinonyssus tringae. The 5.8S rDNA gene ranged from 189 bp in Ptilonyssus hirsti to 177 in Rhinonyssus echinipes. The ITS2 fragment was less variable in size, with all species showing values very close to 90. These values are very similar to those found by de Rojas et al. [ 64 ] and Úbeda et al. [ 66 ]. In addition, according to the GenBank database, members of the Dermanysoidea superfamily (Dermanyssus longipes FM179377 and Androlaelaps casalis AM903317) showed similar values for ITS1 and for the 5.8s rDNA gene, with the ITS2 region being significantly shorter (73 and 62 bp, respectively). The contents of the different bases are shown in Supplementary Table S4 as percentages. As can be seen in Table S4, our results were practically identical to those obtained by de Rojas et al. [64] for a smaller number of rhinonyssid species. Likewise, the lengths of the markers considered in the different species studied herein, as shown in Table S4, were also similar to those obtained by Úbeda et al. [ 66 ] for species of the genus Rhinonyssus, ranging from 468 bp for R. himantopus to 506 in R. neglectus to 540 bp for Sternostoma boydi [ 64 ], as well as to those of other representatives of the superfamily Dermanysoidea, such as Dermanyssus longipes (accession number FM179377). With regard to the variability of the different fragments (Table 1), as expected, the 5.8S rDNA gene was the most conserved region, while the ITS1 region was the longest and most variable. Finally, ITS2 was the shortest and, unusually, quite preserved fragment, at least among species from the same genus. This fact was observed by Navajas et al. [ 36 ] in mites of the family Phytoseiidae, and by de Rojas et al. [ 64 ] in representatives of the Rhinonyssidae. 4.3. Analysis of Phylogenetic and Genetic Distances It was not until 1979 that Pence attempted to address phylogeny in this family of mites. Pence, on the basis of Castro’s observations [ 13 ], hypothesized that the chelicerae would have fairly stable morphological characteristics, since they are related to nutrition. According to this hypothesis, the rhinonyssid mites would come from some ancestor of the Macronysidae in which the morphology of the chelicerae had some similarity to those of the genus Tinaminyssus [ 67 ]. Figure 2shows drawings of the chelicerae of all species included in this study. Thus, according to the phylogenetic tree created as explained in the Materials and Methods sections and shown in the results (Figure 1), several questions can be answered. The topology of the tree shown in Figure 1corresponds to that created via the Bayesian inference method, and is similar to that obtained via the maximum likelihood method. However, in the latter case, the bootstrap values were slightly lower than the Bayesian probabilities. In both cases, the four genera considered in this study were shown to be in different clades. This observation coincides with the morphological proposal of Pence (1979) [ 68 ] regarding the classification of genera based on the morphology of the chelicerae. However, species of the genus Sternostoma appeared in a separate branch, and the species of the other three genera were gathered together in a polytomy, which means that a more conserved marker must be tested to resolve the phylogeny at the genus level. The clade that includes the species of the genus Tinaminyssus appears in a common branch with Bayesian posterior probabilities (PP) and ML support (BS) of 100/99. Within this group, two different branches can be distinguished, depending on the morphological characteristics: the “melloi” complex (T. melloi, T. streptopelioides, and T. streptopeliae) and the “columbae” complex (T. minisetosum and T. columbae) [ 18 ]. However, Tinaminyssus bubulci, a parasite of pelecaniform birds, appears in the “melloi” group with the species parasitizing columbiform birds (100/99). Furthermore, two of the morphological criteria for including a species in the “columbae” group are a lack of postanal setae and barely developed opisthosomal setae. T. bubulci shows numerous well developed opisthosomal setae (typical of the “melloi” group), but lacks the postanal setae. Thus, this apparent morphological and ecological contradiction must be tested by assaying more isolated or concatenated molecular markers in order to resolve it.