531
Phylogenomic analyses o a e Neo opical lineages
e eal he independen loss o an ennal ami in ail oad-
wo m bee les (Coleop e a: Phengodidae)
Felipe F ancisco Ba bosa1,*, And é Sil a Roza1,*, José Rica do M. Me mudes1, Michael F. Geise 2,
Ji i Hodecek3,4, La a-Sophie Dey5, Michael A. I ie6, Vi idiana Vega-Badillo7, Vinicius S. Fe ei a5,
Robin Kund a a8
1 Labo a ó io de En omologia, Depa amen o de Zoologia, Ins i u o de Biologia, Uni e sidade Fede al do Rio de Janei o, A1–107, Bloco A,
A . Ca los Chagas Filho, 373, Cidade Uni e si á ia, Ilha do Fundão, Rio de Janei o, B azil
2 Na u al His o y Museum, London SW7 5BD, Uni ed Kingdom
3 Swiss Human Ins i u e o Fo ensic Taphonomy, Uni e si y Cen e o Legal Medicine, CH-1000, Lausanne, Swi ze land
4 Musée Can onal de Zoologie, Palais de Rumine, Place de la Riponne 6, CH-1014, Lausanne, Swi ze land
5 Senckenbe g Deu sches En omologisches Ins i u , Ebe swalde S aße 90, 15374 Münchebe g, Ge many
6 Mon ana En omology Collec ion, Mon ana S a e Uni e si y, Bozeman, MT, 59717, USA
7 Colección En omológica IEXA “D . Miguel Ángel Mo ón Ríos,” Ins i u o de Ecología A.C. (INECOL), Ca e e a An igua a Coa epec 351,
El Haya, 91073 Xalapa, Ve ac uz, México
8 Depa men o Zoology, Facul y o Science, Palacky Uni e si y, 17. lis opadu 50, 77900, Olomouc, Czech Republic
* These au ho s con ibu ed equally.
h ps://zoobank.o g/D427C481-B132-498B-91E5-390C699A2E56
Co esponding au ho s: Vinicius S. Fe ei a ([email p o ec ed]), Robin Kund a a ([email p o ec ed])
Recei ed 06 July 2025
Accep ed 09 Sep embe 2025
Published 10 Oc obe 2025
Academic Edi o Klaus-Die e Klass
Ci a ion: Ba bosa FF, Roza AS, Me mudes JRM, Geise MF, Hodecek J, Dey L-S, I ie MA, Vega-Badillo V, Fe ei a VS, Kund a a R (2025) Phy-
logenomic analyses o a e Neo opical lineages e eal he independen loss o an ennal ami in ail oad-wo m bee les (Coleop e a: Phengodidae).
A h opod Sys ema ics & Phylogeny 83: 531–542. h ps://doi.o g/10.3897/asp.83.e164315
Abs ac
Phengodidae, o ail oad-wo m bee les, a e bioluminescen so -bodied bee les wi h ligh capable adul males and highly paedo-
mo phic la i o m emales. They a e well accep ed as pa o he “lampy oid” clade wi hin Ela e oidea, and hei sup agene ic
ela ionships ha e been ecen ly s udied using a phylogenomic app oach. Howe e , he placemen o axa cu en ly classi ied in he
sub amily Penicillopho inae emained un es ed. Penicillopho inae o m an assemblage o mo phologically modi ied, a ely collec ed
Neo opical gene a ha a e unique among Phengodidae. They a e pa icula ly cha ac e ized by hei monili o m, se a e o uni amose
an ennae, in con as o he ypically bipec ina e an ennae p esen in all o he membe s o he amily. To in es iga e he phylogene ic
posi ion o Penicillopho inae, we implemen ed a low-co e age whole genome sequencing app oach o p oduce genomic da a o
Acladoce a and Wal e ius, wo ou o i e gene a classi ied in his sub amily. The esul ing phylogenomic analyses con i med he
monophyly o Phengodidae; howe e , Penicillopho inae we e no ound as a monophyle ic g oup. Ou esul s eco e ed he opology:
Cydis inae + (Phengodinae + (Acladoce a + (Cenophenginae + Mas inoce inae including Wal e ius))). The e o e, we sugges ha he
an ennal double ami we e los a leas wice among Phengodidae: once in he newly ci cumsc ibed Mas inoce inae (wi h Wal e ius),
and once in Acladoce a, which we en a i ely keep in Penicillopho inae. Fu he , we discuss he mo phological modi ica ions o o he
gene a cu en ly classi ied in Penicillopho inae. Fu u e phylogenomic esea ch should ocus on cla i ying he bounda ies and compo-
si ion o phengodid sub amilies, pa icula ly by including addi ional gene a om Penicillopho inae and Mas inoce inae.
Key wo ds
Acladoce a, bioluminescence, neo eny, paedomo phosis, so -bodied bee le, Wal e ius
A h opod Sys ema ics & Phylogeny 83, 2025, 531–542 | DOI 10.3897/asp.83.e164315
Copy igh Felipe F ancisco Ba bosa e al.: This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY 4.0), which pe mi s un es ic ed
use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Ba bosa FF e al.: Phylogenomic placemen o Penicillopho inae
532
1. In oduc ion
Phengodidae a e a small so -bodied ela e oid amily,
cu en ly con aining app oxima ely 300 species in 45
gene a (Kund a a e al. 2019; Fe ei a e al. 2024; Roza
2025; Uchima-Tabo da e al. 2025). The g oup is p esen
in he Ame icas and in Wes Asia, wi h mos o i s di e si-
y concen a ed in he Neo opical ealm (Kund a a e al.
2019; Fe ei a e al. 2024; Roza 2022; Za agoza-Caballe-
o and Pé ez-He nández 2014). The amily is o pa icu-
la in e es o en omologis s and e olu iona y biologis s
due o he p esence o bioluminescence, which is ound
in bo h imma u es and adul s (Cos a and Za agoza-Ca-
balle o 2010), and he paedomo phosis synd ome, which
highly a ec s all known emales ha emain la i o m
in hei adul hood (Cos a e al. 1999; Fe ei a and I ie
2022). Based on cu en phylogene ic hypo heses, Phen-
godidae a e a monophyle ic amily well nes ed wi hin he
Ela e oidea, as pa o he “lampy oid” clade (Fe ei a e
al. 2024; Kund a a 2025). Thei placemen in Ela e oidea
is co obo a ed by mul igene Sange -based analyses (e.g.
Kund a a e al. 2014; McKenna e al. 2015) as well as
by all phylogenomic s udies ha include he g oup (e.g.
Zhang e al. 2018; McKenna e al. 2019; Douglas e al.
2021; Cai e al. 2022).
The phylogeny o Phengodidae has ecei ed inc eased
a en ion in he las decade, wi h h ee esea ch pape s
(Za agoza-Caballe o and Zu i a-Ga cía 2015; Kund a a
e al. 2019; Fe ei a e al. 2024) and wo doc o al dis-
se a ions (Quin ino 2017; Roza 2022) published on
he subjec . The amily was adi ionally hough o be
endemic o he New Wo ld, and di ided in o h ee sub-
amilies: Mas inoce inae, Phengodinae, and Penicillo-
pho inae (e.g. Wi me 1976; Za agoza-Caballe o and
Pé ez-He nández 2014). The only published mo pholo-
gy-based phylogeny o he amily (Za agoza-Caballe o
and Zu i a-Ga cía 2015), which was deemed p elimina y
by he au ho s hemsel es, ound almos no suppo o
mos o he ela ionships he ein eco e ed. Thei s udy
ound sub amilies o be ei he polyphyle ic (Mas inoce i-
nae and Phengodinae) o nes ed in Telegeusidae (Penicil-
lopho inae), ou side o Phengodidae ( o a syn hesis, see
Fig. 2 in Fe ei a e al. 2024).
Mo e ecen ly, molecula -based phylogenies ha e
made impo an addi ions o he amily sys ema ics and
classi ica ion. These include he ecogni ion o he Asian
Cydis inae as phengodids (Kund a a e al. 2019), and
he ecogni ion and desc ip ion o he ou h New Wo ld
sub amily, Cenophenginae (Fe ei a e al. 2024). The
la e s udy is he la es and mos comp ehensi e phy-
logene ic hypo hesis o he amily Phengodidae. Using
an ancho ed hyb id en ichmen (AHE) phylogenomics
app oach, he au ho s eco e ed ou sub amilies in he
ollowing opology: Cydis inae + (Phengodinae + (Ceno-
phenginae + Mas inoce inae)) (Fe ei a e al. 2024). De-
spi e ecen ad ances on he high-le el phylogene ics o
Phengodidae, he placemen o axa cu en ly classi ied
in he sub amily Penicillopho inae (Figs 1–3) emains
un esol ed and un es ed using a molecula -based ap-
p oach.
The Penicillopho inae a e a g oup o mo phological-
ly modi ied gene a ha a e unique among Phengodidae.
Cu en ly composed o i e gene a and se en species
(Roza e al. 2024), hey di e signi ican ly om o he
Phengodidae in ha ing monili o m, se a e, o sho -
ly uni amose an ennae, which lack he double b anches
p esen in all he o he known gene a o he amily (Figs
1, 2). In addi ion o modi ica ions o he an ennae, he
mo phology among hese gene a is qui e dispa a e. This
a ia ion includes he numbe s o en o ial pi s, gula
su u es, and labial palpome es, he wing ena ion, and
he p esence/absence o en al a sal combs on bo h
he i s p o- and meso a some e (see Roza e al. 2024).
Va iabili y in a numbe o labial palpome es and a wing
ena ion (Fig. 3) migh be an e ec o minia u iza ion
(Za agoza-Caballe o and Pé ez-He nández 2014; Roza e
al. 2024).
Figu e 1. Gene a adi ionally placed in Penicillopho inae. A Penicillopho us sp. (Venezuela), B Acladoce a sp. (Pue o Rico),
C Adend oce a la ula Wi me , 1976 (Gua emala) (modi ied om Roza e al. 2024), D Wal e ius sp. (Belize). Scale ba s: 1 mm.
A h opod Sys ema ics & Phylogeny 83, 2025, 531–542 533
Figu e 2. Mo phology o gene a adi ionally placed in Penicillopho inae. A, B Head en al: A Pe ni cil lo pho us sp. B Acladoce a
hispaniola Wi me , 1981. C– E Righ an enna: C Penicillopho us sp., D Acladoce a sp., E Wal e ius sp. Scale ba s: 0.2 mm (A,
B), 0.5 mm (C–E).
Figu e 3. Mo phology o gene a adi ionally placed in Penicillopho inae. A–D Righ p o a sus: A Pe ni cil lopho us sp., B Acla-
doce a sp., C Adend oce a ca meli a Roza e al., 2024 (modi ied om Roza e al. 2024), D Wal e ius sp. E–H Le hind wing:
E Penicillopho us sp. F Acladoce a sp. G Aden d oce a la ula Wi me , 1976 (modi ied om Roza e al. 2024), H Wal e ius sp.
Scale ba s: 0.2 mm (A–D), 0.5 mm (E–H).
Ba bosa FF e al.: Phylogenomic placemen o Penicillopho inae
534
P e ious mo phological phylogene ic assessmen s o
Phengodidae conside ed Penicillopho inae o be ou side
he amily, o ming a clade wi h Telegeusidae (cu en -
ly in Ela e oidea: Ome hidae) (Za agoza-Caballe o and
Zu i a-Ga cía 2015), o well nes ed wi hin Phengodidae:
Mas inoce inae (Quin ino 2017; Roza 2022). Howe e ,
hose hypo heses ob ained low s a is ical suppo (i.e.
nodal suppo ) o no suppo a all and we e based main-
ly on homoplas ic ea u es, such as an ennome e shapes
and he absence o educ ion o some hindwing eins.
Rep esen a i es o Penicillopho inae a e a ely collec ed,
and only known om hei ype se ies. To da e, he e is
no published molecula da a o any membe o his sub-
amily. The ecen collec ion o axa cu en ly classi ied
as Penicillopho inae by MFG and JH allowed he DNA
ex ac ion and p oduc ion o he i s genomic da a o
wo ep esen a i es o Penicillopho inae. We ook his
oppo uni y o es hei phylogene ic placemen wi hin
Phengodidae.
2. Ma e ial and Me hods
2.1. Taxon sampling, iden i ica ion, and
mo phology
Ou axon sampling included a o al o 13 e minals, o
which i e (including Acladoce a Wi me , 1981 and
Wal e ius Za agoza-Caballe o, 2008) we e newly se-
quenced. Fo ull in o ma ion on he analyzed da ase ,
see Table S1. In he p esen s udy we included ewe e -
minals han Fe ei a e al. (2024) did; gi en he high le el
o missing da a in he da ase by Fe ei a e al. (2024),
we op ed o a smalle bu mo e comple e gene-sampled
app oach o es he phylogene ic posi ion o Penicillo-
pho inae gene a. The e o e, we used a s aigh o wa d
and simple p ocedu e o phylogenomic subsampling (see
Edwa ds 2016; Koch 2021). Wi h his p ocedu e, we ha e
ob ained much mo e sequence da a due o he quali y o
he specimens (i.e. specimens which yielded mo e ge-
nomics da a, bo h in olume and quali y, see Table S1
and he sequencing me hod.
The wo Penicillopho inae samples we e ini ially iden-
i ied by hei collec o s as Acladoce a sp. (SDEIC003)
(by JH), and as Wal e ius sp. Za agoza-Caballe o, 2008
(SDEIC035) (by MFG). Thei iden i ica ions we e la e
co obo a ed by ASR and MFG based on cu en li e -
a u e on Phengodidae (Wi me 1976; Za agoza-Cabal-
le o and Pé ez-He nández 2014; Roza e al. 2024), and
by di ec compa ison wi h ype specimens o Acladoc-
e a hispaniola Wi me , 1981 and Wal e ius caballe oae.
Sampled Acladoce a sp. was collec ed in he Dominican
Republic, and will be deposi ed in he Na u al His o y
Museum, London, The Uni ed Kingdom. Wal e ius sp.
was collec ed in Belize, and will be deposi ed in he
Na u éum - Muséum can onal des sciences na u elles,
Lausanne, Swi ze land. The mo phological e minol-
ogy ollows Cos a and Za agoza–Caballe o (2010) and
Roza (2023). Fo hind wings, we ollow Law ence e al.
(2021). The examined hind wings we e glued on ca ds
and pe manen ly placed unde each espec i e specimen.
Pho og aphs and measu emen s we e aken wi h he Lei-
ca DFC450 and Applica ion Sui e CV3 so wa e. The
pho og aphs we e edi ed using Adobe Pho oshop CC, and
he igu e pla es and d awings we e designed wi h Adobe
Illus a o CC.
Ou inal da ase s (50-NT, 70-NT, 50-AA, and 70-
AA, see esul s sec ion) consis ed o 13 samples. Ing oup
consis ed o 10 axa ep esen ing all i e cu en ly ecog-
nized sub amilies o Phengodidae: Cydis inae (Mic ocy-
dis us mino (Bolí a and Piel ain, 1913)); Phengodinae
(Za hipis in eg ipennis (LeCon e, 1874)); Penicillopho i-
nae (Acladoce a sp. and Wal e ius sp.); Cenophenginae
(Cenophengus debilis LeCon e, 1881); and Mas inoce i-
nae (B asiloce us obe hu i (Pic, 1955), Cephaloph ixo-
h ix sp., Dis emocephalus opacu lus (Ho n, 1895), Oxy-
mas inoce us pe uanus (Wi me , 1956), and Ph ixo h ix
hi us E. Oli ie , 1909). We adop ed he Phengodidae
classi ica ion p oposed by Fe ei a e al. (2024). As ou -
g oup, we sampled one Sinopy opho idae (Sinopy opho-
us schimmeli Bi and Li, 2019; as he mos dis an ou -
g oup, = oo ), one Lampy idae [Lamp ohiza splendidula
(Linnaeus, 1767)], and one Rhagoph halmidae [Meng-
huoius gigan eus (Fai mai e, 1888); as a ep esen a i e
o he sis e -g oup o Phengodidae].
2.2. DNA ex ac ion, sequencing, and
genome assembly
Genomic ex ac ions ollowed Fe ei a e al. (2024). Ex-
ac ions we e done using he DNeasy Blood and Tissue
ki (Qiagen, Ge man own, MD, USA). The manu ac-
u e ’s p o ocol o issue samples was ollowed wi h a
p olonged o e nigh (10–14 hou s) lysis pe iod. The inal
DNA was elu ed in bu e EB a e a 10-min incuba ion.
This s ep was epea ed wice, wi h 30 µl each ime, o a
inal elu ion olume o 60 µl. Ex ac ions we e done in
he molecula labo a o y a he Senckenbe g Deu sches
En omologisches Ins i u (SDEI). The ull genomic ex-
ac ions a e pe manen ly deposi ed a -80 °C a he DNA
and issue collec ions a he SDEI.
Quali y con ol, no maliza ion, lib a y p epa a ion,
and sequencing o samples we e done by No ogene Ge-
nomics (Munich, Ge many). A DNA lib a y o 350 bp in-
se size was cons uc ed o he sample and sequenced in
a No aSeq X Plus Se ies wi h a pai -end 150 bp sequenc-
ing s a egy. Abou 10Gb eads we e ob ained om each
sample.
Sequence eads we e ini ially p ocessed using he
CAPTUS pipeline (O iz e al. 2023): aw eads we e
adap e - immed and il e ed in wo consecu i e ounds
using BBDUK om BBTOOLS (Bushnell 2022; O iz e
al. 2023), and de no o assembled using MEGAHIT (Li
e al. 2015), wi h CAPTUS de aul p ese “CAPSKIM,”
which is op imized o hyb idiza ion cap u e o genome
skimming da a (o a combina ion o bo h) (O iz e al.
2023).
A h opod Sys ema ics & Phylogeny 83, 2025, 531–542 535
2.3. O holog sea ch
A e ini ial da a quali y con ol and genome assembly,
ou o holog sea ch was conduc ed using CAPTUS (O iz
e al. 2023). We used CAPTUS modules “ex ac ” and
“align” o iden i y o hologous genes om he da ase
used o p oduce he cus om Ela e idae O holog Ta ge
En ichmen Bai s (“Ela e Bai s”), which we e o iginally
p esen ed in Douglas e al. (2021). This o holog da abase
was o iginally designed o be used o Ela e idae (Doug-
las e al. 2021), bu i was also success ully used o Phen-
godidae by Fe ei a e al. (2024). CAPTUS pipeline was
implemen ed ollowing O iz e al. (2023) and CAPTUS
u o ial a ailable a h ps://edga domo iz.gi hub.io/cap-
us.docs/ u o ials/assembly/basic (las access on 9 Sep-
embe 2025).
Since we did no implemen an in i o hyb idiza ion
s a egy o da a acquisi ion (see he p e ious sec ion),
ins ead o using he “Bai -100-85_Ela e idae. as a” as
ou e e ence ile o he o holog sea ch in CAPTUS, we
used he o iginal ile “Ela e idae_Submission. as a” used
o hei p obe design. Full de ails abou he p oduc ion
o he “Ela e Bai s” a e a ailable a Douglas e al. (2021),
and o iginal iles can be ound a h ps://gi hub.com/
AAFC-BICoE/Ela e idae-o holog-bai se (las access on
9 Sep embe 2025).
CAPTUS ex ac s ep was pe o med in ou p e iously
assembled genomes. We implemen ed he CAPTUS “Nu-
clea p o eins” sea ch s a egy. This app oach uses Scipio
(Ha je e al. 2011) o pe o m p o ein ex ac ions and au-
oma ically co ec ameshi s o downs eam analyses
(see de ails in O iz e al. 2023). Du ing he “align” s ep,
CAPTUS ex ac ed he copies (hi s) o ma ke s ound in
assembly iles and anked hem by hei simila i y o he
e e ence sequences (O iz e al. 2023). Sequences we e
hen aligned wi h MAFFT Ve sion 7 (Ka oh and S andley
2013), and pa alogs il e ed using CAPTUS nai e me hod
and in o med me hod (see O iz e al. 2023 o de ails).
A e pa alogs we e il e ed, alignmen s we e immed
wi h ClipKIT (S eenwyk e al. 2020).
2.4. Phylogenomic analyses
Phylogene ic ela ionship econs uc ion and o he es s
we e pe o med on ou e sions o ou mul iple se-
quence alignmen s (MSAs) composed o loci wi h ≥50%
o ≥70% o axa p esen (“comple eness”). Fo de e min-
ing he comple eness o each da ase , we used Geneious
11.1.5. The mul iple sequence alignmen s we e conca -
ena ed using AMAS (Bo owiec 2016). The phyloge-
ne ic analyses we e conduc ed unde wo ee-building
me hods (op imali y c i e ia) as ollows: 1) conca ena ed
analyses pe o med using he maximum likelihood (ML)
me hod (Felsens ein 1973, 1981) ia IQ-TREE2 so wa e
(Minh e al. 2020a), and 2) a summa y me hod o species
ee in e ence om gene ees, modeled unde he mul i-
species coalescen model (MSCM; Pamilo and Nei 1988;
Rannala and Yang 2003) ia ASTRAL-P o2 so wa e
(Mi a ab e al. 2014; Zhang and Mi a ab 2022), wi h in-
pu gene ees being econs uc ed indi idually unde ML
ia IQ-TREE2 so wa e.
Phylogene ic analyses we e pe o med in ou da ase s
(Table 1), i.e. a he nucleo ide le el 50-NT (50% com-
ple eness ma ix) and 70-NT (70% comple eness ma ix),
and amino acid le el 50-AA (50% comple eness ma ix)
and 70-AA (70% comple eness ma ix). All analyses ca -
ied ou wi h hese da ase s we e pa i ioned by locus. We
de e mined he e olu iona y models o he pa i ioned
and indi idual gene ees analyses (MFP + MERGE
command o IQ-TREE2) ia ModelFinde so wa e
(Kalyaanamoo hy e al. 2017). The esul ing ees we e
isualized ia he FigT ee 1.4.0 (Rambau 2012) and
iTOL: In e ac i e T ee O Li e (Le unic and Bo k 2016)
so wa e packages.
The nodal suppo in ML analyses was measu ed
h ough he ollowing me ics: he “ul a as ” boo s ap
(UFBoo ; Hoang e al. 2018), he Shimodai a–Hasega-
wa-like app oxima e likelihood a io es (SH-aLRT;
Guindon e al. 2010), and he Bayesian-like ans o ma-
ion o aLRT (aBayes; Anisimo a e al. 2011). Fo coales-
cen ASTRAL analyses, suppo s we e calcula ed as local
pos e io p obabili ies (Local PP; Mi a ab e al. 2014;
Zhang and Mi a ab 2022). Nodes wi h suppo alues
abo e ≥95% / ≥0.95 ( o UFBoo , aBayes, and Local PP)
and abo e ≥80% ( o SH-aLRT) we e conside ed s ong-
ly suppo ed (Guindon e al. 2010; Anisimo a e al. 2011;
Hoang e al. 2018). Suppo measu es we e compu ed ia
IQ-TREE2 so wa e, excep o he Local PP, calcula ed
ia ASTRAL-P o2 so wa e.
Conco dance ac o s a e impo an ools in phyloge-
nomics. These measu es help o summa ize in o ma ion
om di e en gene ees and si es ha a ise due o he
bounda y be ween e icula e and di e gen e olu ion
(Baum 2007). Two conco dance ac o s a e widely used
in phylogenomics s udies: 1) he gene conco dance ac o
(gCF; Baum 2007; Minh e al. 2020b), which compu es
he p opo ion o indi idual gene ees ha suppo each
speci ic node, co ec ed o a iable e minal co e age;
and 2) he imp o ed e sion o he si e conco dance ac-
o (sCF; Minh e al. 2020b; Mo e al. 2023), which com-
pu es he p opo ion o alignmen si es conco dan wi h
each speci ic node in a maximum likelihood amewo k
using he p obabili y dis ibu ions o ances al s a es a
in e nal nodes. Al hough conco dance ac o s ha e been
in e p e ed as measu es o s a is ical suppo o nodes,
Lan ea and Hahn (2024) indica ed ha his in e p e a ion
is no adequa e.
The conco dance ac o s (gCF and sCF) a e mo e
p ope ly de ined as “desc ip o s o opological a ia ion”
and “es ima es o biological pa ame e s,” in which hey
can be used o measu e he p opo ion o he da ase o
which a gi en spli /clade is conside ed ue (see Lan ea
and Hahn 2024). In his in e p e a ion, conco dan ac o s
a e complemen a y measu es in ela ion o nodal suppo
measu es in a phylogenomic con ex ; hey can p o ide
impo an in o ma ion ega ding he p edic i e powe
o he in e ed phylogene ic species ee ha may no be
clea ly con ained in egula s a is ics ega ding node sup-
po (Minh e al. 2020b; Lan ea and Hahn 2024). Since,
Ba bosa FF e al.: Phylogenomic placemen o Penicillopho inae
536
by de ini ion, i is no adequa e o in e p e conco dance
ac o s as measu es o nodal suppo , we canno de ine
h eshold le els o signi icance o he gCF and sCF,
as we do o SH-aLRT, aBayes, UFBoo , and Local PP.
The e o e, in his s udy, we do no p esen gCF and sCF
alues.
2.5. Fou -clus e likelihood mapping
To u he in es iga e he s a is ical suppo o al e na-
i e hypo heses, in which he sampled Penicillopho inae
gene a Acladoce a and Wal e ius clus e ed oge he , we
applied he me hod o ou -clus e qua e -likelihood
mapping analysis (FcLM; S imme and on Haesele
1997; Niesel -S uwe and on Haesele 2001). We e alu-
a ed he posi ion o he ollowing gene a a) Acladoce a,
b) Wal e ius, c) Cenophengus, and d) Dis emocephalus.
Using his p ocedu e, we p ope ly es ed h ee compe ing
hypo heses: 1) (Acladoce a + Wal e ius)-(Cenophengus
+ Dis emocephalus); 2) (Acladoce a + Dis emocepha-
lus)-(Wal e ius + Cenophengus); and 3) (Acladoce a +
Cenophengus)-(Wal e ius + Dis emocephalus). In his
me hod, hypo hesized g oups a e o ganized in o qua e s
( ou - e minal se s), ep esen ing a simpli ied opology o
he ela ionships o be es ed. This p ocedu e ou pu s a
wo-dimensional simplex plo ha displays he s a is ical
suppo o compe ing hypo heses ia he p opo ions o
qua e s eco e ed om each possible opology o e en
inconclusi e ela ionships.
This analysis was designed o di ec ly assess he phy-
logene ic signal o all es ed da ase s (nucleo ides, amino
acids, 50% and 70% comple eness), which a e suppo ing
he pu a i e non-monophyly o Acladoce a and Wal e ius
in he analyses. A he same ime, we es ed he cu en
pu a i e opology ega ding he sub amilies Penicillo-
pho inae, Cenophenginae, and Mas inoce inae (Fe ei a
e al. 2024). Wi h his p ocedu e, we included all possible
ela ionships, ega ding he wo sampled Penicillopho i-
nae gene a and hei pu a i e mos closely ela ed sub-
amilies. Fou -clus e qua e likelihood mapping analy-
ses we e pe o med using IQ-TREE2 so wa e.
Figu e 4. A Phylogenomic hypo hesis o Phengodidae. All opologies ende ed he same opology wi h he analyses pe o med a
he nucleo ide le el 50-NT (50% comple eness ma ix) and 70-NT (70% comple eness ma ix) and amino acid le el 50-AA (50%
comple eness ma ix) and 70-AA (70% comple eness ma ix). All b anches a e suppo ed wi h maximum suppo le els o he
Shimodai a–Hasegawa-like app oxima e likelihood a io es (SH-aLRT), he Bayesian-like ans o ma ion o aLRT (aBayes), and
he “ul a as ” boo s ap (UFBoo ). B ou -clus e likelihood mapping (FcLM) es o al e na i e phylogene ic hypo heses showing
he placemen o Acladoce a and Wal e ius in ela ionship o o he Phengodidae g oups.
A h opod Sys ema ics & Phylogeny 83, 2025, 531–542 537
Table 1. S a is ical suppo o in e nal b anches measu ed o each phylogene ic econs uc ion based on nucleo ide (NT) and amino acid (AA) da ase s wi h 50% and 70% comple eness. Suppo le els
o maximum likelihood (ML) analyses e e o he Shimodai a–Hasegawa-like app oxima e likelihood a io es (SH-aLRT; in %), he Bayesian-like ans o ma ion o aLRT (aBayes); and he “ul a as ”
boo s ap (UFBoo , in %). Suppo le els o coalescen ASTRAL analysis e e o he local pos e io p obabili ies (Local PP).
T ee/
Analysis Suppo s
(Rhago ph halmidae
+
Phengodidae)
Phengo didae
(Phengodinae + (Acla-
doce a
+
(Cenophenginae +
Mas inoce inae includ-
ing Wal e ius)))
(Acladoce a
+
(Cenophenginae +
Mas inoce i nae includ-
ing Wal e ius))
(Cenophenginae
+
Mas inoce inae includ-
ing Wal e ius)
Mas inoce inae includ-
ing Wal e ius
(Oxymas ino ce us
+
Cephaloph ixo h ix)
01-ML-50-
AA
SH-aLRT
/ aBayes/
UFBoo
100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100
02-ML-70-
AA
SH-aLRT
/ aBayes/
UFBoo
100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100
03-ML-50-
NT
SH-aLRT
/ aBayes/
UFBoo
100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100
04-ML-70-
NT
SH-aLRT
/ aBayes/
UFBoo
100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100 100/1/100
05-AS-
TRAL-50-
AA
Local PP 1 1 1 1 0.7109 0.9996 0.8796
06-AS-
TRAL-70-
AA
Local PP 1 1 1 1 0.6642 0.9997 0.9064
07-AS-
TRAL-50-
NT
Local PP 1 1 1 1 1 1 1
08-AS-
TRAL-70-
NT
Local PP 1 1 1 1 1 1 1
Ba bosa FF e al.: Phylogenomic placemen o Penicillopho inae
538
3. Resul s
3.1. O holog sea ch, indi idual loci e-
co e ed, and da ase s assembly
A e o holog sea ch wi h CAPTUS, ou loci eco e y
le els anged om 839 (Za hipis in eg ipennis) o 2239
(Lamp ohiza splendidula) om each sample o a chi ed
da a sou ce (Table S1). A e p ocessing and conca ena-
ion o single-locus alignmen s wi h AMAS, ou inal
da ase s consis ed o he ollowing numbe o loci, bp,
and deg ee o missing da a: 50-NT (2180 loci, 1.300.931
bp, deg ee o missing da a: 2.59%–84.65%), 70-NT
(1697 loci, 1.011.955 bp, deg ee o missing da a: 1.76%–
81.45%), 50-AA (2180 loci, 433.626 aa, deg ee o miss-
ing da a: 2.59%–84.66%) 70-AA (1697 loci, 337.309 aa,
deg ee o missing da a: 1.76%–81.46%). The inal da a-
se s a e a ailable in File S1.
3.2. Phylogene ic analyses and ou -
clus e likelihood mapping
Rhagoph halmidae we e eco e ed as a sis e -g oup o
Phengodidae, and Phengodidae as a monophylum in all
analyses, wi h maximum suppo . All analyses (Fig. 4;
Table 1; File S1) ende ed iden ical opologies in he
ollowing con o ma ion: Cydis inae + (Phengodinae +
(Acladoce a + (Cenophenginae + Mas inoce inae in-
cluding Wal e ius))). All maximum likelihood analy-
ses eco e ed all b anches wi h maximum suppo , and
ASTRAL analyses eco e ed almos all g oups wi h
maximum suppo (see de ails below). The monophyly
o he wo sampled gene a adi ionally placed in Peni-
cillopho inae, i.e. Acladoce a and Wal e ius, was ejec -
ed in all analyses. Acladoce a was eco e ed in a basal
posi ion in ela ion o Cenophenginae + Mas inoce inae
including Wal e ius in all analyses, wi h maximum sup-
po . The g oup Cenophenginae + Mas inoce inae includ-
ing Wal e ius was eco e ed in all analyses (Local PP
o 50-AA-ASTRAL = 0.7109; Local PP o 70-AA-AS-
TRAL= 0.6642; all o he suppo s a maximum le el).
Mas inoce inae including Wal e ius we e eco e ed
monophyle ic in all analyses, wi h almos maximum sup-
po (Local PP o 50-AA-ASTRAL = 0.9996; Local PP
o 70-AA-ASTRAL= 0.9997; all o he suppo s a maxi-
mum le el). Inside Mas inoce inae, Wal e ius was eco -
e ed as a sis e -g oup o Dis emocephalus in all analy-
ses, always wi h maximum suppo . These ela ionships
we e suppo ed by he explo a ion o he phylogene ic
signal con en (i.e. a p opo ion o qua e s eco e ed)
ia a ou -clus e likelihood mapping using bo h nucleo-
ide and amino acid da ase s. The educed qua e (Acla-
doce a + Cenophengus)-(Wal e ius + Dis emocephalus)
p esen ed maximum suppo (100%) o bo h nucleo ide
and amino acid da ase s (Fig. 4).
4. Discussion
A e he mos comp ehensi e phylogenomic hypo hesis
o Phengodidae by Fe ei a e al. (2024), he monophy-
ly and phylogene ic placemen o a ely collec ed and
mo phologically modi ied Penicillopho inae emained
he bigges challenge in he amily’s sys ema ics (Kun-
d a a 2025). Al hough placed ou side he Phengodidae
by he mo phology-based analysis o Za agoza-Cabal-
le o and Zu i a-Ga cía (2015), Penicillopho inae we e
u he ea ed as phengodids by mos au ho s (Roza
2022, Fe ei a e al. 2024, Roza e al. 2024). He e, us-
ing a phylogenomic app oach, we con i med ha Acla-
doce a and Wal e ius, bo h hi he o classi ied in Pen-
icillopho inae, a e indeed phengodids. Howe e , hei
monophyly was ejec ed in all o ou analyses (Fig. 4;
File S1). Al hough hese wo gene a sha e simpli ied,
non-bipec ina e an ennae, hei mo phology o he wise
a ies conside ably.
Ou analyses eco e ed Acladoce a as a sis e -g oup
o Cenophenginae + Mas inoce inae, and Wal e ius nes -
ed inside Mas inoce inae. These esul s a e co obo a ed
by mo phology. Acladoce a has wo gula su u es (Fig.
2B) and he hindwing ein CAS (Fig. 3F), ea u es sha ed
wi h Cydis inae and wi h Cenophenginae. Besides ha ,
Acladoce a does no ha e he cha ac e is ic en al a -
sal combs ha occu in se e al gene a ac oss he di e se
Mas inoce inae. Wal e ius, on he o he hand, has a sin-
gle gula su u e and lacks he CAS ein. I also has he
en al a sal combs on bo h he i s p o- and meso a -
some e (Fig. 3D), a s a e sha ed wi h se e al New Wo ld
Phengodidae (Za agoza-Caballe o and Pé ez-He nández
2014; Roza 2022). In his con ex , i makes sense ha
Wal e ius is mo e closely ela ed o Mas inoce inae han
o Acladoce a.
The placemen o he emaining Penicillopho inae (i.e.
Adend oce a Wi me , 1976, Penicillopho us Paulus,
1975, and Ta sakan hos Za agoza-Caballe o, 2008), al-
hough no in es iga ed in ou s udy, can be hypo hesized
based on hei mo phology. Adend oce a and Penicillo-
pho us a e mo e simila o Wal e ius han o Acladoce a,
based on a numbe o cha ac e s like he p esence o a sin-
gle gula su u e, en al a sal combs on he i s p o- and
meso a some e, al hough o a iable leng h in ela ion o
he a some e (Figs 3A, C, D), and by he p esence o an
open adial cell, wi h a long ein 3 pa allel o he adial
cell (Figs 3E, G, H). The e a e some di e ences be ween
Wal e ius and wo abo e-men ioned gene a, like he p es-
ence o wo en o ial pi s in Wal e ius (one en o ial pi in
Adend oce a and Penicillopho us), and he sho ly uni-
amose an ennae in Wal e ius (despi e being desc ibed as
monili o m by Za agoza-Caballe o 2008), wi h he ami
sho and s ou , closely posi ioned in ela ion o he an-
ennome e (Fig. 2E; se a e in Adend oce a and se a e o
uni amose, wi h ami small, o med by an ema gina ion
o he se a e apex in Penicillopho us; Fig. 2C).
A h opod Sys ema ics & Phylogeny 83, 2025, 531–542 539
Acladoce a, in u n, has he mos abe an mo phology
when compa ed wi h o he gene a cu en ly placed in he
Penicillopho inae. The only simila i y wi h Adend oce a
and Penicillopho us is in he an ennae. O he wise, Acla-
doce a has wo gula su u es (Fig. 2B), no a sal combs
(Fig. 3B), and absen hind-wing adial cell (Fig. 3F). The
emaining genus in Penicillopho inae, Ta sakan hos, is
mos likely no a phengodid (ASR and MAI pe s. ob-
se .), and i s sys ema ic placemen will be ea ed in
de ail elsewhe e. To sum up, i is p obable ha Adend o-
ce a and Penicillopho us a e mo e ela ed o Wal e ius
o o he Mas inoce inae gene a, which would p omp he
sub amily Penicillopho inae o be conside ed a synonym
o Mas inoce inae.
The an ennae o Phengodidae a e ypically bipec ina e
o bi labella e, i.e. wi h mos an ennome es ha ing a pai
o sho o long ami. Such an ennae occu in all phen-
godid sub amilies bu Penicillopho inae, and we e also
p esen on he only ossil phengodid ecen ly desc ibed
om he C e aceous Bu mese ambe (Roza e al. 2023).
Fu he , bi amose an ennae a e also known in Rhagoph-
halmidae ( he sis e -g oup o Phengodidae; Kund a a e
al. 2022) and in C e ophengodidae, which a e pu a i e
ex inc ela i es o Phengodidae known only om Bu -
mese ambe (Li e al. 2021). Ou esul s con i med ha
an ennal simpli ica ion (i.e. loss o educ ion o he ple-
siomo phic double ami) happened a leas wice in he
e olu iona y his o y o Phengodidae, and we canno ule
ou he possibili y ha u u e analyses including Aden-
d oce a and Penicillopho us show e en mo e cases. This
is no su p ising, since ami modi ica ions a e a he com-
mon in he highly di e se Mas inoce inae: used ami a e
ound in se e al species o Akamboja Roza e al., 2017
and Eu yopa Go ham, 1881 (Wi me 1996; Coelho e al.
2024), he enla gemen o ce ain an ennome es and si-
mul aneous loss o ami a e ound in Eu ymas inoce us
Wi me , 1976 and Eu yogna hus Wi me , 1976 (Wi -
me 1976), and he p esence o a hi d amus, a ached
apically on he an ennome e body, is p esen in all spe-
cies o Pa ap o hodius Schae e , 1904 and some spe-
cies o Ph ixo h ix Oli ie , 1807 (e.g. Ph ixo h ix i i-
anii Wi me , 1996) (Za agoza-Caballe o 1999; Wi me
1996). The e o e, he an ennal simpli ica ion o Wal e ius
only con ibu es o a la ge eco d on an ennal a ia ion
in Mas inoce inae. I should be no ed ha a he ex eme
a iabili y in he shape o an ennae among closely ela ed
axa is a common phenomenon in many o he so -bodied
ela e oids, including Rhagoph halmidae (Kund a a e al.
2022) o Ela e idae: D ilini (Kund a a & Bocak 2019).
As we ailed o sequence Penicillopho us in ou s udy,
he sub amily s a us o Penicillopho inae emains dubi-
ous, and he only change we p oposed in his s udy is he
ans e o Wal e ius o Mas inoce inae. Fu u e s udies
including mo e Penicillopho inae (especially he ype
genus, Penicillopho us) and a b oade sampling o o he
sub amilies, in pa icula mo e Mas inoce inae gene a,
a e necessa y o unde s and and ede ine he bounda ies
and composi ion o hese sub amilies, and u he eluci-
da e he phylogene ic his o y o he amily Phengodidae.
5. Conclusions
Wi h a low-co e age whole genome sequencing ap-
p oach, we success ully p oduced genomic da a o wo
species o he highly mo phologically modi ied gene a
adi ionally placed in Penicillopho inae (Acladoce a
sp. and Wal e ius sp.) allowing us o es hei phylo-
gene ic placemen wi hin Phengodidae. Penicillopho i-
nae we e consis en ly eco e ed as a non-monophyle ic
g oup, ende ing he ollowing ela ionship: Cydis inae +
(Phengodinae + (Acladoce a + (Cenophenginae + Mas-
inoce inae including Wal e ius))). Al hough we did no
ha e access o he DNA-g ade ma e ial o he ype genus
o Penicillopho inae, we we e able o es he placemen
o o he supe icially simila gene a wi hin he b oade
con ex o Phengodidae phylogenomics. These esul s
su e om he absence o he ype genus o he Peni-
cillopho inae, which is mo phologically qui e di e gen
om he wo gene a included he e (Figs 2–3). The e o e,
he only axonomic change p oposed in ou s udy is he
ans e o Wal e ius o Mas inoce inae. Disco e y o a
DNA-g ade specimen o ha genus emains a p io i y.
Fu u e esea ch is essen ial o cla i y he limi s o Peni-
cillopho inae and o he phengodid sub amilies, especial-
ly Mas inoce inae.
6. Decla a ions
Au ho con ibu ions. Concep ualiza ion: FFB, ASR, VSF, RK. Fund-
ing acquisi ion: ASR, JRMM, VSF. Da a cu a ion: ASR, VSF, LSD.
Fo mal analysis: FFB, VSF. In es iga ion: FFB, ASR, JRMM, MFG,
JH, LSD, MAI, VVB, VSF, RK. Me hodology: FFB, LSD, VSF. W i -
ing—o iginal d a : FFB, ASR, VSF, RK. W i ing— e iew & edi ing:
FFB, ASR, JRMM, MFG, JH, LSD, MAI, VVB, VSF, RK. P ojec ad-
minis a ion: FFB, ASR, VSF, RK. Valida ion: FFB, ASR, VSF, RK.
Visualiza ion: ASR, VSF.
Compe ing in e es s. The au ho s decla e ha hey ha e no con lic s o
in e es in ela ion o his wo k.
7. Acknowledgemen s
MFG would like o hank M. F ances Kelle and Da id Wya o he
oppo uni y o join hei esea ch ip o Belize, and he Belize Fo es
Depa men (Minis y o Sus ainable De elopmen , Clima e Change
and Risk Managemen , Belmopan) o issuing pe mi s. JH would like o
hank Jiří Pi kl o his collabo a ion and assis ance wi h he expedi ion
in he Dominican Republic, Michel Sa o i and Nadi Al a ez o hei
suppo , Gab iel de los San os o his help in he Dominican Republic,
and he Minis e io de Medio Ambien e y Recu sos Na u ales o issu-
ing he necessa y collec ing and expo pe mi s (VAPB-08862, VAPB-
10887, VAPB-12695). ASR acknowledges he Fundação Ca los Chagas
Filho de Ampa o à Pesquisa do Es ado do Rio de Janei o (FAPERJ) o
he pe sonal unding (g an numbe 205.818/2022 and 205.819/2022)
and o he pho og aphic sys em acqui ed h ough g an (g an numbe
110.040/2014). JRMM was suppo ed by a ellowship om he Con-
selho Nacional de Desen ol imen o Cien í ico e Tecnológico (CNPq,
