Mitogenome of a new Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered in rock pools along with its temperature and desiccation-related proteins repertoire

Vol.:(0123456789)
O ganisms Di e si y & E olu ion (2025) 25:119–135
h ps://doi.o g/10.1007/s13127-024-00662-x
ORIGINAL ARTICLE
Mi ogenome o anew Ramazzo ius species (Ta dig ada: Eu a dig ada:
Ramazzo iidae) disco e ed in ock pools alongwi hi s empe a u e
anddesicca ion‑ ela ed p o eins epe oi e
Ma eoVecchi1 · DanielS ec1
Recei ed: 6 Augus 2024 / Accep ed: 9 Oc obe 2024 / Published online: 31 Oc obe 2024
© The Au ho (s) 2024
Abs ac
Ramazzo ius is a widesp ead genus o a dig ades wi h ex eme c yp obio ic capabili ies. Thanks o i s abili y o su i e
desicca ion and eezing, his genus is usually eco ded om ha sh habi a s such as exposed mosses and lichens and ock
pools. In he las yea s, esea ch ocused on bo h desc ibing Ramazzo ius di e si y and e ealing he molecula mechanisms
behind hei c yp obio ic capabili ies. Despi e he esea ch e o s in hese ields, much s ill emains o be disco e ed. He e
we desc ibe a new Ramazzo ius species om an I alian ock pool by means o in eg a i e axonomy (mo phology, mo pho-
me y, and DNA sequencing) and sequenced i s genome wi h Nanopo e echnology o p o ide an assembled mi ogenome
and anno a e i s Tempe a u e and Desicca ion Resis ance P o eins (TDPR) epe oi e. The new gonocho ic species is phy-
logene ically close o he pa henogene ic R. a ieo na us, a s ain o which (YOKOZUNA-1) has been adop ed as model
o ganism o he s udy o c yp obiosis. The mi ogenome o he new species shows pe ec syn eny wi h R. a ieo na us
and sha es wi h i mos o he TDPR genes. The ela i e gene ic simila i y o he new species o he model R. a ieo na us,
combined wi h unique biological ai s ( o example he di e ence in ep oduc i e mode and he unique habi a i colonizes),
makes he new species a po en ial new addi ion o he ange o model a dig ade species.
Keywo ds Ramazzo ius· Anhyd obiosis· Nanopo e· New species· Mi ogenome
In oduc ion
Ta dig ades (also known as wa e bea s) a e a g oup o
mic oscopic animals o ming a phylum included in he clade
Pana h opoda (Schill, 2018). These meio aunal animals
li e in a wide a ie y o habi a s: om aqua ic o e es-
ial en i onmen s, om seas o moun ains (Nelson e al.,
2018), whe e hey can be ound in di e en subs a es such
as sedimen s, soil, b yophy es, lichens, and e en in c yo-
coni e holes on glacie s (Nelson e al., 2018; Zawie ucha
e al., 2016). Thanks o hei abili y o en e in o ame a-
bolic li e s ages (c yp obiosis), some a dig ades can su i e
ex eme condi ions like desicca ion and eezing (Henghe
& Schill, 2018; Rebecchi e al., 2007; Schill & Henghe ,
2018; Zawie ucha e al., 2023).
Ramazzo ius (Binda & Pila o, 1986) is a genus o lim-
no e es ial a dig ades cha ac e ized by he p esence o
apophyses o he inse ion o he s yle muscles (AISM)
in he shape o blun hooks, a icula ed ex e nal claws, and
some imes pai ed ellip ical o gans on he head. Rep esen a-
i es o Ramazzo ius a e conside ed xe ophilic, o en ound
in habi a s exposed o sunligh , o en dwelling in b yophy es
and lichens (Ba els e al., 2011; Bise o , 1997; Guide i
e al., 2022), bu su p isingly also in mo e aqua ic habi a s
like ock pools sedimen (Vecchi e al., 2022). The genus
Ramazzo ius includes di e en species complexes wi h
gene ally un esol ed phylogene ic ela ionships among and
inside hem (Dey e al., 2024; Kihm e al., 2023). The mos
common and widesp ead g oup is he polyphyle ic obe -
haeuse i mo pho-g oup, cha ac e ized by eggs wi h hemi-
sphe ical p ocesses and ampan c yp ic and pseudoc yp-
ic specia ion (Guide i e al., 2022; S ec e al., 2018). The
monophyle ic baumanni and szep yckii complexes om he
opics a e cha ac e ized by peculia o namen ed cu icle and
* Ma eo Vecchi
ma eo. [email p o ec ed]; m. [email p o ec ed]ow.pl
1 Ins i u e o Sys ema ics andE olu ion o Animals, Polish
Academy o Sciences, Sławkowska 17, 31-016, K aków,
Poland
120 M.Vecchi, D.S ec
hei eggs a e unknown (Dey e al., 2024). C yoconicus,
ecen ly sepa a ed om Ramazzo ius and cha ac e ized by
peculia AISM and claws mo phology and by an in ense
body pigmen a ion (Guide i e al., 2019; Zawie ucha e al.,
2018), has been sugges ed o no be alid (Dey e al., 2024),
and hus be pa o Ramazzo ius. Addi ionally, species ha
do no all in o any o hese g oups a e p esen and a e gen-
e ally cha ac e ized by body wi h ela i ely plain sculp u e
(compa ed o he baumanni and szep yckii complexes) and
wi h egg p ocesses usually in he shape o spines and cones
(Be olani & Kinchin, 1993; Bise o , 1997; Guide i e al.,
2019; Kihm e al., 2023; S ec e al., 2016).
The genus Ramazzo ius made i s way in o becoming
a model axon o he s udy o ex eme esis ance mecha-
nisms in a dig ades and hei biological in e ac ions (see
o example Emdee e al., 2024; Ho ikawa, 2008; Ho ikawa
e al., 2008, 2013); howe e , a his ime, expe imen al and
genomic da a a e a ailable o only one species o he genus
(R. a ieo na us Be olani & Kinchin, 1993 s ain YOKO-
ZUNA-1; Ho ikawa e al., 2008), p ecluding a compa a i e
app oach ac oss ela ed species.
Ta dig ades om ock pools ha e been in es iga ed ho -
oughly only ecen ly (Vecchi e al., 2022); howe e , wo new
species om hese habi a s ha e been disco e ed and al eady
desc ibed (Vecchi e al., 2023a, 2023b) showing hei po en-
ial as sou ce o a dig ades biodi e si y. Ramazzo ius om
ock pools ha e been shown o possess an inc edible ole -
ance o eeze– haw cycles (Zawie ucha e al., 2023), e en
highe han a dig ades axa adap ed o li e on glacie s, high-
ligh ing he ex eme adap a ions encoun e ed in a dig ades
om his habi a .
Ramazzo ius claudii sp. no . om a ock pool in he
I alian Apennines is desc ibed he e by means o in eg a i e
axonomy using ligh mic oscopy (LM), scanning elec on
mic oscopy (SEM), and DNA sequencing. To p o ide he
communi y wi h mo e genomic da a o help un a el he
esis ance mechanisms o a dig ades, we also p o ide o
he i s ime in a a dig ade species desc ip ion, i s anno-
a ed whole mi ochond ial genome and i s epe oi e o Tem-
pe a u e and Desicca ion-Rela ed P o eins (Fleming e al.,
2024) sequenced wi h long- ead hi d gene a ion sequencing
echnology (Nanopo e).
Ma e ials andme hods
Sampling and a dig ades ex ac ion
A ock pool sedimen sample was collec ed on 27/06/2020 in
Co niglio, Pa ma, I aly (44.3960680 10.00437) by sc aping
he sedimen wi h a clean plas ic spoon in o a plas ic ube.
The sample was kep desicca ed and ozen a − 20°C un il
p ocessing. The sample was p ocessed o ex ac a dig ades
as in (Vecchi e al., 2023a). The sample was collec ed unde
sampling pe mi N.0001671/2020 om Pa co Nazionale
Appennino Tosco-Emiliano (I aly).
Addi ional ma e ial
Indi iduals o Hebesuncus conjungens (Thulin, 1911) we e
ex ac ed om a moss collec ed on 15/06/2023 in Co niglio,
Pa ma, I aly (44.3800370 10.0414130) o DNA sequencing
(Sample collec ed unde sampling pe mi N.0003526/2021
om Pa co Nazionale Appennino Tosco-Emiliano, I aly).
P e iously ex ac ed Ramazzo ius indi iduals (S66.01 and
S132.02 om Vecchi e al., 2022), and al eady sequenced
o COI, we e used o sequencing addi ional ma ke s (18S,
28S and ITS2).
Mic oscopy andimaging
Specimens o ligh mic oscopy we e moun ed on mic o-
scope slides in a small d op o Hoye ’s medium, secu ed
wi h a co e slip and d ied a 50°C o a week. Slides we e
examined unde a Leica DMLB ligh mic oscope wi h phase
con as (PCM), associa ed wi h a digi al came a. Fo s uc-
u es ha could no be sa is ac o ily ocused on a single ligh
mic oscope pho og aph, a s ack o 2–5 images we e aken
wi h an equidis ance o ca. 0.2μm and assembled manu-
ally in o a single deep- ocus image in GIMP .2–10 (GIMP
De elopmen Team, 2019). Specimens o scanning elec-
on mic oscopy we e p epa ed acco ding o he p o ocol
o (Cama da e al., 2023). Specimens we e examined unde
high acuum in a Ve sa 3D Dual Beam Scanning Elec on
Mic oscope a he ATOMIN acili y o he Jagellonian Uni-
e si y, K aków, Poland. Fo s uc u es ha could no be
sa is ac o ily ocused in a single pho og aph, a s ack o 2–6
images we e aken wi h an equidis ance o ca 0.2μm and
assembled manually in o a single deep- ocus image. All ig-
u es we e assembled in Figu e J (Mu e e & Zinck, 2013).
Mo phome ics andmo phological nomencla u e
All measu emen s a e gi en in mic ome e s (μm). S uc u es
we e measu ed only i hei o ien a ion was sui able. Body
leng h was measu ed om he an e io ex emi y o he pos-
e io end o he body, excluding he hind legs. Buccal ube
leng h and he le el o he s yle suppo inse ion poin we e
measu ed acco ding o (Pila o, 1981). The p index is he
a io o he leng h o a gi en s uc u e o he leng h o he
buccal ube (Pila o, 1981). Claws we e measu ed acco d-
ing o (S ec e al., 2018) and a ios a e p o ided acco ding
o (Vecchi e al., 2023b). Mo phome ic da a we e handled
using he “Pa achela” e . 1.7 empla e a ailable om he
Ta dig ada Regis e (Michalczyk & Kaczma ek, 2013).
121
Mi ogenome o anew Ramazzo ius species (Ta dig ada: Eu a dig ada: Ramazzo iidae)…
The aw mo phome ic da a a e p o ided as Supplemen a y
Ma e ials (Online esou ces 01).
Geno yping
DNA was ex ac ed om indi idual animals ollowing a
Chelex® 100 esin (BioRad) ex ac ion me hod by Casque
e al. (2012) wi h modi ica ions desc ibed in de ail in S ec
e al. (2020). B ie ly, animals we e indi idually placed in
40μl o a 4% Chelex® solu ion wi h he addi ion o 3μl
o p o einase K (20mg/ml, ac i i y ≥ 30 U/mg) and incu-
ba ed in a he momixe a 56°C and 750 pm o 30min.
The solu ion was hen hea ed a 70°C o 10min and he
supe na an was ans e ed o a new mic ocen i uge ube
and used as inpu o PCR. We sequenced ou DNA ag-
men s, h ee nuclea (18S RNA, 28S RNA, and ITS2) and
one mi ochond ial (COI). All agmen s we e ampli ied and
sequenced acco ding o he p ime s and p o ocols desc ibed
in S ec e al. (2020). Sequencing p oduc s we e ead wi h he
ABI 3130xl sequence a he Genomed company (Wa saw,
Poland).
Phylogene ic econs uc ion
A phylogene ic econs uc ion was made using he conca -
ena ed ma ke s 18S RNA + 28S RNA + COI + ITS2 om
Ramazzo ius clade A (sensu Dey e al., 2024), wi h he addi-
ion o sequences om Ramazzo ius k e schmanni Guide i
e al., 2022, Ramazzo ius g oenlandensis Kihm e al., 2023
and he sequences newly gene a ed o his s udy. Sequences
o H. conjugens we e used as ou g oup. The GenBank acces-
sion numbe s o he sequences and hose used in he phylo-
gene ic econs uc ion a e p esen ed in Table1.
The 18S RNA, 28S RNA, and ITS2 sequences we e
aligned wi h MAFFT e . 7 (Ka oh, 2002; Ka oh & Toh,
2008) wi h he G-INS-i me hod ( h ead = 4, h ead b = 5,
h eadi = 0, eo de , adjus di ec ion, anysymbol, maxi e -
a e = 1000, e ee 1, globalpai inpu ). The COI sequences
we e aligned acco ding o hei amino acid sequences ( ans-
la ed using he in e eb a e mi ochond ial code) wi h he
MUSCLE algo i hm (Edga , 2004) in MEGA7 wi h de aul
se ings (all gap penal ies = 0, max i e a ions = 8, clus e ing
me hod = UPGMB, lambda = 24). Alignmen s we e isually
inspec ed and immed in MEGA7. Sequences we e conca -
ena ed wi h he R package “conca ipede” 1.0.0 (Vecchi &
B uneaux, 2021).
Model selec ion and maximum likelihood (ML) phy-
logene ic econs uc ion we e pe o med wi h IQTREE
(T i inopulos e al., 2016) on he pa i ioned da ase on he
IQTREE web se e (h p:// iq ee. cibi . uni ie. ac. a /) wi h
de aul pa ame e s and ul a as boo s ap wi h 1000 epli-
ca es. The conca ena ed alignmen is a ailable as Supple-
men a y Ma e ials (DATA block o Online esou ces 02).
Model selec ion esul s a e a ailable as Supplemen a y
Ma e ial (Online esou ces 03).
Fo Bayesian In e ence (BI) phylogene ic econs uc-
ion, model selec ion was pe o med o each alignmen
pa i ion (6 in o al: 18S RNA, 28S RNA, ITS-2 and h ee
COI codons) using Pa i ionFinde 2 (Lan ea e al., 2016).
Bayesian in e ence (BI) phylogene ic econs uc ion was
pe o med using M Bayes 3.2.6 (Ronquis e al., 2012).
Two uns (one cold chain and h ee hea ed chains each) o
20 million gene a ions we e used wi h a bu n-in o 2 mil-
lion gene a ions, sampling a ee e e y 1000 gene a ions.
Pos e io dis ibu ion sani y was checked using T ace 1.7
(Rambau e al., 2018). The M Bayes inpu ile wi h he
inpu alignmen is a ailable as Supplemen a y Ma e ials
(Online esou ces 02); model selec ion esul s a e a ailable
as Supplemen a y Ma e ial (Online esou ces 03). Bo h he
IQTREE ee and he M Bayes consensus ee a e a ailable
as Supplemen a y Ma e ials (Online esou ces 04). The
phylogene ic ees we e isualized wi h FigT ee (Rambau ,
2007) and edi ed wi h Inkscape (Inkscape P ojec , 2020).
Whole‑genome ampli ica ion andsequencing
One indi idual Ramazzo ius o he new species (Ram_
IT.158_WGA_1) was s a ed in s e ile dis illed wa e o
24h a 18°C and hen washed wice in s e ile dis illed wa e .
The indi idual was hen dissec ed in a 0.5-μl d op o s e ile
dis illed wa e wi h a s e ilized en omological needle and
used as s a ing ma e ial o a whole-genomic ampli ica-
ion (WGA) eac ion using he REPLI-g Mini Ki (Ca . No.
150023, Qiagen) acco ding o he manu ac u e p o ocol.
The eac ion p oduc was pu i ied wi h a GeneMAGNET
PCR/DNA Clean-Up Pu i ica ion Ki (Ca . No. E3420,
EURx) and he dsDNA was quan i ied using a Qubi Fluo-
ome ic assay. App oxima ely 2μg o ampli ied DNA
we e deb anched o 1h wi h T7 Endonuclease I (Ca . No.
M0302, New England Biolabs) acco ding o he manu ac-
u e p o ocol. The eac ion p oduc was again pu i ied wi h
GeneMAGNET and quan i ied wi h Qubi . The dsDNA was
used as inpu o lib a y p epa a ion wi h he Ox o d Nanop-
o e Na i e Ba coding Ki 24 14 (Ca . No. SQK-NBD114.24,
Ox o d Nanopo e Technologies—ONT) ollowing he ONT
communi y p o ocol NBE_9169_V114_REVP_15SEP2022
and sequenced on pa o a FLO-MIN114 R10 low cell
using a MinION Mk1B o 48h. Basecalling, demul i-
plexing, and adap o s imming we e done wi h he so -
wa e MinKNOW (ONT) using a as basecalling s a egy.
Sequences o he DNA con ol sample (DCS) in oduced
du ing lib a y p epa a ion we e emo ed wi h choppe (De
Cos e & Rademake s, 2023). The aw eads we e submi -
ed o NCBI SRA (BioP ojec PRJNA1082523, accession
numbe SRR28173874).
122 M.Vecchi, D.S ec
Mi ogenome assembly andanno a ion
The clean eads we e blas ed agains a a dig ades mi o-
chond ial p o eins da abase (compiled om a dig ade mi o-
chond ial p o ein sequences p esen in GenBank; Online
esou ces 05) using DIAMOND (Buch ink e al., 2015;
op ions: –sensi i e –max- a ge -seqs 1) and he ma ching
que y sequences we e ex ac ed wi h seq k (h ps:// gi hub.
com/ lh3/ seq k) and assembled wi h Flye 2.9.3 (Kolmogo-
o e al., 2019; Lin e al., 2016) wi h 5 polishing ound
(op ions: -i 5). The assembled sequences we e isualized
wi h Bandage (Wick e al., 2015) and he iden i y o he
mi ogenome con ig con i med by blas n agains he Ramaz-
zo ius a ieo na us YOKOZUNA-1 mi ogenome (GenBank
NC_031407). A inal polishing o he e ie ed mi ogenome
con ig was done wi h medaka 1.11.3 (h ps:// gi hub.
Table 1 GenBank accession numbe s o he sequences used in he phylogene ic econs uc ion
SSU (18S) LSU (28S) COI ITS2 Sou ce
Ramazzo ius claudii n.sp. IT.158.1 PQ108467 PQ108476 PQ109084 PQ110585 This s udy
Ramazzo ius claudii n.sp. IT.158.3 PQ108468 PQ108477 PQ109085 PQ110586 This s udy
Ramazzo ius claudii n.sp. S62 1 MW306836 Vecchi e al. (2022)
Ramazzo ius claudii n.sp. S62 2 MW306835 Vecchi e al. (2022)
Ramazzo ius claudii n.sp. S66 1 PQ108470 PQ108475 MW306833 PQ110584 This s udy; Vecchi e al. (2022)
Ramazzo ius claudii n.sp. S66 2 MW306834 Vecchi e al. (2022)
Ramazzo ius claudii n.sp. S67 1 MW306832 Vecchi e al. (2022)
Ramazzo ius AT.002 b MG432811 MG432814 S ec e al. (2018)
Ramazzo ius DE.002 a MG573257 S ec e al. (2018)
Ramazzo ius DE.002 b MG432817 MG432812 MG432815 Zawie ucha e al. (2018)
Ramazzo ius g oenlandensis 1 OR600266 OR600265 OR596527 Kihm e al. (2023)
Ramazzo ius g oenlandensis 2 MG432810 Zawie ucha e al. (2018)
Ramazzo ius g oenlandensis 3 EU251381 Fau by e al. (2008)
Ramazzo ius g oenlandensis 4 EU251382 Fau by e al. (2008)
Ramazzo ius k e schmanni T1 OM370801 OM402517 Guide i e al. (2022)
Ramazzo ius k e schmanni T2 OM370802 OM402518 Guide i e al. (2022)
Ramazzo ius k e schmanni T3 OM370803 OM402519 Guide i e al. (2022)
Ramazzo ius k e schmanni T4 OM370804 OM402520 Guide i e al. (2022)
Ramazzo ius obe haeuse i FR.12 MG573241 MG573242 MG573244 MG573243 S ec e al. (2018)
Ramazzo ius PL.023 MG573253 S ec e al. (2018)
Ramazzo ius PL.140 1 MG573248 S ec e al. (2018)
Ramazzo ius PL.140 2 MG573249 S ec e al. (2018)
Ramazzo ius PL.141 MG573247 S ec e al. (2018)
Ramazzo ius PT.010 1 MG573245 S ec e al. (2018)
Ramazzo ius PT.010 2 MG573246 S ec e al. (2018)
Ramazzo ius Rama1 EU251380 Fau by e al. (2008)
Ramazzo ius Rama2 EU251379 Fau by e al. (2008)
Ramazzo ius Ro-Ni aa-1 EF620418 EF620419 Møbje g e al. (2007)
Ramazzo ius S132 1 PQ108469 PQ108474 MW306838 PQ110583 This s udy; Vecchi e al. (2022)
Ramazzo ius S132 2 MW306837 Vecchi e al. (2022)
Ramazzo ius S49 1 MW306839 Vecchi e al. (2022)
Ramazzo ius S49 2 MW306840 Vecchi e al. (2022)
Ramazzo ius S67 2 MW306841 Vecchi e al. (2022)
Ramazzo ius subanomalus 1 MF001997 MF001998 MF001999 MG432819 S ec e al. (2017)
Ramazzo ius subanomalus 2 KU900021 KU900019 S ec e al. (2016)
Ramazzo ius Ta 398 FJ435728 FJ435799 Guil and Gi ibe (2012)
Ramazzo ius Ta 400 FJ435727 FJ435800 Guil and Gi ibe (2012)
Ramazzo ius a ieo na us YOKOZUNA-1 MG432818 MG432813 MG432816 Zawie ucha e al. (2018)
Hebesuncus conjugens IT.148.2 PQ108465 PQ108478 PQ109086 PQ110587 This s udy
Hebesuncus conjugens IT.148.3 PQ108466 PQ108479 PQ109087 PQ110588 This s udy
123
Mi ogenome o anew Ramazzo ius species (Ta dig ada: Eu a dig ada: Ramazzo iidae)…
com/ nanop o e e ch/ medaka) using all he o iginal eads.
The mi ochond ial genome was anno a ed wi h MITOS2
(Be n e al., 2013; Dona h e al., 2019) on he Galaxy
Eu ope web se e (h ps:// usega laxy. eu/). The anno a ion
was inspec ed and cu a ed by hand and a GenBank la ile
was p oduced using he sc ip s mi os2 as a.py and aln2 bl.
py (h ps:// gi hub. com/ IMEDEA/ mi og enomi cs), and
able2asn (h ps:// p. ncbi. nlm. nih. go / asn1- con e e s/ by_
p og am/). The mi ogenome was isualized wi h OGDRAW
(G eine e al., 2019). Co e age da a was ex ac ed om he
medaka BAM iles using he R package “gmo iz” (Zeglinski
e al., 2021). Di e gence and syn eny o he mi ochond ial
genomes o he new species and R. a ieo na us YOKO-
ZUNA-1 (NC_031407) we e isualized using he R pack-
ages “ggplo 2” (Wickham, 2016), “genoPlo R” (Guy e al.,
2010), “gggenes” (Wilkins, 2024), and “pa chwo k” (Ped-
e sen, 2023). The newly assembled mi ogenome sequence is
a ailable in GenBank (Accession numbe PP419898).
Tempe a u e anddesicca ion‑ ela ed p o ein genes
anno a ion
Genes in he new Ramazzo ius species we e iden i ied om
a genome assembly o all clean eads ob ained wi h Flye and
Medaka as explained abo e. The comple eness o he assem-
bly was e alua ed wi h BUSCO (Simão e al., 2015) (line-
age me azoa_odb10) on he gVolan e webse e (Nishimu a
e al., 2017). Con igs con aining po en ial Tempe a u e and
Desicca ion-Rela ed P o eins (TDRPs) sequences we e
ex ac ed wi h DIAMOND (op ions: –sensi i e) using he
eu a dig ade TDRPs (CAHS, MAHS, MRE11, SAHS)
sequences p o ided by Fleming e al. (2024), wi h he addi-
ion o Dsup (GenBank LC050827). The iden i ied con ings
we e anno a ed abini io on he AUGUSTUS webse e
(Ho & S anke, 2013; S anke e al., 2008) ained on he
genome and p o eome o R. a ieo na us YOKOZUNA-1
(h p:// kumam ushi. o g/). TDRPs we e iden i ied among he
p edic ed p o eins wi h BLASTp (e- alue = 10e − 10) agains
he TDRPs p o ein sequences om Fleming e al., (2024).
The iden i ied p o ein sequences (and hei ela i e coding
sequences) we e manually cu a ed o con i m hei iden-
i y. Maximum Likelihood phylogenies o he main TDRPs
g oups we e ob ained as in Fleming e al. (2024), wi h he
addi ion o he sequences p oduced in his s udy.
Resul s
Phylogene ic econs uc ion
The phylogene ic econs uc ion was pe o med wi h bo h
Bayesian in e ence (BI) and maximum likelihood (ML)
me hods wi h he models p o ided in Online Resou ces
03. Bo h me hods e ie ed cong uen opologies, e en
hough ML p o ided a gene ally less esol ed ee (Fig.1;
Online Resou ce 03). In bo h econs uc ion opologies,
he new Ramazzo ius species was ound in a clade (pp = 1,
BS = 98%) wi h R. a ieo na us YOKOZUNA-1 and Ramaz-
zo ius a . a ieo na us om I aly.
Genome sequencing andassembly
The sequencing un esul ed in 3.69Gb o sequences. The
summa y s a is ics o he aw eads a e p esen ed in Table2.
The assembly me ics and he BUSCO sco e we e compa ed
o hose o he R. a ieo na us YOKOZUNA-1 assembly
gene a ed by Hashimo o e al. (2016) (a ailable a h p://
www. kumam ushi. o g/ da a/ YOKOZ UNA-1. sca olds. a).
The assembly has a compa able size o R. a ieo na us
YOKOZUNA-1 (Table2), bu i is mo e agmen ed. The
Table 2 Raw eads and
assembly me ics R. claudii
sp. no .. aw
eads
R. claudii sp. no . assembly R. a ieo na us
YOKOZUNA-1 assembly
Size 3.69Gb 62.89Mb 55.82Mb
N o sequences 4,817,658 1468 199
N o sequences > 1kb 738,949 1415 198
N o sequences > 10kb 28,279 800 36
GC con en (%) 47.4 47.8 47.2
N50 1399 157,755 4,740,345
L50 485,725 109 4
N90 280 18,551 1,295,620
L90 3,074,768 549 15
BUSCO C: 71.6% [S: 69.4%, D: 2.2%]
F: 7.4%
M: 21.0%
n: 954
C: 74.4% [S: 73.2%, D: 1.2%]
F: 7.0%
M: 18.6%
n: 954

124 M.Vecchi, D.S ec
BUSCO sco e o he new assembly (71.6%) is compa able
o he one o R. a ieo na us YOKOZUNA-1 (74.4%). The
assembly o R. claudii sp. no . genome gene a ed in his
s udy can be ound a h ps:// doi. o g/h ps:// doi. o g/ 10. 6084/
m9. igsh a e. 27061 267.
Mi ogenome sequencing
O all he aw eads, abou 1.75% we e o mi ochon-
d ial o igin. The mi ogenome assembly is 14,437bp in
leng h (Fig.1) wi h an a e age co e age o 4486 × (min
1506 × – max 6785 ×). Two ibosomal RNAs ( nS an nL),
22 RNAs (wi h wo nL and nS), 2 ATP syn hase genes,
and he genes o he complexes I, III, and IV a e p esen .
A pu a i e unanno a ed con ol egion is p esen be ween
nL and nL. The o de o he genes is iden ical o he
mi ogenome o Ramazzo ius a ieo na us YOKOZUNA-1
(Fig.2), wi h an a e age nucleo idic di e gence o 20.6%.
TDRP iden i ica ion
CAHS
A o al o 15 CAHS genes we e iden i ied (Table3, Online
esou ces 06). The phylogene ic econs uc ions based o
he CAHS p o ein sequences a e simila o he ones ound
by (Fleming e al., 2024), wi h he excep ions o he CAHS
amilies 2, 3, and 5 which ha e been ound pa aphyle ic
(Online esou ces 06).
0.03
Ramazzo ius sp. DE.002 2
Ramazzo ius g oenlandensis 2
Ramazzo ius g oenlandensis 1
Ramazzo ius sp. Ta 398
Ramazzo ius claudii n. sp. S66 2
Ramazzo ius claudii n. sp. S66 1
Ramazzo ius sp. Rama1
Ramazzo ius subanomalus 1
Ramazzo ius a ieo na us YOKOZUNA-1
Ramazzo ius sp. PL.141
Hebesuncus conjugens IT.148.3
Ramazzo ius sp. Ro-Ni aa-1
Ramazzo ius claudii n. sp. IT.158.1
Ramazzo ius g oenlandensis 4
Ramazzo ius sp. PL.140 2
Ramazzo ius sp. DE.002 1
Ramazzo ius sp. Ta 400
Ramazzo ius sp. AT.002
Ramazzo ius a . a ieo na us S132 1
Ramazzo ius sp. PT.010 1
Ramazzo ius a . a ieo na us S132 2
Ramazzo ius k e schmanni T4
Ramazzo ius a . a ieo na us S49 1
Ramazzo ius g oenlandensis 3
Ramazzo us sp. PL.023
Ramazzo ius k e schmanni T1
Ramazzo ius a . a ieo na us S49 2
Hebesuncus conjugens IT.148.2
Ramazzo ius sp. Rama2
Ramazzo ius claudii n. sp. S62 2
Ramazzo ius sp. PL.140 1
Ramazzo ius claudii n. sp. S67 2
Ramazzo ius k e schmanni T2
Ramazzo ius k e schmanni T3
Ramazzo ius claudii n. sp. IT.158.3
Ramazzo ius a . a ieo na us S67 1
Ramazzo ius subanomalus 2
Ramazzo ius claudii n. sp. S62 1
Ramazzo ius obe haeuse i FR.124
Ramazzo ius sp. PT.010 2
0.95/-
0.99/98
1/91
1/98
1/100
0.87/-
1/100
1/100
0.94/98
0.99/98
0.99/99
1/100
0.95/99
1
1/100
1/100
1/100
1/100
0.8/-
1/98
0.98/96
0.71/98
0.91/-
1/100
Ramazzous claudii n. sp.
mi ochond ial genome
14 437 bp
AB
Fig. 1 Ramazzo ius claudii sp. no . mi ogenome and phylogene ic
posi ion. A Mi ogenome isualiza ion: inne ci cle ep esen s GC
con en . Di ec ion o ansc ip ion is clockwise o genes in on he
inne side, whe eas i is an iclockwise o he genes on he ex e nal
side. B Topology o he Bayesian In e ence (BI) phylogene ic econ-
s uc ion o Ramazzo ius clade A (sensu (Dey e al., 2024)): alues
abo e b anches ep esen BI pos e io p obabili ies/ML boo s ap (“-”
indica ed boo s ap alue < 70% o node no p esen in ML phyloge-
ne ic econs uc ion), scale ba indica es subs i u ions/si e
125
Mi ogenome o anew Ramazzo ius species (Ta dig ada: Eu a dig ada: Ramazzo iidae)…
SAHS–FABP
A o al o 8 SAHS genes we e iden i ied (Table3, Online
esou ces 07). The Mac obio idae SAHS and he SAHS sub-
amily 1 we e eco e ed as monophyle ic (Online esou ces
07), while SAHS sub amily 2 was eco e ed as pa aphyl-
e ic. 5 FABP ( a y acid binding p o eins, see (Fleming e al.,
2024) o hei phylogene ic ela ion wi h SAHS) genes we e
also eco e ed and included in he phylogeny.
MAHS
A single MAHS gene was ound (Table3, Online esou ces
09).
MRE11
A single MRE11 gene was iden i ied (Table3, Online
esou ces 09).
Fig. 2 Di e gence and syn eny
o Ramazzo ius claudii sp. no .
and R. a ieo na us YOKO-
ZUNA mi ogenomes. In black:
nucleo idic di e gence; in ed:
indel di e gence. Di e gence
was calcula ed on 250bp slid-
ing windows; A ow di ec ions
indica e ansc ip ion di ec ion
Di e gence
Ramaz
zo us
claudii n.sp.
Ramaz
zo us
a ieo na us
YOKOZUNA
Table 3 GenBank accession numbe s o he TDRPs genes iden i ied in Ramazzo ius claudii sp. no . classi ied acco ding o hei homology wi h
he ones om R. a ieo na us YOKOZUNA-1
Gene in R. a ieo na us
YOKOZUNA-1
R. claudii sp. no . Gene in R. a ieo na us
YOKOZUNA-1
R. claudii sp. no . Gene in R. a ieo na us
YOKOZUNA-1
R. claudii sp.
no .
CAHS SAHS Sub amily 1 MAHS
CAHS 1a PQ117601 SAHS 2 PQ117620 MAHS 1 PQ117614
CAHS 1b PQ117608 SAHS 7 PQ117618 MRE11
CAHS 2a PQ117603 SAHS 12 MRE11 A PQ117615
CAHS 3a PQ117609 SAHS 8 PQ117619 MRE11 B
CAHS 3b PQ117599 SAHS 9 No ound MRE11 C
CAHS 3c PQ117607 SAHS 10 No ound MRE11 D
CAHS 4a PQ117602 SAHS 11 PQ117615 FABP
CAHS 5a PQ117610 SAHS Sub amily 2 FABP 1 No ound
CAHS 5b PQ117604 SAHS 1 PQ117617 FABP 2 PQ117628
CAHS 5c PQ117606 SAHS 3 PQ117621 FABP 3 PQ117625
CAHS 6a PQ117612 SAHS 4 PQ117616 FABP 4 PQ117627
CAHS 6b PQ117613 SAHS 5 PQ117623 FABP 5 PQ117626
CAHS 6c No ound SAHS 6 PQ117622 FABP 6 PQ117624
CAHS 6d PQ117611 SAHS 13 No ound FABP 7 No ound
CAHS 7a PQ117605 Dsup
Dsup PQ356341
126 M.Vecchi, D.S ec
Dsup
A single Dsup gene was iden i ied (Table3, Online esou ces
10).
Taxonomic accoun
Ramazzo ius claudii sp. no . Vecchi & S ec, 2024
Zoobank egis a ion: u n:lsid:zoobank.o g:ac :0B3D4075-
0C0B-4552-B7B6-73EC10711ED4
Ramazzo ius sp. A in (Vecchi e al., 2022)
Ramazzo ius sp. I aly in (Zawie ucha e al., 2023)
Type locali y. Rock pool sedimen om icini y o Sella
del Ma magna, Co niglio, Pa ma, I aly (44°23′45.8″N
10°00′15.7″E).
Ma e ial examined. Holo ype (Slide IT.158.7), 23 pa a ypes
(Slides IT.158.4 – IT.158.10) and 20 eggs (Slides IT.158.01
– IT.158.03) moun ed on slides in Hoye ’s medium. 4 animals
(S ub TAR.2.06) and 5 eggs (S ub TAR.2.07) moun ed on
s ubs o SEM. 3 animals used o DNA sequencing (Ram.
sp.IT.158.01, Ram.sp.IT.158.03 and Ram_IT.158_WGA_1).
Ma e ial eposi o y. Ta dig ada collec ion o he Ins i u e
o Sys ema ics and E olu ion o Animals (Polish Academy
o Sciences), Sławkowska 17, 31–016, K aków, Poland.
E ymology. This species is dedica ed o Claudio Fe a i
(Uni e si y o Pa ma, I aly), o acknowledge his cons an
suppo and help in explo ing he I alian No he n Apen-
nines in sea ch o ock pools.
(Tables4 and 5, Figs.3, 4, 5, 6, 7, 8, and 9, Online esou ces
01).
Desc ip ion. Animals (mo phome ics in Table4, aw meas-
u emen s in Online esou ces 01): Eyes absen in li e indi-
iduals. Pigmen a ion o he cu icle ed-b own, dis ibu ed
in one ing a ound he mou h, a do so-caudal con inuous
band, and 8 la e al- en al bands (Fig.3). Cu icle wi h ain
sculp u e, mo e isible in he do so-caudal pa o he body
(Figs.4 and 5). A pai o senso y o gans p esen on he head
(Fig.5A). The sculp u e is composed by ounded polygonal
g anules; howe e , in some indi iduals (in pa icula in he
caudal po ion o bigge indi iduals), he g anules ha e an
elonga ed “spindle” shape (Figs.4 and 5).
Bucco-pha yngeal appa a us o he Ramazzo ius- ype
(Fig.6). Mou h opening an e o- en al. O al ca i y a ma u e
isible unde PCM (Fig.6B and C). The a ma u e is com-
posed o one band o ee h, loca ed in he pos e io o al ca i y
(Fig.6B and C). The band composed o a single ow o la ge
and egula ly spaced g anula ee h. In en al iew, he ee h
a e less isible and seem connec ed o he pos e io edge o
he o al ca i y (Fig.6C). Unde PCM, o he s uc u es wi hin
o al ca i y no isible (Fig.6B and C). Apophyses o he
inse ion o s yle muscles (AISM) in he shape o blun hooks
and asymme ical in size and shape wi h espec o he on al
plane (Fig.6D). S yle u cae wi h ounded ends. Buccal ube
wi h a pos e io bend and hickened walls pos e io ly om he
s yle suppo inse ion poin . Pha yngeal bulb (bulbus) almos
o al, wi h apophyses and wo clea ly sepa a ed mac opla-
coids. Pha yngeal apophyses iangula , smalle han mac o-
placoids (Fig.6E). Mac oplacoid leng h con igu a ion 2 < 1.
Mic oplacoid and sep ulum absen (Fig.6E). Mac oplacoids
a e usually oundish; howe e , in bigge specimens, hey look
sligh ly mo e elonga ed. Cons ic ions in he mac oplacoids
a e p esen . In he i s mac oplacoid a cen al cons ic ion is
p esen , whe eas in he second mac oplacoids a cons ic ion
in cen al-pos e io posi ion is p esen .
Claws o he Ramazzo ius- ype. P ima y b anches o
ex e nal and pos e io claws long and hin. A non-scle o ized
ligh e ac ing uni (LRU) is p esen be ween he claw base
and he p ima y b anch, which a e connec ed by a couple o
hin cu icula ilamen s (Fig.7A, B).
In e nal and an e io claws much smalle and o a di e -
en shape han ex e nal claws (Fig.7). Claws wi h smoo h
pseudolunules (Fig.7B). Accesso y poin s on p ima y
b anches o all claws p esen (Fig.7D-E). Ba s and o he
cu icula hickenings on legs, absen . A papilla is p esen on
he ex e nal side o legs IV, bu no always isible depending
on he animal posi ioning (Fig.7C).
Eggs (mo phome ics in Table5, aw measu emen s
in Online esou ces 01): Laid eely, whi e and sphe ical,
co e ed in p ocesses wi h shape anging om spike-like o
ilamen ous (Fig.8). The p ocesses exhibi ex eme di e -
si y in size, wi h mos o hem being wi hin he ange o
4.6–14.4μm, bu wi h some o hem eaching up o 27.9μm
(Fig.8C). Egg p ocesses and su ace be ween p ocesses do -
ed unde PCM, smoo h unde SEM (Fig.8I).
Males Indi iduals wi h gonad illed wi h de eloping spe -
ma ids we e ound, indica ing he p esence o males (Fig.9).
DNA sequences.
SSU (18S): PQ108467 – 8, PQ108470;
LSU (28S): PQ108475 – PQ108477;
COI: MW306832 – MW306836, PQ109084 – 5;
ITS-2: PQ110584 – 6.
Di e en ial diagnosis
By ha ing eggs wi h do ed cho ion and elonga ed p ocesses,
R. claudii sp. no . is simila o six o he species, howe e i
di e s om:
127
Mi ogenome o anew Ramazzo ius species (Ta dig ada: Eu a dig ada: Ramazzo iidae)…
Table 4 Summa y o animals mo phome ics o Ramazzo ius claudii sp. no . cb and p a ios acco ding o Vecchi e al. (2023b)
Cha ac e NRange Mean SD Holo ype
μm p μm p μm p μm p
Body leng h 20 292 – 488 934 – 1205 391 1061 55 67 379 1120
Buccal ube
 Buccal ube leng h 20 29.7 – 43.9 – 36.7 –3.9 –33.8 –
 S yle suppo inse ion poin 20 19.9 – 30.0 65.9 – 70.4 25.0 68.1 2.8 1.3 22.9 67.8
 Buccal ube ex e nal wid h 20 2.5 – 3.6 7.1 – 9.8 3.0 8.3 0.3 0.6 3.3 9.8
 Buccal ube in e nal wid h 18 1.0 – 2.0 3.0 – 6.0 1.4 3.9 0.2 0.7 2.0 6.0
Placoid leng hs
 Mac oplacoid 1 19 3.7 – 6.7 11.2 – 16.6 4.9 13.2 0.9 1.5 3.8 11.3
 Mac oplacoid 2 19 3.0 – 5.9 9.6 – 14.6 4.3 11.5 0.8 1.2 3.6 10.7
 Mac oplacoid ow 19 7.0 – 12.9 22.9 – 32.0 9.9 26.6 1.8 2.5 8.2 24.3
Claw 1 leng hs
 Ex e nal base 19 8.0 – 13.1 22.9 – 32.2 10.0 27.6 1.3 2.6 9.4 27.7
 Ex e nal p ima y b anch 18 12.5 – 19.1 38.9 – 48.9 16.0 43.9 2.1 2.6 15.1 44.8
 Ex e nal seconda y b anch 17 6.9 – 11.4 23.4 – 32.6 9.4 26.1 1.3 2.2 9.4 27.7
 Ex e nal cb a io 18 49.0 – 73.8 – 63.1 –6.0 –61.9 –
 Ex e nal b a io 16 49.8 – 67.4 59.1 3.9
 Ex e nal o al 17 18.8 – 31.0 60.6 – 78.0 24.8 68.5 3.3 5.2 24.3 71.8
 In e nal base 13 4.5 – 8.5 13.8 – 22.6 7.2 19.6 1.2 2.6 7.5 22.2
 In e nal p ima y b anch 12 6.7 – 12.5 20.1 – 31.1 9.8 26.7 1.7 3.0 8.8 25.9
 In e nal seconda y b anch 13 4.4 – 10.3 13.3 – 25.5 7.5 20.7 1.4 3.2 7.3 21.7
 In e nal cb a io 12 47.0 – 93.4 – 74.2 –13.4 –85.5 –
 In e nal b a io 12 63.4 – 93.9 76.9 8.4
 In e nal o al 10 10.8 – 16.6 32.4 – 41.0 13.2 36.4 1.7 2.6 ??
Claw 2 leng hs
 Ex e nal base 19 8.3 – 13.5 24.8 – 35.2 10.8 29.5 1.5 2.6 10.4 30.8
 Ex e nal p ima y b anch 18 11.1 – 21.4 37.2 – 52.8 16.4 45.0 2.3 3.5 16.5 48.7
 Ex e nal seconda y b anch 19 7.5 – 11.7 24.4 – 30.7 10.0 27.4 1.1 1.8 9.6 28.4
 Ex e nal cb a io 18 56.9 – 77.3 – 65.9 –7.5 –63.3 –
 Ex e nal b a io 18 51.2 – 75.1 61.4 5.7
 Ex e nal o al 14 22.2 – 33.1 61.8 – 82.3 26.8 73.5 3.5 5.4 25.0 73.8
 In e nal base 15 3.6 – 9.5 10.3 – 22.2 7.1 19.5 1.4 2.9 7.5 22.2
 In e nal p ima y b anch 15 7.7 – 14.4 24.0 – 37.7 10.4 28.6 1.7 3.4 8.8 25.9
I n e nal seconda y b anch 11 6.1 – 9.8 18.2 – 25.6 8.2 22.7 1.1 2.4 8.7 25.6
 In e nal cb a io 15 42.9 – 85.6 – 68.6 –11.7 –85.6 –
 In e nal b a io 11 59.0 – 98.9 80.0 12.1
 In e nal o al 14 10.1 – 17.7 29.9 – 44.2 14.0 38.3 2.4 3.6 10.1 29.9
Claw 3 leng hs
 Ex e nal base 19 7.6 – 13.9 25.4 – 34.3 10.8 29.2 1.7 2.3 9.3 27.5
 Ex e nal p ima y b anch 20 13.6 – 20.0 44.3 – 53.2 17.6 47.8 1.9 2.3 17.4 51.6
 Ex e nal seconda y b anch 18 6.8 – 12.1 22.8 – 29.9 9.8 26.8 1.4 1.8 8.7 25.8
 Ex e nal cb a io 19 52.9 – 73.3 – 61.2 –5.7 –53.4 –
 Ex e nal b a io 18 49.3 – 62.8 56.0 4.2
 Ex e nal o al 15 20.6 – 34.8 64.7 – 85.8 26.6 72.6 4.2 6.0 25.5 75.5
 In e nal base 17 5.8 – 11.8 17.6 – 28.2 7.9 21.6 1.3 2.6 6.8 20.2
 In e nal p ima y b anch 18 7.3 – 12.9 21.2 – 33.6 10.3 28.6 1.8 3.6 9.6 28.4
 In e nal seconda y b anch 17 5.9 – 10.9 16.2 – 30.1 8.5 23.3 1.5 3.3 8.3 24.4
 In e nal cb a io 17 59.1 – 106.1 – 75.8 –12.9 –71.1 –
 In e nal b a io 17 58.3 – 141.6 82.1 18.1
 In e nal o al 15 12.5 – 17.3 35.0 – 43.0 14.5 39.6 1.5 2.6 13.8 40.8
134 M.Vecchi, D.S ec
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