Academic Edi o s: Ra ael
Gu ié ez-López and Alazne
Díez-Fe nández
Recei ed: 12 Decembe 2024
Re ised: 27 Janua y 2025
Accep ed: 30 Janua y 2025
Published: 6 Feb ua y 2025
Ci a ion: Sánchez-Ca ión, S.A.;
Má quez, F.J.; de Rojas, M. U ili y o
28S Ribosomal RNA Gene Domains
o Molecula Classi ica ion and
Phylogeny o Rhinonyssid Mi es.
Pa hogens 2025,14, 156. h ps://
doi.o g/10.3390/pa hogens14020156
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Licensee MDPI, Basel, Swi ze land.
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A icle
U ili y o 28S Ribosomal RNA Gene Domains o Molecula
Classi ica ion and Phylogeny o Rhinonyssid Mi es
Susana A. Sánchez-Ca ión 1, F ancisco J. Má quez 2and Manuel de Rojas 1,*
1Depa men 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 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]
Abs ac : The amily Rhinonyssidae includes endopa asi ic, blood- eeding mi es ha
a e pa asi ic on bi ds and ha emain la gely uns udied despi e hei po en ial ole as
ec o s o ese oi s o a ious pa hogens, like o he De manyssoidea. T adi ionally, he
axonomy o he g oup has been based on mo phome ic cha ac e is ics, which makes
iden i ica ion e y di icul in many g oups o closely ela ed species. On he o he hand,
s udies on he phylogene ic ela ionships wi hin his g oup o mi es ha e been neglec ed
un il he ea ly yea s o he p esen cen u y. In his s udy, wel e species belonging o i e
di e en species complexes we e iden i ied, and domains D1–D3 28S RNA o each one
we e sequenced, o he i s ime, o in es iga e he sequence a ia ion and i s axonomic
implica ions o phylogene ic in e ence. Ou da a indica e ha his molecula ma ke
can e ec i ely di e en ia e be ween species wi hin he “mo acillae”, “sai ae”, “pa i”, and
“hi s i” complexes o he genus P ilonyssus and he “melloi” and “columbae” complexes o
he genus Tinaminyssus. Fu he mo e, he phylogene ic ee ha can be de i ed om he
domain D1–D3 28S RNA sequences p esen ed in his s udy is cong uen wi h he cu en
axonomy o he Rhinonyssidae. This esea ch calls o a eassessmen o he axonomic
s a us o some g oup o species.
Keywo ds: mi es; Rhinonyssidae; molecula sys ema ic; 28S RNA; phylogeny
1. In oduc ion
Rhinonyssid mi es a e haema ophagous endopa asi es o he nasal passages o bi ds,
which a e damaged by he eeding ac i i y o he mi es (Rhinonyssidosis a ium disease) [
1
].
The amily Rhinonyssidae comp ises 11 gene a, wi h a la ge numbe o species. T adi-
ionally, he classi ica ion o he species in his g oup has been ca ied ou acco ding o
mo phome ic cha ac e is ics [
2
–
6
]. Howe e , such mo phological cha ac e is ics a y
wi hin he same species, and con inuous g aded a ia ion may e en o e lap be ween indi-
iduals o di e en species [
7
]. Fu he mo e, he axonomic alue o some mo phological
cha ac e is ics changes acco ding o he genus o species g oup conside ed [3,7].
Thus, he sys ema ics o hinonyssid mi es p ima ily ely on he mo phome ic
cha ac e is ics o adul emales, wi h he mo phome ic ai s o males and p eimagi-
nal s ages being la gely o e looked in he de elopmen o he hinonyssid classi ica ion
sys em [
2
,
8
–
14
]. O e he yea s, he p inciples o cons uc ing he na u al hinonyssid
sys em ha e e ol ed signi ican ly, leading o se e al majo e isions in he sup aspeci ic
classi ica ion. One o he mos challenging aspec s has been es ablishing c i e ia o axa a
he gene ic and sub amilial le els. The ini ial sup aspeci ic classi ica ion o Rhinonyssidae,
Pa hogens 2025,14, 156 h ps://doi.o g/10.3390/pa hogens14020156
Pa hogens 2025,14, 156 2 o 9
which ca ego ized hem as he sub amily Rhinonyssinae wi hin he amily De manyssidae
and included h ee gene a (Rhinonyssus,P ilonyssus, and S e nos omum), was p oposed
by T ouessa in 1895 [
15
]. This classi ica ion was based on mo phological ea u es o he
s igma, gna hosoma, and he scle o iza ion o he emale idiosoma. In 1935, Vi z hum
ele a ed hinonyssids o amily s a us and ecognized se en gene a, using c i e ia such
as he p esence o absence o pe i emes, he numbe o do sal shields on he podosoma
and opis hosoma, and he shape o he mou hpa s o dis inguish gene a. In he ollowing
decades, he hinonyssid axonomic sys em was expanded signi ican ly due o ex ensi e
s udies o hei auna ac oss a ious egions and he desc ip ion o many new species [
16
].
Du ing his ime, addi ional cha ac e is ics such as he shape and size o idiosomal shields,
he p esence o one o wo chelice al inge s, he p esence o he i os e num, and body
chae o axy began o be inco po a ed in o he sys ema ics [2,9,11,13,17–20].
In 1967, B ege o a belie ed ha he o igin o he p ope hinonyssids ook place be o e
he di ision o bi ds in o main phylogene ic lineages [
21
]. Acco ding o he poin o iew,
wo amilies mus be conside ed in his g oup o mi es: he amily Rhinonyssidae and a
second g oup o mi es, conside ed o be an independen and phylogene ically younge
amily, he P ilonyssidae.
Howe e , Fain [
22
,
23
] soon abandoned he a angemen o gene a in o sub amilies,
and cu en ly, no sub amilies a e dis inguished wi hin he amily Rhinonyssidae.
In his pape , Dom ow’s axonomic sys em has been adop ed, bu wi h conside a ion
o mos o Bu enko’s addi ions. The ollowing 11 gene a a e ecognized in he amily Rhi-
nonyssidae: La inyssus,Locus ellonyssus,Mesonyssus,P ilonyssoides,P ilonyssus,Rallinyssus,
Rhinoecius,Rhinonyssus,S e nos oma,Tinaminyssus, and Vi znyssus [14,24].
The high le els o in e gene ic mo phological a iabili y (e.g., be ween Tinaminyssus
and P ilonyssus) and he insigni ican di e ences among some g oups o species o he
same genus (e.g., some species o Rhinonyssus,Tinaminyssus,S e nos oma, o P ilonyssus)
in hinonyssid mi es make he classi ica ion o his amily complica ed. Thus, o e he
yea s, axonomis s ha e es ablished species complexes o pa ially sol e hese p oblems.
Molecula da a analysis seems o be a use ul ool o esol e axonomic ques ions and
iden i ica ion a he species le el.
Phylogene ic analysis p o ides impo an in o ma ion on biodi e si y and axonomy.
Cu en ly, mos mode n axonomic s udies ake a comp ehensi e app oach inco po a ing
bo h mo phology and DNA sequencing [
25
,
26
]. Thus, Dowling e al., in 2010 [
27
], epo ed
he i s la ge-scale phylogene ic ela ionships wi hin De manyssoidea and he e olu ion
o pa asi ic lineages wi hin he supe amily using he 28S RNA egion (domains 1–3).
Mo eo e , Zhao e al. (2020) used 28S DNA domains o e alua e hese ma ke s o DNA
ba coding [28].
The aim o he p esen s udy was o de e mine he le el o a ia ion be ween he
sequences o he D1–D3 domains o he 28S RNA gene o di e en gene a and species o
hinonyssid mi es and o e alua e hei use ulness in phylogene ic analysis and axonomic
s udies wi hin his g oup. The esul ing phylogene ic ee was examined in ligh o he
cu en axonomy o he Rhinonyssidae amily. Fu he mo e, ou da a we e used o conside
some axonomic ques ions, such as he s a us o axa wi hin species complexes and he
u ili y o chelice al mo phology o genus iden i ica ion.
2. Ma e ials and Me hods
2.1. Biological Ma e ial
Twel e species o hinonyssid mi es belonging o wo di e en gene a o he amily
Rhinonyssidae om he nasal passages o Spanish bi ds we e analysed (Table 1). The
ex ac ion o pa asi ic aca o auna om hese bi ds, as well as he ea men o localized
Pa hogens 2025,14, 156 3 o 9
mi es, was ca ied ou acco ding o he me hodology desc ibed by Sánchez Ca ión e al.
(2023) [8].
Table 1. Mi e species conside ed in his s udy showing hei hos bi ds, geog aphic loca ions, and
GenkBank accession numbe s om 28S RNA sequences. The species conside ed o be an ou g oup
only show he GenkBank access numbe .
Mi e Species Hos Bi ds Geog aphic Loca ion Genbank Accession
Numbe
P ilonyssus eu o u di Tu dus me ula Spain PQ726434
P ilonyssus muscicapae Muscicapa s ia a Spain: Cádiz PQ726436
P ilonyssus syl iae Cu uca melanocephala Spain: Mon ellano: Se illa PQ726435
P ilonyssus mo acillae Mo acilla alba Spain: Mon ellano: Se illa PQ726433
P ilonyssus chlo is Chlo is chlo is Spain: Mon ellano: Se illa PQ726430
P ilonyssus hi s i Passe domes icus Spain: Se illa PQ726432
P ilonyssus ingillae F ingilla coelebs Spain: Mon ellano: Se illa PQ726431
Tinaminyssus bubulci Bubulcis ibis Spain: Mon ellano: Se illa PQ726429
Tinaminyssus columbae Columba li ia Spain: Za agoza PQ726426
Tinaminyssus melloi Columba li ia Spain: Za agoza PQ726425
Tinaminyssus s ep opeliae S ep opelia decaoc o Spain: Se illa PQ726427
Tinaminyssus s ep opelioides
S ep opelia u u Spain PQ726428
S ea onyssus occiden alis - GU440594.1
De manyssus gallinae - FJ911771.1
O ni honyssus bu sa - FJ911789.1
Hos s we e acqui ed h ough a ious me hods. Game bi ds, such as S ep opelia u u ,
we e supplied o us by local hun e s. The “Cen o Zoosani a io de Se illa”, esponsible o
he ci y’s bi d popula ion con ol p og am, cap u ed indi idual bi ds and p o ided hem
o us ozen o pa asi e analysis (e.g., S ep opelia decaoc o,Passe domes icus). Many o he
emaining bi d specimens we e ound dead unde di e en condi ions: along oadsides,
due o high- ol age powe lines, o as a esul o ex eme wea he condi ions. Th ee
addi ional sequences, ob ained om GenBank and ep esen ing wo species in he amily
Mac onyssidae and one species in he amily De manyssidae, a e lis ed in Table 1and we e
used as an ou g oup in he phylogene ic analyses.
2.2. Species Iden i ica ion
In his s udy, we conside ed species belonging o wo di e en gene a, Tinaminyssus
and P ilonyssus, and di e en species complexes in each o hem (Table 1). Once he mi es
we e clea ed and moun ed in Hoye ’s medium, a comple e mo phome ic s udy o each
one was ca ied ou based on he scien i ic li e a u e on he amily Rhinonyssidae.
2.3. Molecula S udy
To isola e DNA om indi idual mi es, he NucleoSpin
®
Tissue XS DNA ex ac ion ki
om Mache ey-Nagel (GmbH & Co. KG, Dü en, Ge many) was used. As a p elimina y
s ep, mi es we e c ushed in a 1.5 mL mic ocen i uge ube con aining 20
µ
L o ex ac ion
bu e om he ki , and he manu ac u e ’s p o ocol was ollowed. The p esence o DNA
was checked by pe o ming 0.8% aga ose gel elec opho esis.
Fo ampli ica ion o he D1–D3 28S DNA egion, he ini ial dena u a ion s ep was
ca ied ou a 95
◦
C o 5 min, ollowed by 35 cycles o 30 s a 95
◦
C o dena u a ion, 30 s
a 53
◦
C o p ime annealing, and 45 s a 72
◦
C o p ime ex ension. The inal ex ension
was ca ied ou o 5 min a 72
◦
C. The p ime s used we e as desc ibed by Dowling
e al., (2010) [
27
]: o wa d, 5
′
-GCTGCGAGTGAACTGGAATCAAGCCT-3
′
e e se, and
5′-AGGTCACCATCTTCTTTCGGGTC-3′.
Pa hogens 2025,14, 156 4 o 9
PCR p oduc s we e wo-way sequenced by he Sange me hod by Allgene ics (A
Co uña, Spain).
2.4. Sequence Alignmen s and Phylogene ic Analysis
The sequences o he D1–D3 28S DNA egion we e aligned using MEGA .5.2 and
manually adjus ed, while DAMBE .5.0 [
29
,
30
] was used o op imize he alignmen s. Pai -
wise dis ance ma ices we e de e mined using he wo-pa ame e Kimu a (K2P) model [
31
].
Phylogene ic ees we e cons uc ed om he nucleo ide da a using wo me hods. Maxi-
mum likelihood (ML) ees we e gene a ed using PHYML .3.0 [
32
], and Bayesian in e ence
(BI) ees we e cons uc ed wi h M . Bayes .3.2.7 [
33
]. The subs i u ion models selec ed by
JMODELTEST 2.1.10 included a gene al ime- e e sible model wi h a gamma-dis ibu ed
a e o a ia ion in GTR + G + I o he D1–D3 28S DNA egion. These subs i u ion models
we e used o analyse phylogene ic ela ionships wi h PHYML .3.0. Topology suppo
was examined by boo s apping he o iginal da ase 1000 imes (heu is ic op ion) [
34
,
35
].
Bayesian phylogene ic analyses we e conduc ed using M Bayes 3.2.7 [
33
]. Two indepen-
den Ma ko Chain Mon e Ca lo (MCMC) uns we e pe o med, each wi h ou chains,
o 10,000,000 gene a ions. T ees we e sampled e e y 100 gene a ions. A GTR +
Γ
+ I
model o nucleo ide subs i u ion (nse = 6; a es = in gamma) was employed. A 50%
majo i y- ule consensus ee was cons uc ed a e disca ding he ini ial 25% o ees as
bu n-in (bu nin ac = 0.25). Pos e io p obabili ies we e used o assess nodal suppo . The
phylogene ic ee (Figu e 1) was displayed using he Fig ee 1.3.1 so wa e [36].
Pa hogens 2025, 14, x FOR PEER REVIEW 4 o 9
a 53 °C o p ime annealing, and 45 s a 72 °C o p ime ex ension. The inal ex ension
was ca ied ou o 5 min a 72 °C. The p ime s used we e as desc ibed by Dowling e al.,
(2010) [27]: o wa d, 5′-GCTGCGAGTGAACTGGAATCAAGCCT-3′ e e se, and 5′-AG-
GTCACCATCTTCTTTCGGGTC-3′.
PCR p oduc s we e wo-way sequenced by he Sange me hod by Allgene ics (A
Co uña, Spain).
2.4. Sequence Alignmen s and Phylogene ic Analysis
The sequences o he D1-D3 28S DNA egion we e aligned using MEGA .5.2 and
manually adjus ed, while DAMBE .5.0 [29,30] was used o op imize he alignmen s. Pai -
wise dis ance ma ices we e de e mined using he wo-pa ame e Kimu a (K2P) model
[31]. Phylogene ic ees we e cons uc ed om he nucleo ide da a using wo me hods.
Maximum likelihood (ML) ees we e gene a ed using PHYML .3.0 [32], and Bayesian
in e ence (BI) ees we e cons uc ed wi h M . Bayes .3.2.7 [33]. The subs i u ion models
selec ed by JMODELTEST 2.1.10 included a gene al ime- e e sible model wi h a
gamma-dis ibu ed a e o a ia ion in GTR + G + I o he D1-D3 28S DNA egion. These
subs i u ion models we e used o analyse phylogene ic ela ionships wi h PHYML .3.0. To-
pology suppo was examined by boo s apping he o iginal da ase 1000 imes (heu is ic op-
ion) [34,35]. Bayesian phylogene ic analyses we e conduc ed using M Bayes 3.2.7 [33]. Two
independen Ma ko Chain Mon e Ca lo (MCMC) uns we e pe o med, each wi h ou
chains, o 10,000,000 gene a ions. T ees we e sampled e e y 100 gene a ions. A GTR + Γ + I
model o nucleo ide subs i u ion (nse = 6; a es = in gamma) was employed. A 50% majo i y-
ule consensus ee was cons uc ed a e disca ding he ini ial 25% o ees as bu n-in (bu nin-
ac = 0.25). Pos e io p obabili ies we e used o assess nodal suppo . The phylogene ic ee
(Figu e 1) was displayed using he Fig ee 1.3.1 so wa e [36].
Figu e 1. Phylogene ic ee o di e en gene a and species o he amily Rhinonyssidae based on he
D1-D3 28S RNA agmen . The phylogeny was in e ed using he Bayesian (B) and maximum like-
lihood (ML) me hods and shows a Bayesian opology. The pe cen age o eplica e ees in which he
Figu e 1. Phylogene ic ee o di e en gene a and species o he amily Rhinonyssidae based on
he D1–D3 28S RNA agmen . The phylogeny was in e ed using he Bayesian (B) and maximum
likelihood (ML) me hods and shows a Bayesian opology. The pe cen age o eplica e ees in which
he associa ed axa clus e ed in he boo s ap es (1000 eplica es) is shown in he b anches (B/ML).
Bayesian pos e io p obabili ies (BPPs) ha e been con e ed in o pe cen ages.
Pa hogens 2025,14, 156 5 o 9
3. Resul s
3.1. Species Iden i ica ion
Fo he iden i ica ion o each species, mo phome ic ea u es desc ibed by di e en
au ho s we e conside ed. A comple e mo phome ic desc ip ion o each o he species
conside ed can be ound in Sanchez-Ca ión e al., 2023 [8].
3.2. Molecula Analysis
In he analysis o he aligned sequences, o which we used he p og am MEGA
e sion 5.2, an alignmen o he D1–D3 28S DNA egion o 791 base pai s was ob ained,
including 414 conse ed posi ions and 373 a iable posi ions, as shown in Supplemen a y
Ma e ials Table S1. A low a iabili y was obse ed be ween he leng hs o he di e en
sequences, which ange be ween 777 bp in P ilonyssus hi s i and 791 bp in Tinaminyssus
s ep opeliae,T. columbae,T. s ep opelioides, and T. melloi, wi h a mean o 788 bp. These
leng hs a e sligh ly sho e han hose ound in GenBank o o he De manyssoidea species
such as Laelaps je ma i (832 bp), De manyssus hi su us (824), O ni honyssus we necki (786), o
And olaelaps sp. (819).
The bounda ies o he D1, D2, and D3 domains we e de e mined by compa ison wi h
he 28S DNA domains o he 28S DNA o O ni honyssus baco i, yielding an a e age leng h
o 207 bp o D1, 431 o D2, and 140 o D3.
As o he nucleo ide composi ion desc ibed, we ound highe con en s o adenine,
hymine, and guanine—27.51%, 27.7%, and 27.23%, espec i ely—while he igu e o
cy osine was he lowes a 17.56%. The end o a lowe pe cen age o cy osine and a
pe cen age o be ween 26 and 30% o he es o bases was main ained in o he species o
De manyssoidea species (Supplemen a y Ma e ials Table S1).
3.3. Phylogene ic and Gene ic Dis ance Analyses
The da a on gene ic dis ances and pe cen ages o simila i y among all he species
conside ed in his s udy a e shown in Supplemen a y Ma e ials Table S2.
The ee opology shown in Figu e 1co esponds o he one c ea ed by he Bayesian
in e ence me hod and is simila o he one ob ained by he maximum likelihood me hod.
In bo h me hods, he di e en clades p esen a high deg ee o suppo .
In his ee, we can obse e wo well-suppo ed clades wi h Bayesian p obabili ies
(PP) and ML suppo (BS) o 100/100, each o which includes one o he wo gene a ha
we e conside ed in his s udy. In addi ion, he clade co esponding o he genus P ilonyssus
esol es in o wo o he subclades wi h suppo s o 100/95 and 99.99/100 ha include
g oups o species wi h mo phological di e ences in hei chelice ae. This obse a ion
ag ees wi h he mo phological p oposal o Pence (1979) [
37
] on he classi ica ion o gene a
based on hei chelice al mo phology.
The clade, including species o he genus Tinaminyssus, appea s on a common b anch
wi h Bayesian p obabili ies (PP) and ML suppo (BS) o 100/99. Wi hin his g oup, wo
di e en b anches a e dis inguished, based on hei mo phological and ecological cha ac-
e is ics: species ha pa asi ize columbi o m bi ds and a b anch ha includes T. bubulci, a
species ha is pa asi ic on pelecani o mes.
In u n, he clade g ouping he pa asi ic species o columbi o mes p esen s wo sub-
clades (98/77) g ouping wo species complexes, espec i ely: he “melloi” complex (T.
melloi,T. s ep opelioides, and T. s ep opeliae) and he “columbae” complex (T. columbae) [
38
].
Pa hogens 2025,14, 156 6 o 9
4. Discussion
Phylogene ic s udies o he Rhinonyssidae amily ha e p ima ily elied on mo pho-
logical cha ac e is ics since Pence’s pionee ing wo k in 1979 [
37
]. The i s molecula
phylogene ic s udy was conduc ed by de Rojas e al. (2001), ma king a signi ican ad ance-
men in ou unde s anding o his g oup [26].
Ou analysis, al hough limi ed o a single indi idual pe species due o sample a ail-
abili y, demons a ed he high esol ing powe o he 28S RNA gene, e ec i ely delinea ing
a clea phylogene ic s uc u e among he 15 species examined. This inding suppo s he
selec ion o 28S as a sui able ma ke , which is consis en wi h p e ious s udies ha epo ed
limi ed in aspeci ic a ia ion in a ian in anasal aca i using ma ke s such as ITS1-5.8S-ITS2
and COI [39].
The choice o he 28S RNA gene as a molecula ma ke in his s udy is u he sup-
po ed by a obus body o esea ch. P e ious s udies ha e highligh ed he u ili y o he
nuclea ibosomal DNA 28S, pa icula ly he D3 egion, in esol ing phylogene ic ela-
ionships [
40
,
41
]. Fo ins ance, in O iba ida mi es, Lehmi z and Decke (2017) ound ha
93% o he species s udied (83/89) exhibi ed a unique 28S D3 sequence [
41
]. Howe e , he
D1–D3 egion o he LSSU should be used wi h cau ion, as i may exhibi low in e speci ic
polymo phism and homoplasy in ce ain aca ian g oups, such as Rhipicephalus icks [
42
].
Ne e heless, in he p esen s udy, he esul s ob ained clea ly delinea e he phylogene ic
s uc u e o he 15 species ha we e compa ed. An ono skaia (2018) p o ides a comp ehen-
si e o e iew o he ad an ages and disad an ages o commonly used nuclea (18S,5.8S,
28S,ITSI, and ITSII) and mi ochond ial (COI,NADI,NADII, and cy b) ma ke s [
43
]. In he
case o T ombiculidae, COI was shown o di e en ia e closely ela ed species o Lep o om-
bidium by compa ing 16 amino acid posi ions. In conclusion, he combined esul s o ou
s udy and he li e a u e suppo he use o 28S as a obus and disc imina o y molecula
ma ke o esol ing phylogene ic ela ionships among he species examined.
Fu he mo e, an analysis o he a ailable 28S RNA sequences in GenBank, encom-
passing a b oad ange o aca i axa and di e se geog aphic o igins, sugges s ha his gene
egion exhibi s lowe le els o in apopula ion a ia ion. A compa ison o a 28S RNA
agmen (posi ions 25–822) among De manyssidae species, using D. gallinae (accession
MT813465) as a e e ence, e ealed a high deg ee o simila i y (99.87–99.25%) among D.
gallinae indi iduals. This simila i y dec eased o 95.86% when compa ed wi h D. hi undinis
and d opped below 91% o mo e dis an ly ela ed axa.
In he ee opology shown in Figu e 1(Bayesian opology), we can obse e wo
well-suppo ed clades wi h Bayesian p obabili ies (PP) and ML suppo (BS) o 100/100,
each o which includes one o he wo gene a ha we e conside ed in his wo k. The ML
opology is shown in Supplemen a y Ma e ials Figu e S1.
In addi ion, he clade co esponding o he genus P ilonyssus esol es in o wo o he
subclades wi h suppo s o 100/95 and 99.99/100 ha include g oups o species wi h
mo phological di e ences in hei chelice ae. This obse a ion coincides wi h he mo pho-
logical p oposal o Pence (1979) [
37
] on he classi ica ion o gene a based on hei chelice al
mo phology (Supplemen a y Ma e ials Figu e S2).
The clade, including species o he genus Tinaminyssus, appea s on a common b anch
wi h Bayesian p obabili ies (PP) and ML suppo (BS) o 100/99. Wi hin his g oup, wo
di e en b anches a e dis inguished based on hei mo phological and ecological cha ac-
e is ics: species ha pa asi ize columbi o m bi ds and a b anch ha includes T. bubulci, a
species ha is pa asi ic on Pelecani o mes.
In u n, he clade g ouping he pa asi ic species o columbi o mes p esen s wo sub-
clades (98/77) g ouping wo species complexes, espec i ely: he “melloi” complex (T.
melloi,T. s ep opelioides, and T. s ep opeliae) and he “columbae” complex (T. columbae) [7].
Pa hogens 2025,14, 156 7 o 9
These esul s a e compa ible wi h hose ob ained by Sánchez-Ca ión e al. in 2023 [
8
]
based on he s udy o he ITS1-5.8S-ITS2 agmen .
On he o he hand, he genus P ilonyssus includes he la ges numbe o species wi hin
he amily Rhinonyssidae. The species ha a e included in his genus a e e y simila om
a mo phome ical poin o iew, which has o ced axonomis s o g oup hem in o species
complexes such as “hi s i”, “lanii”, “mo acillae”, “o honychus”, “pa i”, and “sai ae” [
42
].
In his s udy, h ee species o he “hi s i” complex, h ee o he “sai ae” complex, and one
o he “mo acillae” complex we e included.
The opology o he Bayesian phylogene ic ee o he species clade o he genus
P ilonyssus g oups wo sis e b anches wi h high suppo (100/100). One con ains species o
he “hi s i” complex (P. hi s i and P. chlo is) and “pa i” (P. ingillae), and he o he con ains
species o he “sai ae” complex (P. eu o u di,P. muscicapae, and P. syl iae) and “mo acillae”
(P. mo acillae).
The i s o he b anches men ioned abo e, wi h suppo o 99.99/95, g oups species
ha p esen chelice ae wi hou a widened base. The second includes species wi h e y
simila mo phologies and p esen ing chelice ae wi h a la ed base, whose clade is also well
suppo ed wi h 100/95 alues.
I is impo an o no e ha all species ha a e included in his clade a e e y sim-
ila om a mo phological poin o iew, no only in he basal widening o hei che-
lice ae bu also in he numbe o do sal scu es and hei shape, as well as do sal and
en al chae o axia.
Thus, he gene ic dis ances be ween hese species (P. eu o u di,P. muscicapae,P. syl iae,
and P. mo acillae) a e in he same o de o magni ude as hose ob ained be ween T. s ep-
opeliae and T. s ep opelioides, which can be conside ed c yp ic species. On he o he hand,
i is impo an o no e ha he dis ances be ween species o P ilonyssus belonging o ei he
clade o his genus a e simila o hose ob ained be ween species o di e en gene a, which
poin s o he possibili y ha hey a e closely ela ed gene a. This ac con i ms he obse -
a ions o Sánchez-Ca ión e al., 2023 [
8
], who ob ained simila esul s by analysing he
ITS1-5.8S-ITS2 agmen .
These esul s sugges he need o es ablishing c i e ia o delimi ing species and
de e mining disc imina o y cha ac e is ics in g oups o closely ela ed species o he amily
Rhinonyssidae.
5. Conclusions
F om da a ob ained in his s udy, i can be concluded ha , al hough he analysis o
he D1–D3 28S RNA agmen sol es di e en axonomic and phylogene ic p oblems a
he genus and species le els, i would be necessa y o es new molecula ma ke s ha can
co obo a e in e gene ic ela ionships o iden i y closely ela ed species ha a e included
in species complexes. In addi ion, a ho ough e ision o he classi ica ion o his amily
and a speci ic mo phome ic and molecula iden i ica ion ha allows o he de e mina ion
o he disc imina o y ai s o his g oup o mi es is essen ial.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps:
//www.mdpi.com/a icle/10.3390/pa hogens14020156/s1: Table S1: Nucleo ide composi ion o he
species sequences desc ibed in his s udy; Table S2: Ma ix o gene ic dis ances (lowe le co ne ) and
pe cen ages o simila i y be ween hem (uppe igh co ne ) be ween he di e en species conside ed
in his s udy; Figu e S1: Phylogene ic ee o di e en gene a and species o he amily Rhinonyssidae
based on he D1–D3 28S RNA agmen . Figu e S2: Mo phology o he chelice ae o he species
iden i ied in his s udy. Scales a e shown in µm (modi ied om Sánchez-Ca ión, 2023 [8]).
Pa hogens 2025,14, 156 8 o 9
Au ho Con ibu ions: F.J.M. and M.d.R. concei ed his s udy., S.A.S.-C. and M.d.R. analysed he
da a. M.d.R. and S.A.S.-C. w o e he i s d a , which all o he au ho s e ised. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch was unded by a V Plan P opio de In es igación de la Uni e sidad de Se illa,
Spain, g an o he Depa men o Mic obiology and Pa asi ology, Uni e si y o Se ille.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Sequences o each o he species analysed in his s udy a e a ailable
om NCBI (GenBank accession numbe s: om PQ726425 o PQ726436).
Acknowledgmen s: We hank Cen o Municipal Zoosani a io de Se illa, and especially F ancisco
Peña Fe nández and Ra ael Cuad ado Nie o, o p o iding some o he samples. We also hank
Miguel Fe nández Almena a o bi d iden i ica ions.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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