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

Ci a ion: Sánchez-Ca ión, S.A.; Dimo , I.; Má quez Jiménez, F.J.; de Rojas Ál a ez, M. Mo phome ical Iden i ica ion and Phylogene ic Analysis o Rhinonyssidae (Aca i: Mesos igma a) Pa asi izing A ian Hos s: New Molecula Da a. Mic oo ganisms 2023,11, 1783. h ps://doi.o g/10.3390/ mic oo ganisms11071783 Academic Edi o : Ki ill Sha sho Recei ed: 2 June 2023 Re ised: 5 July 2023 Accep ed: 6 July 2023 Published: 10 July 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic oo ganisms A icle Mo phome ical Iden i ica ion and Phylogene ic Analysis o Rhinonyssidae (Aca i: Mesos igma a) Pa asi izing A ian Hos s: New Molecula Da a Susana A. Sánchez-Ca ión1, I an Dimo 2, F ancisco J. Má quez Jiménez 3and Manuel de Rojas Ál a ez 1,* 1Depa amen o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se illa, P o eso Ga cía González 2, 41012 Se illa, Spain; [email p o ec ed] 2Depa men o Clinical Ana omy and Ope a i e Su ge y Named a e P o esso M.G. P i es, Pa lo Fi s Sain Pe e sbu g S a e Medical Uni e si y, L’ a Tols ogo S . 6-8, Sain Pe e sbu g 197022, Russia; [email p o ec ed]u 3Depa men o Animal Biology, Vege al Biology and Ecology, Facul y o Expe imen al Sciences, Uni e sidad de Jaén, 23071 Jaén, Spain; [email p o ec ed] *Co espondence: de [email p o ec ed]; Tel.: +34-954-55-64-50 Abs ac : Membe s o he amily Rhinonyssidae a e iny hema ophagous endopa asi ic mi es ha inhabi he nasal ca i ies o bi ds and can cause auma o hei hos s. T adi ionally, iden i ying species in his g oup has elied on obse ing hei mo phome ical cha ac e is ics. Ne e heless, de e mining species wi hin his pa icula g oup has become mo e challenging due o he ising numbe o newly disco e ed species. Mo eo e , he mo phome ical ai s a y depending on he speci ic genus o g oup o species being s udied. In his s udy, he comple e in e nal ansc ibed space ITS1, 5.8S DNA, and ITS2 egions o he ibosomal DNA om eigh een species o hinonyssid mi es belonging o ou gene a we e sequenced o assess he u ili y o his genomic egion in esol ing axonomic ques ions in his g oup and o es ima e he phylogene ic ela ionships among he species. Mi es we e collec ed by dissec ing he nasal ca i ies o bi ds unde a s e eomic oscope. Specimens used o mo phome ical analyses we e clea ed in 85% lac ic acid o 1–48 h and moun ed in Hoye ’s medium. O he specimens we e p ese ed a − 20 ◦ C o molecula s udies. F om he da a ob ained in his s udy, i can be concluded ha a ho ough e iew and an accu a e mo phome ical iden i ica ion and de e mina ion o he disc imina o y ai s a e needed in his g oup o mi es. Mo eo e , al hough he ITS1–5.8S–ITS2 agmen sol es di e en axonomic and phylogene ic p oblems a he species le el, i would be necessa y o es new molecula ma ke s, o e en a combina ion o nuclea and mi ochond ial ma ke s o di e en domains o he nuclea 28S DNA, o disco e a eliable axonomic si ua ion o hinonyssids. Keywo ds: mi es; molecula sys ema ics; Rhinonyssidae; mo phome ical; phylogene ic 1. In oduc ion Rhinonyssid mi es a e pe manen hema ophagous endopa asi es ha mo e slowly and inhabi he nasal ca i ies o bi ds, aking ad an age o he mos ascula ized a eas o eed [ 1 ]. Usually, hese mi es do no cause signi ican pa hological p oblems in hei hos s, al hough hei way o eeding could cause di ec damage o bi ds (Rhinonyssidosis a ium disease) [ 2 ] and can cause auma and up u e o he nasal issues. The amily Rhinonyssidae includes a ound 600 species desc ibed wo ldwide, which a e cu en ly g ouped in o 11 gene a [ 3 ]: La inyssus [ 4 ], Locus ellonyssus [ 5 ], Mesonyssus [ 6 ], P ilonys- soides [ 7 ], P ilonyssus [ 8 ], Rallinyssus [ 9 ], Rhinoecius [ 10 ], Rhinonyssus [ 11 ], S e nos oma [ 8 ], Tinaminyssus [12], and Vi znyssus [13]. The i s s udies on hese pa asi es da e back o he la e 19 h cen u y, du ing which se e al Eu opean esea che s men ioned he p esence o nasal pa asi es in bi ds [ 14 ]. Mic oo ganisms 2023,11, 1783. h ps://doi.o g/10.3390/mic oo ganisms11071783 h ps://www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2023,11, 1783 2 o 14 Be lesse and T ouessa de ined he genus P ilonyssus and he sub amily Rhinonyssinae in 1889 [ 8 ]. I was no un il 1935 ha new species we e included, and, yea s la e , he wo ks o Cas o and Pe ei a pionee ed a la ge and mo e comple e compendium [ 13 ]. S and mann, in he middle o he 20 h cen u y, inc eased he mo phological in o ma ion on his amily o mi es [ 15 ]. Fain began his s udies on hinonyssids om Eu ope and A ica in 1957, ollowed by a huge scien i ic wo k on hinonyssids [ 16 ]. Se e al au ho s con inued o make impo an con ibu ions o ou knowledge o he amily Rhinonyssidae in he second hal o he las cen u y. Fu he mo e, he classi ica ion o he amily Rhinonyssidae has always been based on he mo phome y o he mi es ound. Howe e , many o he mo phological cha ac e is ics ha ha e been used o disc imina o y pu poses a e based on cha ac e is ics ha g ad- ually change among closely ela ed species, wi h g ea a iabili y obse ed in g oups o species ha a e axonomically and ecologically close. This e en ually leads o ambiguous iden i ica ions, as well as g oupings o species ha may be closely ela ed, such as he “sai ae” g oup o P ilonyssus [ 17 ], he “melloi” g oup among he Tinaminyssus [ 18 ], o he “coni en is” g oup in he genus Rhinonyssus [19] The new echniques o molecula biology, h ough he s udy o di e en molecula ma ke s, ha e allowed us o es ablish ela ionships be ween he gene ics o popula ions and dis inc aspec s o mo phology [ 20 ], p o iding a complemen a y app oach o he accu a e iden i ica ion o species in di e en g oups o pa asi es and es ablishing phylogene ic ela ionships among hem [21,22]. Despi e hese ac s, ew molecula s udies on hinonyssid mi es ha e been ca ied ou . The small size, he g ea di icul y o iden i ying he huge numbe o mo phologically simila species, he lack o adequa e desc ip ions and illus a ion o he species [ 23 ], and he disag eemen among aca ologis s ega ding he disc imina o y mo phological ea u es o iden i ying gene a and species may explain he high numbe o synonymies published by di e en au ho s. Thus, in he case o he amily Rhinonyssidae, he e ha e been ew molecula s udies and ew molecula ma ke s ha e been es ed as nuclea ma ke s, such as he ITS1–5.8S–ITS2 agmen and he 28S egion, o as mi ochond ial DNA (e.g., cy och ome oxidase I (COI) and 16S). This means ha molecula da a in he GenBank da abase ega ding di e en ma ke s and/o he numbe o hinonyssid species a e sca ce [17,24,25]. The aim o he p esen s udy was o de e mine he le el o a ia ion among he ITS1–5.8S–ITS2 sequences o se e al di e en species and popula ions, and o analyze he phylogene ic ela ionships among hinonyssid mi es. Fo his pu pose, ou da ase was aligned wi h o he ITS1–5.8S–ITS2 sequences ha we e ob ained p e iously [25,26] The esul ing phylogene ic ee was analyzed acco ding o he cu en axonomy o he amily Rhinonyssidae. Ou da a add essed some axonomic ques ions, such as he s a us o he axa included in species complexes ha comp ise e y closely ela ed species, and he use ulness o chelice al mo phology o iden i ying gene a. 2. Ma e ials and Me hods Eigh een species o hinonyssid mi es belonging o ou gene a we e analyzed using new samples collec ed o his s udy in Spain and Russia. Th ee di e en popula ions belonging o he same species and wo gene a we e included, alongside some sequences e ie ed om GenBank (Table S1). Mi es we e collec ed by dissec ing he nasal ca i ies o bi ds unde a s e eomic oscope. The specimens used o mo phome ical analyses we e clea ed in 85% lac ic acid o 1–48 h (depending on he o iginal opaci y) and moun ed in Hoye ’s medium. O he specimens we e p ese ed a −20 ◦C o he molecula s udies (Table S2). Hos s we e ob ained in di e en ways. Hun ing bi ds like S ep opelia u u and Columba palumbus we e p o ided o us by hun e s. The “Cen o Zoosani a io de Se illa”, which is in cha ge o he bi d popula ion con ol p og am de eloped in he ci y o Se ille, cap u ed he indi idual bi ds and p o ided hem o us ozen o pa asi e examina ion Mic oo ganisms 2023,11, 1783 3 o 14 (e.g., Columba li ia,S ep opelia decaoc o,Passe domes icus). Mos o he emaining su eyed bi d species we e ob ained dead unde a ious ci cums ances: on he oads, because o high- ol age ansmission lines, o e en due o ad e se wea he (ele a ed empe a u e and lack o wa e du ing he summe , e c.) (Table S2). 2.1. Mo phome ical S udy and Iden i ica ion Fo an accu a e iden i ica ion o each o he species and popula ions which was conside ed, illus a ions om he do sal and he en al iews we e made on pape using a Nikon Eclipse Mic oscope wi h a came a lucida. All specimens om each popula ion we e analyzed using he disc imina o y mo pho- logical ea u es conside ed by di e en au ho s. In addi ion, a b ie desc ip ion o each genus is epo ed he e. Fo Tinaminyssus spp., he mos impo an mo phome ical ea u es o disc imina ing he species a e he shape o he chelice ae, a leng h o 450–630 µ m, he p esence and shape o he pos s igma al shields, chae o axy and he shape o he do sal podosomal shield, he a angemen o he z6 se ae, he leng h o he J 1 and J 2 se ae, he gna hosomal o mula, he numbe o en al se ae and con exi as coxae, and he p esence o spina coxae [ 27 – 29 ]. P ilonyssus spp. a e mi es wi h oblong bodies 380–760 µ m long. The do sal side o he podosoma has only a podosomal shield, he do sal side o he opis hosoma has only one shield (ei he opis hosomal o pygidial), and he s igma has oblong pe i emes and is loca ed do sola e ally. The e can be p esence o absence o he mesosomal shield, an absen pos s igmal shield, e minally loca ed gna hosoma, p esence o absence o he deu os e nal den icles, absence o he i os e num, p esence o absence o he s e nal shield, p esence o geni al and anal shields, an anus wi h he anal shield loca ed en ally, he p esence o aspe os, and absence o he con exi as and spina coxae [3,7,13,16,27,28,30,31]. The Rhinonyssus species consis s o mi es wi h gene ally ounded idiosomes, a leng h o 430–970 µ m, he p esence o a podosomal shield, he absence o opis hosomal and pygidial shields, do sola e ally loca ed s igma a wi hou pe i ems, p esence o absence o mesosomal shields, he absence o a pos s igmal shield, e minally loca ed gna hosoma, he absence o deu os e nal den icles, he absence o a i os e num, p esence o absence o he p ima y scu um, he p esence o a geni al shield, an anal shield ha is p esen o s ongly educed, an anus loca ed on he en al o do sal side o he opis hosoma, he p esence o absence o aspe os, and an absence o con exi ies and spines on he coxae [7,15,16,27,28,30,32,33]. The S e nos oma species is cha ac e ized by an o al body and a leng h o 330– 630 µm . On he idiosoma, he wo do sal shields a e podosomal and opis hosomal. Pygidial shields a e absen . The s igma a a e loca ed do sola e ally, and he pe i emes a e absen . Mesosomal shields a e p esen o absen . Pos s igmal shields a e absen . The gna hosoma a e loca ed en ally, and only he mos dis al pa can ex end beyond he on al edge o he idiosoma. Deu os e nal den icles a e p esen o absen . The i os e num is absen . S e nal, geni al, and anal shields a e p esen . The anus has an anal shield si ua ed en ally o e minally. Aspe os can be p esen o absen [3,7,16,27,28,31,32,34]. Mo eo e , wel e mo phome ical measu es and wo a ios be ween he measu es (LOS/WOS and LPS/WPS) we e e ie ed. Mo phome ical measu emen s o he do sal and en al sides o Tinaminyssus spp., P ilonyssus spp., Rhinonyssus spp., and S e nos oma spp. we e made, namely, he leng h o he body (including palps) (LB), he wid h o he idiosoma (WI), he leng h o he podosomal shield (LPS), he wid h o he podosomal shield (WPS), he leng h o he opis hosomal shield (LOS), he wid h o he opis hosomal shield (WOS), he leng h o he anal shield (LAS), he wid h o he anal shield (WAS), he leng h o he en al iew o he gna hosoma (including palps) (LG), and he wid h o he gna hosoma (WG) [26]. Fu he mo e, in 2016, Dimo [ 35 ] in oduced abb e ia ions and La in e ms o he se ae and some o he s uc u es, as ollows: MS, mesosomal shield (mesosomal scu ulum); LMSL, leng h o he le mesosomal shield; LMSR, leng h o he igh mesosomal shield; Mic oo ganisms 2023,11, 1783 4 o 14 WMSL, wid h o he le mesosomal shield; WMSR, wid h o he igh mesosomal shield; PSS, pos s igmal shield; PgS, pygidial shield; Dd, deu os e nal den icles; Cc, con exi y on he coxa (con exi as coxae); Sc, spines on he coxa (spina coxae); α 1– α 4, mic ophy es o he opis osomal shield; dch, chelice a inge ; bch, base o he chelice a; An, anus; LPgS, leng h o he pygidial shield; WPgS, wid h o he pygidial shield. Some o hese ea u es ha e been conside ed o mo phological iden i ica ion. 2.2. Molecula S udy DNA was isola ed om single mi es using he NucleoSpin ® Tissue XS DNA ex ac ion ki manu ac u ed by Mache ey-Nagel (GmbH & Co. KG, Dü en, Ge many). B ie ly, he mi es we e coa sely c ushed wi h a plas ic pes le in a 1.5 mL mic ocen i uge ube con aining 20 µ L o an ex ac ion bu e acco ding o he manu ac u e ’s p o ocol. Each sample was checked on SYBR Sa e s ained wi h 2% T is-bo a e-e hylenediamine e aace ic acid and 0.8% aga ose gel. The PCR mix o ampli ying he ITS egion consis ed o 10 µ L o 10 × PCR bu e , 1 µ L o 10 mM dNTP, 6 µ L and 50 mM MgCl 2 , 1.5 µ L o 10 µ M o wa d p ime , 1.5 µ L o 10 µ M e e se p ime , 10 µ L o empla e DNA, and 0.5 µ L o Taq DNA polyme ase (2.5 uni s). The samples we e au ocla ed wi h double-dis illed wa e o a inal olume o 50 µL. Fo ampli ica ion o he ITS egion, he ini ial dena u a ion s ep was ca ied ou a 94 ◦ C o 2 min, ollowed by 35 cycles o 20 s a 94 ◦ C o dena u a ion, 30 s a 53 ◦C o annealing he p ime s, and 45 s a 72 ◦ C o p ime ex ension. A inal ex ension s ep was ca ied ou o 7 min a 72 ◦ C. The p ime s we e as ollows: o wa d, 5- AGAGGAAGTAAAAGTCGTAACAAG-3 0 ; e e se, 5-ATATGCTTAAATTCAGGGGG- 3 [36]. The PCR p oduc s we e sequenced in wo di ec ions using he Sange me hod by he comme cial company Allgene ics (A Co uña, Spain). GenBank accession numbe s o he sequenced samples a e p o ided in Supplemen a y Table S1. Sequence Alignmen s and Phylogene ic Analyses The sequences o he ITS egion we e aligned using MEGA .5.2 [ 37 ] and adjus ed manually, whe eas DAMBE .5.0 [ 38 ] was used o op imize he alignmen s. Pai wise dis ance ma ices we e gene a ed using he Kimu a wo-pa ame e model (K2P) [39]. Phylogene ic ees we e in e ed on he basis o he nucleo ide da a and buil using wo me hods. Maximum likelihood (ML) ees we e gene a ed using PHYML .3.0 [ 40 ], and Bayesian in e ence (BI) ees we e buil using M . Bayes .3.2.6 [ 41 ]. JMODELTEST 2.1.10 [ 42 ] was used o selec he bes - i ing model o nucleo ide subs i u ion o he alignmen o he ITS egion. Models o sequence e olu ion we e chosen o he subsequen analyses acco ding o he Akaike In o ma ion C i e ion [ 43 , 44 ]. The bes - i ing nucleo ide subs i u ion models selec ed by JMODELTEST included a gene al ime- e e sible model wi h a gamma-dis ibu ed a e a ia ion o GTR + G o he ITS egion. These nucleo ide subs i u ion models we e used o analyze he phylogene ic ela ionships using PHYML. Suppo o he opology was examined by boo s apping he o iginal da ase 1000 imes ( he heu is ic op ion) [ 45 ]. The bes - i ing nucleo ide models selec ed by JMODELTEST and a ailable o BI in M Bayes we e GTR + G (ns = 6 wi h gamma a es) Fo BI, he s anda d de ia ion o he spli ing equency was used o assess con e gence; he chains we e sampled e e y 500 gene a ions and each da ase was un o 10 million gene a ions. Bu n-in was de e mined empi ically by examining he log-likelihood alues o he chains. Phylogene ic ees based on he ITS egion we e oo ed wi h a species o he supe amily De manyssoidea, namely, De manyssus gallinae. 3. Resul s 3.1. Mo phome ical Iden i ica ion All mi es we e iden i ied s ic ly on he basis o he desc ip ions, measu emen s, and he bes illus a ions and edesc ip ions o emales ha di e en au ho s ha e ca ied ou , Mic oo ganisms 2023,11, 1783 5 o 14 wi h which he specimens conside ed in his s udy we e compa ed. Thus, a summa y o he mos impo an disc imina o y ai s is epo ed in Supplemen a y Table S3. P e ious wo ks we e used o iden i y he species [3,6,7,13,15,16,18,19,27,27,31,32,46–63]. The speci ic publica ions a e p esen ed in Supplemen a y Table S3 wi hin he desc ip ions o each iden i ied species. Molecula Resul s The e was li le a iabili y in he leng hs o he di e en sequences, which anged be ween 449 and 536 bp, wi h an a e age o 506 bp. The nucleo ide composi ion (Supplemen a y Table S4) showed a highe con en o adenine and hymine (29.6% and 31.7%, espec i ely), while cy osine had he lowes concen a ion (14.5%), ollowed by guanine (24.2%). In he collec ion o hese da a, he alues o he ex e nal elemen s we e no conside ed. Table 1shows he agmen leng hs (in base pai s) o he ITS1, 5.8S, and ITS2 agmen s. The bounda ies we e de e mined by using he conse ed a eas lanking he 18S, 5.8S, and 28S sequences acco ding o Na ajas e al. [36] and de Rojas e al. [64]. Table 1. Leng hs o ansc ibed in e nal space s 1 and 2 (ITS1, ITS2) and he DNA gene 5.8 (in base pai s). Species ITS1 5.8S ITS2 Tinaminyssus melloi (Za agoza) Cas o, 1948 218 184 - Tinaminyssus melloi (U e a) Cas o, 1948 218 182 90 Tinaminyssus columbae (U e a) Cas o, 1948 200 182 90 Tinaminyssus columbae (Za agoza) Cas o, 1948 200 182 90 Tinaminyssus minise osum Bu enko, 1984 201 181 90 Tinaminyssus s ep opelioides Bu enko, 1984 218 182 90 Tinaminyssus s ep opeliae Fain, 1962 218 182 94 Tinaminyssus bubulci Zump & Till, 1955 198 181 - P ilonyssus hi s i (Mon ellano) Cas o, 1948 214 189 - P ilonyssus hi s i (U e a) Cas o, 1948 214 189 87 P ilonyssus mo acillae Fain, 1966 214 191 82 P ilonyssus muscicapae B ege o a, 1970 213 189 82 P ilonyssus mo acillae phoenicu i Fain, 1966 213 189 82 P ilonyssus ingillae (Mon ellano) Fain & Sixl, 1971 226 188 87 P ilonyssus pa i (Russia) Fain & Hyland, 1963 226 188 89 Rhinonyssus echinipes Hi s , 1921 206 177 89 Rhinonyssus himan opus S and mann, 1951 197 181 90 Rhinonyssus neglec us Hi s , 1921 210 177 89 Mic oo ganisms 2023,11, 1783 6 o 14 Table 1. Con . Species ITS1 5.8S ITS2 Rhinonyssus ingae Fain, 1963 193 177 89 S e nos oma boydi S and mann, 1951 236 184 90 S e nos oma ulicae Gue a a & Úbeda, 1975 232 184 90 Thus, when he agmen s o he ITS1–5.8S–TS2 egion o he species conside ed in he p esen s udy we e aligned, an alignmen was ob ained wi h 576 posi ions, including gaps, wi h 197 conse ed and 353 a iables. The pai wise dis ance ma ix p oduced by a pai wise compa ison o all conside ed axa is gi en in Supplemen a y Table S5, which also includes he pe cen ages o simila - i y be ween he axa. Table 2shows a summa y o he in e gene ic, in e speci ic, and in apopula ion dis ances, as well as he pe cen age o simila i y. Table 2. Va ia ion in he gene ic dis ances using Kimu a’s wo-pa ame e model and he pe cen age o in e gene ic, in e speci ic, and in apopula ion simila i y (*, gene ic dis ance be ween P. ingillae– P. pa i). Le el Gene ic Dis ance (K2P) % Gene a Tinaminyssus–S e nos oma 0.26–0.34 77.6–81.1 Tinaminyssus–Rhinonyssus 0.33–0.54 70.5–79.5 Tinaminyssus–P ilonyssus 0.22–0.39 73.8–82.8 S e nos oma–Rhinonyssus 0.32–0.42 72.7–77.8 S e nos oma–P ilonyssus 0.23–0.32 76.4–82.5 P ilonyssus–Rhinonyssus 0.31–0.48 71.7–79.3 Species Tinaminyssus 0.05–0.27 82.5–96 S e nos oma 0.1 91.5 P ilonyssus 0.08–0.30 (0.01 *) 72.6–94.1 Rhinonyssus 0.08–0.48 78.3–93.2 Popula ions T. melloi (Za agoza–U e a) 0 100 T. columbae (U e a–Za agoza) 0 100 P. hi s i (Mon ellano–U e a) 0.04 96.7 3.2. Phylogene ic S udy o he Species Included in he Alignmen In he analysis o he sequences aligned wi h Mega e sion 11.01 and Clus al W e sion 2.0, an alignmen was ob ained o he ITS1–5.8S–ITS2 agmen o 576 base pai s, inding 197 p ese ed posi ions and 353 a iables. Phylogene ic ees we e cons uc ed as explained in he Ma e ials and Me hods sec ion. The opology o he Bayesian ee, including all he conside ed axa wi h De manyssus gallinae as an ou g oup, is shown in Figu e 1. Each node in his ee shows he Bayesian pos e io p obabili ies (PP) ollowed by he ML boo s ap alues (BS). Mic oo ganisms 2023,11, 1783 7 o 14 Mic oo ganisms 2023, 11, x FOR PEER REVIEW 7 o 14 gallinae as an ou g oup, is shown in Figu e 1. Each node in his ee shows he Bayesian pos e io p obabili ies (PP) ollowed by he ML boo s ap alues (BS). Figu e 1. Phylogene ic ee (BI opology) o hinonyssid mi es o diffe en species and geog aphical o igins based on he ITS1-5.8S-ITS2 agmen . Values nex o he nodes indica e he Bayesian pos e- io p obabili ies (PP), ollowed by he ML boo s ap alues (BS). Suppo is only indica ed on nodes wi h PP > 0.95 and BS > 75%. Each species shows he GB access numbe and he leng h o he ampli- ied agmen . (*) Sequences we e e ie ed om GenBank. 4. Discussion The ques ion o delimi ing species in molecula gene ics emains a p oblem o be sol ed because he e is no objec i e limi which indica es ha wo species a e dis inc [24]. Howe e , Doble and Fa ell [65] obse ed ha , in he case o pa asi ic o ganisms, “gene low be ween pa asi e popula ions in diffe en hos species may be limi ed, e en i he pa asi es a e no 100% speci ic”. This s uc u e can a o he o ma ion o aces o specia- ion. The amily Rhinonyssidae is belie ed o ha e descended om ec opa asi ic ances o s ela ed o he Mac onyssidae [4]. Rhinonyssid gene a a y in hei deg ees o hos speci- ici y, wi h some gene a being es ic ed o one hos amily and o he s ound in hos s om Figu e 1. Phylogene ic ee (BI opology) o hinonyssid mi es o di e en species and geog aphical o igins based on he ITS1-5.8S-ITS2 agmen . Values nex o he nodes indica e he Bayesian pos e io p obabili ies (PP), ollowed by he ML boo s ap alues (BS). Suppo is only indica ed on nodes wi h PP > 0.95 and BS > 75%. Each species shows he GB access numbe and he leng h o he ampli ied agmen . (*) Sequences we e e ie ed om GenBank. 4. Discussion The ques ion o delimi ing species in molecula gene ics emains a p oblem o be sol ed because he e is no objec i e limi which indica es ha wo species a e dis inc [ 24 ]. Howe e , Doble and Fa ell [ 65 ] obse ed ha , in he case o pa asi ic o ganisms, “gene low be ween pa asi e popula ions in di e en hos species may be limi ed, e en i he pa - asi es a e no 100% speci ic”. This s uc u e can a o he o ma ion o aces o specia ion. The amily Rhinonyssidae is belie ed o ha e descended om ec opa asi ic ances- o s ela ed o he Mac onyssidae [ 4 ]. Rhinonyssid gene a a y in hei deg ees o hos speci ici y, wi h some gene a being es ic ed o one hos amily and o he s ound in hos s om di e en o de s [ 48 ]. In No h Ame ica, gene a wi h b oad hos anges a e P ilonyssus (passe i o m, cap imulgi o m, alconi o m, and apodi o m hos s), Rhinonyssus (anse i- o m, podicipedi o m, and cha ad ii o m hos s), S e nos oma (passe i o m, pici o m, and cha ad ii o m hos s), and Tinaminyssus (ciconii o m and columbi o m hos s). Among he Mic oo ganisms 2023,11, 1783 8 o 14 mo e hos -speci ic hinonyssids, Rhinoecius species pa asi ize owls (S igi o mes), Rallinys- sus species pa asi ize ails (Rallidae), and La inyssus species pa asi ize gulls and e ns (La idae) [3]. 4.1. Mo phological Analysis Th oughou he cou se o hei s udy, he p inciples go e ning he cons uc ion o he na u al hinonyssid sys em ha e unde gone signi ican changes. These changes ha e o en equi ed a comple e e iew o he sup aspeci ic classi ica ion, wi h he mos challenging aspec being he de elopmen o c i e ia o de e mining he gene a and sub amily anks, such as he mo phology o he chelice ae (Figu e 2). Thus, i s ly, an accu a e iden i ica ion o he s udied species is needed. In he Supplemen a y Ma e ials, Table S3, a b ie de- sc ip ion including all disc imina o y cha ac e is ics o he species iden i ied in his s udy is shown. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 8 o 14 diffe en o de s [48]. In No h Ame ica, gene a wi h b oad hos anges a e P ilonyssus (passe i o m, cap imulgi o m, alconi o m, and apodi o m hos s), Rhinonyssus (anse - i o m, podicipedi o m, and cha ad ii o m hos s), S e nos oma (passe i o m, pici o m, and cha ad ii o m hos s), and Tinaminyssus (ciconii o m and columbi o m hos s). Among he mo e hos -speci ic hinonyssids, Rhinoecius species pa asi ize owls (S igi o mes), Rallinyssus species pa asi ize ails (Rallidae), and La inyssus species pa asi ize gulls and e ns (La idae) [3]. 4.1. Mo phological Analysis Th oughou he cou se o hei s udy, he p inciples go e ning he cons uc ion o he na u al hinonyssid sys em ha e unde gone signi ican changes. These changes ha e o en equi ed a comple e e iew o he sup aspeci ic classi ica ion, wi h he mos chal- lenging aspec being he de elopmen o c i e ia o de e mining he gene a and sub am- ily anks, such as he mo phology o he chelice ae (Figu e 2). Thus, i s ly, an accu a e iden i ica ion o he s udied species is needed. In he Supplemen a y Ma e ials, Table S3, a b ie desc ip ion including all disc imina o y cha ac e is ics o he species iden i ied in his s udy is shown. Figu e 2. Mo phology o he chelice ae o he species iden i ied in his s udy. Scales a e shown in µm. I is no ewo hy ha , wi h ega d o he genus Tinaminyssus, in 1948, Cas o [13] de- sc ibed Neonyssus melloi as a new species om some specimens ound in he nasal ca i ies o Columba li ia domes ica L. Acco ding o his au ho , he new species was cha ac e ized by i s e y s ong legs wi h highly de eloped nails, a gna hosome ha was small in size in ela ion o he body, chelice ae o uni o m diame e , and pe i emal scu es su ounding he s igma a. In 1960, Fain [6] c ea ed he genus Mesonyssus o include he Neonyssus species en- dowed wi h long chelice al inge s. The e o e, Neonyssus melloi became pa o he new genus Mesonyssus. Howe e , in 1958, S and mann and Wha on [9] had de ined he genus Figu e 2. Mo phology o he chelice ae o he species iden i ied in his s udy. Scales a e shown in µ m. I is no ewo hy ha , wi h ega d o he genus Tinaminyssus, in 1948, Cas o [ 13 ] desc ibed Neonyssus melloi as a new species om some specimens ound in he nasal ca i ies o Columba li ia domes ica L. Acco ding o his au ho , he new species was cha ac e ized by i s e y s ong legs wi h highly de eloped nails, a gna hosome ha was small in size in ela ion o he body, chelice ae o uni o m diame e , and pe i emal scu es su ounding he s igma a. In 1960, Fain [ 6 ] c ea ed he genus Mesonyssus o include he Neonyssus species en- dowed wi h long chelice al inge s. The e o e, Neonyssus melloi became pa o he new genus Mesonyssus. Howe e , in 1958, S and mann and Wha on [ 9 ] had de ined he genus Tinaminyssus on he basis o a se ies o cha ac e is ics, including all hose assigned by Fain o he genus Mesonyssus. The e o e, he gene ic name p oposed by S and mann and Wha on had p io i y, as indica ed by Dom ow in 1969 [ 27 ], who conside ed ha he co ec name o he genus should be Tinaminyssus. Mic oo ganisms 2023,11, 1783 9 o 14 4.2. Molecula Analysis As shown in he Supplemen a y Ma e ials, Table S4, he leng h o ITS1 a ied be ween 232 bp in S e nos oma ulicae and 193 in Rhinonyssus ingae. The 5.8S DNA gene anged om 189 bp in P ilonyssus hi s i o 177 in Rhinonyssus echinipes. The ITS2 agmen was less a iable in size, wi h all species showing alues e y close o 90. These alues a e e y simila o hose ound by de Rojas e al. [ 64 ] and Úbeda e al. [ 66 ]. In addi ion, acco ding o he GenBank da abase, membe s o he De manysoidea supe amily (De manyssus longipes FM179377 and And olaelaps casalis AM903317) showed simila alues o ITS1 and o he 5.8s DNA gene, wi h he ITS2 egion being signi ican ly sho e (73 and 62 bp, espec i ely). The con en s o he di e en bases a e shown in Supplemen a y Table S4 as pe cen ages. As can be seen in Table S4, ou esul s we e p ac ically iden ical o hose ob ained by de Rojas e al. [64] o a smalle numbe o hinonyssid species. Likewise, he leng hs o he ma ke s conside ed in he di e en species s udied he ein, as shown in Table S4, we e also simila o hose ob ained by Úbeda e al. [ 66 ] o species o he genus Rhinonyssus, anging om 468 bp o R. himan opus o 506 in R. neglec us o 540 bp o S e nos oma boydi [ 64 ], as well as o hose o o he ep esen a i es o he supe amily De manysoidea, such as De manyssus longipes (accession numbe FM179377). Wi h ega d o he a iabili y o he di e en agmen s (Table 1), as expec ed, he 5.8S DNA gene was he mos conse ed egion, while he ITS1 egion was he longes and mos a iable. Finally, ITS2 was he sho es and, unusually, qui e p ese ed agmen , a leas among species om he same genus. This ac was obse ed by Na ajas e al. [ 36 ] in mi es o he amily Phy oseiidae, and by de Rojas e al. [ 64 ] in ep esen a i es o he Rhinonyssidae. 4.3. Analysis o Phylogene ic and Gene ic Dis ances I was no un il 1979 ha Pence a emp ed o add ess phylogeny in his amily o mi es. Pence, on he basis o Cas o’s obse a ions [ 13 ], hypo hesized ha he chelice ae would ha e ai ly s able mo phological cha ac e is ics, since hey a e ela ed o nu i ion. Acco ding o his hypo hesis, he hinonyssid mi es would come om some ances o o he Mac onysidae in which he mo phology o he chelice ae had some simila i y o hose o he genus Tinaminyssus [ 67 ]. Figu e 2shows d awings o he chelice ae o all species included in his s udy. Thus, acco ding o he phylogene ic ee c ea ed as explained in he Ma e ials and Me hods sec ions and shown in he esul s (Figu e 1), se e al ques ions can be answe ed. The opology o he ee shown in Figu e 1co esponds o ha c ea ed ia he Bayesian in e ence me hod, and is simila o ha ob ained ia he maximum likelihood me hod. Howe e , in he la e case, he boo s ap alues we e sligh ly lowe han he Bayesian p obabili ies. In bo h cases, he ou gene a conside ed in his s udy we e shown o be in di e en clades. This obse a ion coincides wi h he mo phological p oposal o Pence (1979) [ 68 ] ega ding he classi ica ion o gene a based on he mo phology o he chelice ae. Howe e , species o he genus S e nos oma appea ed in a sepa a e b anch, and he species o he o he h ee gene a we e ga he ed oge he in a poly omy, which means ha a mo e conse ed ma ke mus be es ed o esol e he phylogeny a he genus le el. The clade ha includes he species o he genus Tinaminyssus appea s in a common b anch wi h Bayesian pos e io p obabili ies (PP) and ML suppo (BS) o 100/99. Wi hin his g oup, wo di e en b anches can be dis inguished, depending on he mo phological cha ac e is ics: he “melloi” complex (T. melloi, T. s ep opelioides, and T. s ep opeliae) and he “columbae” complex (T. minise osum and T. columbae) [ 18 ]. Howe e , Tinaminyssus bubulci, a pa asi e o pelecani o m bi ds, appea s in he “melloi” g oup wi h he species pa asi izing columbi o m bi ds (100/99). Fu he mo e, wo o he mo phological c i e ia o including a species in he “columbae” g oup a e a lack o pos anal se ae and ba ely de eloped opis hosomal se ae. T. bubulci shows nume ous well de eloped opis hosomal se ae ( ypical o he “melloi” g oup), bu lacks he pos anal se ae. Thus, his appa en mo phological and ecological con adic ion mus be es ed by assaying mo e isola ed o conca ena ed molecula ma ke s in o de o esol e i .