REGULAR ARTICLE
Naming he o he cousin: A new goldie ba b (Cyp inidae:
Smiliogas ininae) om he no heas esca pmen in
Sou h A ica, wi h p oposed axonomic ea angemen
o he goldie ba b g oup in sou he n A ica
Ma inus Scheepe s
1,2
| Ped o H. N. B agança
1,3
| Albe Chakona
1,2
1
NRF-Sou h A ican Ins i u e o Aqua ic
Biodi e si y (NRF-SAIAB), Makhanda,
Sou h A ica
2
Depa men o Ich hyology and Fishe ies
Science, Rhodes Uni e si y, Makhanda,
Sou h A ica
3
Depa men o Ich hyology, Ame ican
Museum o Na u al His o y, New Yo k,
New Yo k, USA
Co espondence
Ma inus Scheepe s, NRF-Sou h A ican
Ins i u e o Aqua ic Biodi e si y (NRF-SAIAB),
P. Bag 1015, Makhanda (G ahams own) 6140,
Sou h A ica.
Email: [email p o ec ed] .ac.za
Funding in o ma ion
Na ional Resea ch Founda ion-Founda ional
Biodi e si y In o ma ion P og am REFRESH
p ojec , G an /Awa d Numbe : FBIP-
211006643719; Na ional Resea ch
Founda ion-Founda ional Biodi e si y
In o ma ion P og am TOPOTYPES p ojec ,
G an /Awa d Numbe : IBIP-BS 13100251309;
Wo ld Wildli e Fund, G an /Awa d Numbe :
NRF-SAIAB-WWF 40001528-2019
Abs ac
A g owing body o e idence indica es ha he global di e si y o eshwa e ishes
has no been ully documen ed. S udies o eshwa e ishes ha we e p e iously
hough o be mo phologically a iable ha e e ealed he exis ence o deeply di e -
gen lineages, wi h many dis inc species. In sou he n A ica a numbe o En e omius
species exhibi ei he exceedingly wide o di ided dis ibu ion pa e ns ha should
be a e o eshwa e ishes wi h limi ed dispe sal oppo uni ies be ween i e sys-
ems. One such species is he sidespo ba b, En e omius nee i. As cu en ly de ined,
E. nee i has a disjunc dis ibu ion ha is di ided be ween i e s in he no heas
esca pmen in Sou h A ica and Eswa ini, and ibu a ies o he Uppe Zambezi in
Zambia and sou he n Congo in he Democ a ic Republic o Congo, wi h a la ge geo-
g aphic gap be ween hese wo popula ions. Wi h he use o molecula and mo pho-
logical me hods, he le el o di e gence be ween he wo popula ions was examined,
and a new species was desc ibed om he S eelpoo Ri e in he Limpopo Ri e sys-
em o Sou h A ica. Findings om his s udy p o ide u he e idence o a numbe
o axonomic p oblems wi hin he goldie ba bs o sou he n A ica, and some axo-
nomic ea angemen s a e p oposed o his g oup.
KEYWORDS
colo pa e n, Cyp ini o mes, in eg a i e axonomy, sys ema ics
1|INTRODUCTION
Unde s anding he sys ema ics o ishes in mega-di e se o de s such
as he Cyp ini o mes has imp o ed ma kedly in ecen yea s due o
molecula phylogene ic analyses (Mayden e al., 2008,2009; Sai oh
e al., 2011), and in e amilial ela ionships a e becoming well
es ablished (ShunPing e al., 2008; Wang e al., 2012; Yang
e al., 2015). The amily Cyp inidae consis s o eshwa e ishes
widely dis ibu ed h oughou No h Ame ica, Eu asia, and A ica
(Skel on, 2001). Mo phological simila i y is common among cyp inids,
which led ea ly axonomis s, elying on mo phological cha ac e s
alone, o g oup oge he species in ou sized gene a (Skel on
e al., 2018). One such genus was Ba bus (Daudin 1805), which Mye s
(1960) called a “mons ous agg ega ion,”consis ing o mo e han
u n:lsid:zoobank.o g:pub:7541C64C-65FE-4DAA-B488-35CDFC2FD735.
Recei ed: 6 No embe 2023 Re ised: 20 June 2024 Accep ed: 25 June 2024
DOI: 10.1111/j b.15870
FISH
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© 2024 The Au ho (s). Jou nal o Fish Biology published by John Wiley & Sons L d on behal o Fishe ies Socie y o he B i ish Isles.
J Fish Biol. 2024;105:1137–1150. wileyonlinelib a y.com/jou nal/j b 1137
800 species sp ead ac oss A ica, Asia, and Eu ope (Hayes &
A mb us e , 2017). Howe e , phylogene ic analyses e ealed ha Ba -
bus included many di e en and non ela ed lineages and g oups, indi-
ca ing ha he agg ega ion o la ge numbe s o eshwa e species
sp ead ac oss h ee con inen s in o a single genus made li le sense
(Skel on e al., 2018).
In he la e 1980s a numbe o axonomic and ka yological s udies
ha in es iga ed he di e si y and ela ionships o A ican cyp inids
p o ided e idence ha species placed in Ba bus showed h ee di e -
en ploidy le els, esul ing in he e alida ion o many gene a ha
we e a ha ime conside ed synonyms o Ba bus. The gene a Lucio-
ba bus (Heckel 1843), Cheiloba bus (Smi h 1841), and Pseudoba bus
(Smi h 1841), which we e conside ed o be synonyms o Ba bus,as
well as he ecen ly desc ibed gene a Ama olacyp is Skel on
e al., 2018,Namaquacyp is Skel on e al., 2018, and Sede cyp is
Skel on e al., 2018, a e e aploids (2n=c. 100 ch omosomes). All
hexaploid (2n=c. 150 ch omosomes) species ha e been placed in he
genus Labeoba bus Rüppel 1835, whe eas he A ican diploid species
(2n=c. 50 ch omosomes) we e ini ially designa ed as “Ba bus” o
indica e ha hey a e no closely ela ed o he Eu asian e aploid
Ba bus s.s. (Be ebi e al., 1996; Golub so & K ysano , 1993; Guégan
e al., 1995; Oelle mann & Skel on, 1990; Skel on, 1988; Skel on
e al., 2018; Tsigenopoulos e al., 2002). Following he s udy o Yang
e al. (2015), he A ican diploid species ha e been assigned o he
genus En e omius Cope 1867 in he sub amily Smiliogas inae (Tan &
A mb us e , 2018). This new designa ion was ini ially con es ed
(Ren & Mayden, 2016; Schmid & Ba , 2015; S iassny e al., 2016;
S iassny & Sakha o a, 2016) bu is now widely accep ed as he use o
“Ba bus”would s ill link he small A ican species o he ue Eu asian
Ba bus, when in ac he A ican and Eu asian axa belong o wo di -
e en sub amilies, Smiliogas inae and Ba binae, espec i ely (Skel on
2015,2016; Yang e al., 2015; Skel on e al., 2018; Tan & A mb us e ,
2018). The esu ec ion o En e omius conce ns only hose A ican
diploid species no al eady placed in he gene a Ba boides B üning
1929, Ba bopsis Di Capo iacco 1926, Caecoba bus Boulenge 1929,
Clypeoba bus Fowle 1936, and P olabeops Schul z 1941. The phyloge-
ne ic placemen o Ba bopsis is s ill unknown, and Ba boides,Caecoba -
bus,Clypeoba bus,P olabeops, and he e aploid genus Pseudoba bus
a e nes ed wi hin En e omius (Hayes & A mb us e , 2017; Ren &
Mayden, 2016; Schedel e al., 2022), which ende s En e omius poly-
phyle ic and illus a es he need o u he axonomic changes.
Cu en ly, En e omius consis s o 226 alid species dis ibu ed
ac oss A ica (F icke e al., 2023; Hayes & A mb us e , 2017), wi h
new species con inuously being desc ibed (e.g., Kambikambi
e al., 2021; Ka emo Manda e al., 2020), e alida ed (e.g., Englmaie
e al., 2020; Mae ens e al., 2020; Schmid e al., 2018), o awai ing
o mal desc ip ion (e.g., Popoola e al., 2022). Fo y- wo En e omius
species occu in he sou he n A ican egion as de ined by Skel on
(2001), 23 o which a e endemic o Sou h A ica (F oese &
Pauly, 2022; Kambikambi e al., 2021; Skel on, 2001). Skel on (2001)
placed he sou he n A ican ba bs in h ee g oups based on di e -
ences in he p ima y do sal- in ay mo phology: he so - ayed ba bs
wi h a smoo h and lexible p ima y do sal- in ay, he saw in
ba bs wi h a spinous and se a ed p ima y do sal- in ay, and he spine-
in ba bs wi h a smoo h and spinous p ima y do sal- in ay. Wi hin he
so - ayed ba bs, wo addi ional g oups we e iden i ied, he goldie and
chubbyhead ba bs, based on a dis inc i e b eeding colou a ion and
unique mo phological cha ac e is ics. Cu en ly, only h ee species
belong o he goldie ba b g oup: he sho in ba b En e omius b e ipinnis
(Jubb 1966), he sidespo ba b En e omius nee i (G eenwood, 1962), and
he goldie ba b En e omius pallidus (Smi h 1841). The h ee species in
he goldie ba b g oup a e cha ac e ized by ela i ely small (<70 mm
s anda d leng h [SL]) compac bodies, wo pai s o ba bels, and a b igh
golden colo a ained by males du ing he b eeding season. Despi e pos-
sessing simila cha ac e is ics, En e omius g eenwoodi (Poll, 1967), En e o-
mius lineomacula us (Boulenge 1903), En e omius hamalakanensis
(Fowle 1935), and En e omius i ipa us (Webe 1897) ha e no been
included unde he goldie ba b g oup, bu no jus i ica ion has been p o-
ided o hei exclusion (Skel on, 2001).
E. nee i was desc ibed based on 17 specimens collec ed in 1960
om he Kabompo Ri e , a majo ibu a y o he Uppe Zambezi
Ri e sys em, in Zambia (G eenwood, 1962). A combina ion o cha ac-
e is ic ma kings dis inguish E. nee i om E. b e ipinnis and E. pallidus:
a spo a he base o he anal and pec o al ins, a iable numbe o
la ge da k spo s along he body, and hin wa y pa allel lines along he
op and bo om o each scale ow o he lank, ex ending en ally
beyond he la e al line scale ow (G eenwood, 1962; Jubb, 1968;
Skel on, 2001). Ini ially he Uppe Zambezi and headwa e s o he
Lualaba (sou he n Congo) we e he only known dis ibu ion ange o
he species, un il specimens wi h simila cha ac e is ics we e eco ded
om he O ighs ad and S eelpoo i e s, Limpopo Ri e sys em in
Sou h A ica (Jubb, 1968). These specimens we e assigned o E. nee i,
mainly based on possession o wa y pa allel lines ha a e he key dis-
inguishing ea u e o his species. Subsequen ly mo e popula ions
we e iden i ied in he Le aba, Mu ale, Le uhu, Sabie, C ocodile,
Makondo, and M olozi i e s. This esul ed in he species ha ing a dis-
junc dis ibu ion pa e n, wi h he wo known popula ions sepa a ed
by a la ge geog aphic gap o 1000 km be ween he Uppe Zambezi
Ri e in he no h and he no heas esca pmen o Sou h A ica
(Skel on, 2001). Disjunc dis ibu ion pa e ns ha e been eco ded o
o he sou he n A ica eshwa e ish species, including E. pallidus,
Mesobola b e ianalis (Boulenge 1908), and Amphilius na alensis
(Boulenge 1917). These species, ini ially hough o ep esen widely
dis ibu ed species, bu ollowing applica ion o in eg a i e axonomic
esea ch, we e shown o ep esen deeply di e gen lineages, esul -
ing in he ecen desc ip ion o new species (Chakona e al., 2015;
Mazungula & Chakona, 2021; Riddin e al., 2016).
The aim o he p esen s udy was o use molecula COI (cy o-
ch ome oxidase subuni I) and mo phological da a wi hin an in eg a-
i e axonomy pe spec i e o delimi species bounda ies wi hin
E. nee i. Fu he mo e, he phylogene ic s a us o he “goldie”ba b
g oup was explo ed by acqui ing opo ypic sequences o sou he n
A ican species wi h wo pai s o ba bels and males ha a ain b igh
golden colou a ion du ing he b eeding season. Po en ial axonomic
ea angemen s a e p oposed, and conse a ion implica ions a e
highligh ed.
1138 SCHEEPERS ET AL.
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2|MATERIALS AND METHODS
2.1 |Sample collec ion
Desc ip ion o he new En e omius species is based on 15 specimens
collec ed du ing ield su eys in he S eelpoo Ri e (Limpopo Ri e
sys em), Sou h A ica, in May 2012 and May 2021. Compa a i e opo-
ype specimens o E. nee i we e collec ed du ing ield su eys
be ween 2011 and 2019 om he Kabompo Ri e in Zambia, and he
ype specimens we e measu ed by he au ho Albe Chakona a
he Na ional His o y Museum, London. A o al o 11 new sequences
we e gene a ed o he wo E. nee i popula ions, E. b e ipinnis,
E. g eenwoodi,E. hamalakanensis, and E. i ipa us. Twen y- i e
sequences we e used om BOLD (Ba code o Li e Da a) and a single
sequence om GenBank. The app oaches used o sample collec ion
and p ocessing we e app o ed by he NRF-SAIAB Animal E hics Com-
mi ee ( e e ence no.: 2014/03).
2.2 |Molecula da a
2.2.1 | Ex ac ion, ampli ica ion, and sequencing
DNA was ex ac ed om p ese ed issues using he sal ing-ou p o-
ocol o Sunnucks and Hales (1996), and DNA concen a ions we e
quan i ied using a Nanod op ND-1000 spec opho ome e (Nanod op
Technologies, Inc.). A agmen o he mi ochond ial COI gene was
ampli ied by PCR using uni e sal ish DNA ba coding p ime se s:
FishF1 and FishR1 (Wa d e al., 2005) and VF2-T1 and VR1-T1
(I ano a e al., 2007). PCRs we e pe o med wi h a Ve i i 96-well
he mal cycle (Applied Biosys ems, Fos e Ci y, CA, USA). Each eac-
ion mix u e (25 μL) con ained 100–200 ng o empla e DNA, 12 μL
o Taq DNA Polyme ase 2Mas e Mix RED (Ampliqon PCR
Enzymes & Reagen s), 0.5 μL o each p ime (20 pmol), and 7 μLo
molecula -g ade wa e . PCR ampli ica ion was pe o med using he
ollowing p o ile: 3 min a 95C, ollowed by 12 cycles o 30 s a
95C, 40 s a 62C, dec easing by 0.5C pe cycle o 56.5C, and 50 s
a 72C. This was ollowed by 25 cycles o 30 s a 95C, 30 s a 56C
and 50 s a 72C, wi h a inal ex ension o 72C o 7 min. PCR p od-
uc s we e pu i ied using he ExoSAP me hod wi h a eac ion mix u e
con aining 5 μL o PCR p oduc , 0.5 μL o exonuclease I, and 1 μLo
Fas AP (Applied Biosys ems). Cycle sequencing was pe o med using
he BigDye Cycle Sequencing Ki (Applied Biosys ems) and sequenced
a SAIAB using an ABI 3730xl DNA Analyze (Applied Biosys ems).
Sequences we e edi ed and immed using Geneious P ime 2021.2.2.
2.2.2 | Phylogene ic analysis and species
delimi a ion
To assess he phylogene ic a ini ies o he wo E. nee i popula ions
and e i y he dis inc i eness o he new species, all COI sequences o
A ican Smiliogas inae a ailable in BOLD and GenBank and new
sequences om his s udy we e used o gene a e a phylogene ic ee
(Figu e S1). Sequences we e aligned using MAFFT, e sion 7.450
(Ka oh & Misawa, 2002; Ka oh & S andley, 2013), and inspec ed using
MEGA 11 (Tamu a e al., 2021) o he p esence o s op codons. Iden-
ical sequences we e emo ed p io o phylogene ic analysis. Bayesian
analysis was pe o med using M Bayes, e sion 3.2.6 (Huelsenbeck &
Ronquis , 2001), pa i ioning he da ase by codon posi ion and
employing e e sible-jump Ma ko chain Mon e Ca lo (RJ-MCMC)
sampling, wi h ime- e e sible subs i u ion models and γ-dis ibu ed
a e he e ogenei y (Huelsenbeck e al., 2004). The RJ-MCMC me hod
p ecludes he a p io i selec ion o subs i u ion models o each pa i-
ion and ins ead allows models o be sampled in p opo ion o hei
pos e io p obabili y (Huelsenbeck e al., 2004). Two pa allel analyses
o ou Ma ko chains and 5 million gene a ions we e un, sampling
ees e e y 1000 gene a ions and disca ding he i s 25% o ees as
bu n-in. Resul ing ees we e isualized in FigT ee, e sion 1.4.4
(Rambau , 2009). To assess he con e gence be ween he wo uns,
he a e age s anda d de ia ion o spli equencies was moni o ed in
M Bayes o ensu e i was <0.05. In addi ion, E ec i e Sample Size
(ESS) alues and he po en ial-scale educ ion ac o o all pa ame e s
we e examined using M Bayes and ound o app oach >100 and 1.0,
espec i ely. F om his ee he clade con aining he wo E. nee i
popula ions was e ained o de ailed analysis.
Fou molecula -based species delimi a ion me hods we e used o
iden i y ope a ional axonomic uni s (OTU) and explo e species bound-
a ies be ween axa belonging o he e ained lineage. The i s wo
me hods, “au oma ic ba code gap disco e y”(ABGD) and “assemble
species by au oma ic pa i ioning”(ASAP), a e gene ic dis ance–based
me hods ha ely on he analysis o single-locus sequence alignmen s
o de ine species pa i ions based on pai -wise gene ic dis ances
(Puilland e e al., 2012;Puilland ee al.,2021). Two coalescen
me hods, he “Bayesian implemen a ion o he Poisson ee p ocesses”
(bPTP) (Zhang e al., 2013) and he “gene al mixed Yule coalescen ”
(GMYC) (Fujisiwa & Ba aclough, 2013), we e pe o med. The bPTP
elies on single-locus molecula da a o delimi species based on he
numbe o nucleo ide subs i u ions be ween haplo ypes. In he GMYC
me hod, species a e delimi ed based on b anch leng hs, and i equi es
an u ame ic ee o de ine in aspeci ic and in e speci ic h eshold
pa e ns. (Fujisiwa & Ba aclough, 2013). The ul ame ic ee used o
he GMYC analysis was cons uc ed in BEAST2 (Bouckae e al., 2019)
using he Yule model p io wi h an op imized elaxed clock. The
assump ion o all hese me hods is ha in e speci ic a iabili y will be
subs an ially highe han in aspeci ic di e en ia ion.
2.3 |Mo phological da a
Following Chakona e al. (2014), 15 mo phological and 16 me is ic
cha ac e s we e ob ained o 16 specimens o E. nee i om he
Kabompo Ri e , including he ype se ies. Fo he Limpopo popula-
ion, 15 specimens om he S eelpoo Ri e (Limpopo Ri e sys em)
we e included. Ten specimens o he Limpopo popula ion and 13 spec-
imens o he Kabompo popula ion we e adiog aphed o acili a e he
SCHEEPERS ET AL.1139
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coun ing o skele al ea u es. Measu emen s we e aken using digi al
calipe s o he nea es 0.1 mm. P incipal componen analysis (PCA)
was pe o med using PAST (Hamme e al., 1999) on aw me is ic da a
and no malized mo phome ic da a (Lleona e al., 2000) o explo e
he a iables ha migh assis in dis inguishing bo h species. The no -
maliza ion p ocedu e allows o size- ee compa ison be ween speci-
mens. In a ian cha ac e s (see Table 3) we e excluded om he
analysis.
3|RESULTS
Gene ic and mo phological da a suppo he ecogni ion o he S eel-
poo E. nee i popula ion as a new goldie ba b species and he e
desc ibed as En e omius niggie sp. no . om he Limpopo Ri e sys em
o Sou h A ica.
3.1 |Molecula phylogene ic analyses
A Bayesian phylogene ic ee o all a ailable COI sequences o A ican
Smiliogas ins e ealed ha E. niggie sp. no . and E. nee i clus e ed
wi hin a majo clade con aining eigh mo phologically iden i ied spe-
cies, En e omius a kinsoni (Bailey 1969), En e omius macinensis (Dage
1954), E. i ipa us,E. pallidus,E. b e ipinnis,E. hamalakanensis,
E. lineomacula us, and E. g eenwoodi, as well as wo possible candida e
species En e omius sp. “Cuebe”and En e omius sp. “Bie” om mul iple
d ainages in sub-Saha an A ica (Figu e 1; Table 1; Figu e S1). Fu he -
mo e, wo species, E. b e ipinnis and E. i ipa us, we e each spli in o
a leas wo polyphyle ic lineages.
De ailed analysis using ou species delimi a ion me hods based
on 38 COI sequences ep esen ing E. niggie sp. no ., E. nee i, and he
o he eigh axa men ioned ea lie eco e ed 19 OTUs o candida e
species (Figu e 1). These OTUs we e g ouped in o h ee well-
suppo ed clades (Figu e 1). Clade A con ained E. macinensis om
Bu kina Faso as well as wo OTUs ha we e mo phologically iden i-
ied as E. a kinsoni om Eas A ica. The OTU-con aining specimens
om Malawi and Mozambique a e subs an ially di e en ia ed (3.3%
di e gence) om he opo ype o E. a kinsoni om he Ru iji Ri e sys-
em (Figu e 1). Clade B comp ised wo OTUs om coas al Eas A ica,
lowe Zambezi, and he Incoma i sys ems ha a e cu en ly iden i ied
as En e omius c . i ipa us. These wo OTUs a e subs an ially di e en-
ia ed (9.1%–9.8% di e gence) om and a e dis an ly ela ed o he
opo ypes o E. i ipa us om he Mdlo i Ri e sys em, in Sou h A ica
(Figu e 1). Clade C con ained 14 OTUs, including E. niggie sp. no .
and h ee species cu en ly assigned o he goldie g oup:
E. pallidus,E. nee i, and E. b e ipinnis. Topo ypes o E. i ipa us,
E. hamalakanensis, and E. g eenwoodi as well as specimens ha we e
mo phologically iden i ied as E. lineomacula us we e eco e ed wi hin
his g oup. Th ee OTUs we e eco e ed o specimens ha we e mo -
phologically iden i ied as E. b e ipinnis, whe eas wo OTUs we e iden-
i ied wi hin E. hamalakanensis, and a simila pa e n o
E. g eenwoodi. In e es ingly he disjunc ly dis ibu ed species E. niggie
sp. no . and E. nee i we e ound o be dis an ly ela ed and deeply
di e gen (3.5%) om each o he (Figu e 1).
3.2 |Mo phological da a
PCA pe o med on 15 mo phome ic cha ac e s o E. niggie sp. no .,
and E. nee i exhibi ed a b oad o e lap be ween he wo species, indi-
ca ing ha hey could no be sepa a ed based on hese cha ac e s
(Figu e 2). The i s p incipal componen axis (PCI) accoun ed o
24.3%, PC2 21.2%, and PC3 15.4% o he a ia ion obse ed be ween
he lineages (Table 2). The i s PCA axis (PCI) was included as size di -
e ences we e accoun ed o in he no maliza ion p ocedu e. PCI was
mainly de ined by di e ences in pec o al- o pel ic- in leng h. PCII
con as ed di e ences in caudal peduncle leng h. PCIII highligh ed di -
e ences in pel ic- o anal- in leng h.
PCA pe o med on 10 me is ic cha ac e s showed incomple e
sepa a ion be ween he wo species (Figu e 3). PCI accoun ed o
49.84%, PCII 19.52%, and PCIII 12.69% o he a ia ion be ween line-
ages. Fac o loadings a e p esen ed in Table 2. PCI was de ined by di -
e ences in he numbe o scales along he la e al line. PCII con as ed
wi h di e ences in he numbe o pec o al- in ays. PCII highligh ed
di e ences in he numbe o bo h o al and caudal e eb ae. Despi e
he conside able o e lap in mo phome ic and me is ic cha ac e s
be ween E. niggie sp. no . and E. nee i, he e a e consis en quali a i e
colo pa e n di e ences be ween hese wo disjunc ly dis ibu ed
and gene ically di e gen species ha wa an hei sepa a ion as dis-
inc axonomic en i ies. These cha ac e is ics include he pa e ns o
wa y pa allel lines on he lank and he pa e n o bold spo s on he
do sal midline (Figu e 4). All 16 examined specimens o E. nee i had
he wa y pa allel lines ex ending below he la e al line, and bold spo s
we e p esen on he do sal midline, whe eas in he 15 specimens o
E. niggie sp. no . om he Limpopo, hese a e absen (Figu e 4).
Fo diagnosis o he new species and compa ison wi h E. nee i,
he me is ic and mo phome ic measu emen s ob ained in he p esen
s udy we e used. Fo compa isons wi h he o he species o in e es ,
in o ma ion om he o iginal desc ip ions and o he key e e ences
was used (Ma in & Chakona, 2019; Poll, 1967; Skel on, 2001).
3.3 |Taxonomic accoun s
En e omius niggie sp. no . [niggie: ‘nᶕᶍi] (g/ch om A ikaans/Du ch)
is p onounced wi h a ha d gu u al sound, made a he back o he
h oa .
u n:lsid:zoobank.o g:ac :C24274E8-B384-409F-9A34-A821E9F6
9625.
(Figu es 5and 6; Table 3).
3.4 |P oposed common names
Sou he n sidespo ba b; Suidelike sykol ghieliemien jie (A ikaans).
1140 SCHEEPERS ET AL.
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3.5 |Holo ype
SAIAB 236359, ield numbe : NPEJ21-B080, male, 41.10 mm SL,
S eelpoo Ri e , Limpopo Ri e sys em, 24.80238, 30.11740, col-
lec ed by Ho man A., May 2021 (Figu es 5b and 6b).
3.6 |Pa a ypes
SAIAB 236360, nine unsexed, 33.35–42.21 mm SL, same collec o
and locali y as holo ype. SAIAB 186470, i e unsexed, 35.76–
44.36 mm SL, S eelpoo Ri e , Limpopo Ri e sys em, 24.72907,
30.18468, collec ed by Palme R., May 14, 2012.
3.7 |Addi ional ma e ial
SAIAB 26620, 1 unsexed, 48.00 mm SL, Mohlapi se Ri e , Limpopo
Ri e Sys em, 24.04999, 30.03333, collec ed by J. Engelb ech , Ap il
1, 1986. SAIAB 27269, 1 unsexed, 39.15 mm SL, Le aba Ri e , Lim-
popo Ri e sys em, collec ed by D. Cu le, Sep embe 22, 1986. SAIAB
49504, 6 unsexed, 35.40–45.10 mm SL, ibu a y o he Le u hu
Ri e , Limpopo Ri e sys em, 23.06667, 30.25000, collec ed by
M. Angliss, Ap il 6, 1995. SAIAB 61031, 20 unsexed, 20.00–
39.10 mm SL, Mu ale Ri e , Limpopo Ri e sys em, 22.73444,
30.65861, collec ed by R. Bills, D. Na an, B. Van de Waal, No embe
13, 1999. SAIAB 61044, 1 unsexed, 28.10 mm SL, Mu ale Ri e ,
Limpopo Ri e sys em, 22.70000, 30.64305, collec ed by R. Bills,
FIGURE 1 Bayesian
phylogene ic ee showing he
phylogene ic ela ionships wi hin
he goldie ba b g oup. The lineage
is ex ac ed om he b oade
analysis o a ailable COI
(cy och ome oxidase subuni I)
sequences inco po a ing
95 species cu en ly assigned o
he genus En e omius (Figu e S1).
Bayesian pos e io p obabili ies
a e gi en on he b anches as
pe cen ages; as e isk indica es PP
>99. Ba s show he OTUs
(ope a ional axonomic uni s)
iden i ied by each o he ou
species delimi a ion me hods. Red
OTUs con ain sequences o
opo ypic specimens.
SCHEEPERS ET AL.1141
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D. Na an, B. Van de Waal, 13 No embe 1999. SAIAB 63443,
4 unsexed, 40.70–48.40 mm SL, Wyliespoo , ibu a y o Mu amba
Ri e , Limpopo Ri e sys em, 22.91670, 29.92811, collec ed by
J. Engelb ech , Sep embe 20, 2000. SAIAB 190517, 44 unsexed,
27.50–41.2 mm SL, O ighs ad Ri e , Limpopo Ri e sys em,
24.89027, 30.58833, collec ed by E. Swa z, L. da Cos a, Oc obe
22, 2005. SAIAB 190556, 10 unsexed, 20.60–35.70 mm SL, S e k-
sp ui Ri e , Limpopo Ri e sys em, 25.156944, 30.558611, col-
lec ed by E. Swa z, L. da Cos a, Oc obe 23, 2005. SAIAB 203320,
1 unsexed, 31.00 mm SL, Thabina Ri e , Limpopo Ri e sys em,
TABLE 1 Species names, e e ence numbe s o BOLD/GenBank sequences, and locali y de ails o sequences used o he p esen s udy.
Species Sequence ID
Ca alogue
numbe Coun y Ri e Sys em La i ude Longi ude
En e omius a kinsoni MAFW071-11 SAIAB 119079 Malawi Ruo Zambezi 16.04 35.79
E. a kinsoni MAFW072-11 SAIAB 119079 Malawi Ruo Zambezi 16.04 35.79
E. a kinsoni ACAM028-13 SAIAB 190277 Mozambique Chipembe Dam Mon epuez 13.20 38.62
E. a kinsoni MAFW114-11 SAIAB 118777 Malawi Shi e Zambezi 15.06 35.22
E. a kinsoni MAFW096-11 SAIAB 185654 Malawi Phalombe Lake Chilwa 15.81 35.65
E. a kinsoni MAFW091-11 SAIAB 185654 Malawi Phalombe Lake Chilwa 15.81 35.65
E. a kinsoni GBMNB3511-20 NA Tanzania G ea Ruaha Ru iji 07.63 36.89
En e omius c . i ipa us MPUMA008-12 SAIAB 194032 Sou h A ica Klein-Sand Incoma i 24.66 31.09
En e omius c . i ipa us SAFW912-14 SAIAB 081022 Mozambique Zambezi Zambezi 15.60 32.72
En e omius c . i ipa us ACAM064-13 SAIAB 190265 Mozambique Tshidi Zambezi 15.62 33.67
En e omius c . i ipa us ACAM090-13 SAIAB 190291 Mozambique Tshidi Zambezi 15.69 33.67
En e omius c . i ipa us ACAM005-13 SAIAB 190235 Mozambique Muhukwa Mon epuez 13.43 38.61
En e omius c . i ipa us MAFW095-11 SAIAB 185652 Malawi Phalombe Lake Chilwa 15.81 35.65
En e omius macinensis KP712064 NA Bu kina Faso NA NA NA NA
En e omius b e ipinnis MPUMA015-12 SAIAB 194050 Sou h A ica Mac-Mac Incoma i 25.02 31.00
E. b e ipinnis OR763400
a
SAIAB 206418 Sou h A ica G oo sp ui Limpopo 24.53 27.87
E. b e ipinnis OR763404
a
SAIAB 194786 Sou h A ica Blyde Limpopo 24.90 30.75
En e omius i ipa us OR763397
a
SAIAB 235471 Sou h A ica Umdlo i Umdlo i 29.64 31.09
En e omius nee i OR763394
a
SAIAB 210153 Zambia Kabompo Zambezi 11.89 25.25
E. nee i OR763396
a
SAIAB 210153 Zambia Kabompo Zambezi 11.89 25.25
E. nee i OR763395
a
SAIAB 210153 Zambia Kabompo Zambezi 11.89 25.25
E. nee i SAFW842-13 SAIAB 082862 Democ a ic Republic
o he Congo
Kando Congo 10.80 25.98
En e omius pallidus SAFW824-13 SAIAB 186173 Sou h A ica Baakens Baakens 33.96 25.51
E. pallidus SAFW825-13 SAIAB 186173 Sou h A ica Baakens Baakens 33.96 25.51
En e omius lineomacula us MAFW088-11 SAIAB 185653 Malawi Phalombe Lake Chilwa 15.81 35.64
E. lineomacula us MAFW069-11 SAIAB 119069 Malawi Ruo Zambezi 16.10 35.69
E. lineomacula us MAFW124-11 SAIAB N/A Malawi Nka ha Bay Lake Malawi 11.62 34.23
En e omius hamalakanensis ANGFW230-12 SAIAB 187035 Namibia Oka ango Oka ango 18.12 21.58
E. hamalakanensis ANGFW160-12 SAIAB 186818 Angola Luassingua Oka ango 14.59 18.17
E. hamalakanensis ANGFW195-12 SAIAB 186874 Angola Cui o Oka ango 15.14 19.19
E. hamalakanensis OR763403
a
SAIAB 202855 Bo swana Oka ango Oka ango 19.21 22.75
En e omius g eenwoodi OR763410
a
SAIAB 85020 Angola Cuanza Cuanza 12.03 17.63
E. g eenwoodi SAFW283-08 SAIAB 84816 Angola Cuanza Cuanza 9.80 15.46
En e omius sp. “Cubango”ANGFW046-12 SAIAB 186686 Angola Cubango Oka ango 13.59 16.88
En e omius sp. “Cuebe”ANGFW012-12 SAIAB 186640 Angola Cuebe Oka ango 14.94 17.72
E.niggie sp. no . OR763406
a
SAIAB 236360 Sou h A ica S eelpoo Limpopo 24.80 30.12
E. niggie sp. no . OR763409
a
SAIAB 236359 Sou h A ica S eelpoo Limpopo 24.80 30.12
E. niggie sp. no . OR763407
a
SAIAB 236360 Sou h A ica S eelpoo Limpopo 24.80 30.12
Abb e ia ion: BOLD, Ba code o Li e Da a; NA, no a ailable.
a
No el sequences gene a ed om his s udy.
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24.02706, 30.18103, collec ed by A. Chakona, L. Dlamini, Ma ch
25, 2016. SAIAB 76348, 29 unsexed, 27.3–40.7 mm SL, Bu elskloo -
sp ui , ibu a y o C ocodile Ri e , Incoma i Ri e sys em,
25.40833, 30.46917, collec ed by R. Bills, J. Engelb ech , May
19, 2005. SAIAB 27262, 2 unsexed, 33.00–40.12 mm SL, Mluma i
Ri e , Incoma i Ri e sys em, 25.79999, 31.10000, collec ed by
D. Cu le, July 29, 1986. SAIAB 70742, 3 unsexed, 34.70–57.10 mm
SL, Hou bosloop, ibu a y o C ocodile Ri e , Incoma i Ri e sys em,
25.42222, 30.74333, collec ed by R. Boyco , R. Bills, J. Engelb ech ,
Ap il 29, 2003. SAIAB 67330, 1 unsexed, 33.50 mm SL, ibu a y o
Mkhond o Ri e , Mapu o Ri e sys em, 26.96861, 31.02139, col-
lec ed by J. Msibi, M. Fakudze, R. Boyco , N. Khumalo, No embe
6, 2002. SAIAB 76071, 1 unsexed, 27 mm SL, Bes e sp ui , M olozi
Ri e sys em, 27.75056, 30.83222, collec ed by B. G an , Augus
6, 2005. SAIAB 76077, 3 unsexed, 35.00–45.00 mm SL, ibu a y o
Lenjane Ri e , M olozi Ri e sys em, 27.90083, 31.07472, collec ed
by B. G an , June 8, 2005.
3.8 |Diagnosis
E. niggie sp. no . belongs o he goldie ba b g oup in sou he n A ica,
which is cha ac e ized by species wi h a so p ima y do sal- in ay, a
ela i ely sho compac body (<70 mm SL), he p esence o wo pai s
o well-de eloped ba bels, 24–30 la e al line scales, and a b igh
golden b eeding colou a ion in males. Along wi h E. niggie he goldie
ba b g oup includes he species E. pallidus (Smi h 1841), E. b e ipinnis
(Jubb 1966), E. nee i s.s. (G eenwood, 1962), E. hamalakanensis
(Fowle 1935), E. g eenwoodi (Poll, 1967), E. lineomacula us “Malawi”
(Boulenge 1903), and E. i ipa us (Webe 1897). E. niggie and E. nee i
can be eadily dis inguished om all he a o emen ioned species by
he p esence o dis inc i e pigmen a ion along he ma gins o lank
scales ha a e exp essed as wa y pa allel lines (Figu e 4a,b). Fu he ,
E. niggie can be dis inguished om E. nee i by he lack o wa y pa allel
lines below he la e al line (Figu e 4c) and by he lack o da k bold and
ounded spo s on he do sal midline o he body (Figu e 4d).
FIGURE 2 Sca e plo o PC1 agains
PC2 o a PCA (p incipal componen
analysis) ca ied ou on 16 no malized
mo phome ic cha ac e s o
31 specimens o En e omius niggie
sp. no . and En e omius nee i.
TABLE 2 Fac o loadings o he i s h ee p incipal componen
axes on 16 mo phome ic and 10 me is ic cha ac e s om 31
specimens o En e omius niggie sp. no . and En e omius nee i.
Cha ac e PC1 PC2 PC3
Mo phome ics 24.35% 21.16% 15.36%
Head leng h 0.128 0.106 0.046
Body dep h 0.108 0.546 0.146
P edo sal leng h 0.126 0.174 0.060
Do sal- in base 0.098 0.197 0.246
Pec o al- o pel ic- in leng h 0.838 0.320 0.237
Pel ic- o anal- in leng h 0.213 0.274 0.784
Anal- in base 0.006 0.246 0.289
Caudal peduncle leng h 0.366 0.599 0.160
Caudal peduncle dep h 0.056 0.051 0.002
Head dep h 0.013 0.069 0.237
Snou leng h 0.050 0.050 0.026
O bi diame e 0.026 0.002 0.080
In e -o bi wid h 0.108 0.108 0.084
Pos -o bi leng h 0.214 0.057 0.250
An e io ba bel leng h 0.128 0.106 0.046
Pos e io ba bel leng h 0.108 0.546 0.146
Me is ics 49.84% 19.52% 12.69%
La e al line scales 0.816 0.505 0.034
Ci cumpeduncula scales 0.032 0.064 0.003
P edo sal scales 0.393 0.268 0.387
Pec o al- in ays 0.326 0.756 0.134
Pel ic- in ays 0.069 0.257 0.119
To al e eb ae 0.122 0.122 0.641
P edo sal e eb ae 0.174 0.047 0.319
P e-caudal e eb ae 0.002 0.017 0.026
P e-anal e eb ae 0.002 0.017 0.026
Caudal e eb ae 0.148 0.116 0.551
No e: The mos impo an ac o loadings a e in bold on .
SCHEEPERS ET AL.1143
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FIGURE 3 Sca e plo o PC1 agains
PC2 o a PCA (p incipal componen
analysis) ca ied ou on 9 me is ic
cha ac e s o 31 specimens o E. niggie
sp. no . and En e omius nee i.
FIGURE 4 La e al iew o (a) En e omius nee i (34.6 mm SL) and
(b) En e omius niggie sp. no . (39. 7 mm SL) showing he p esence and
absence o he wa y pa allel lines below he la e al line. Do sal iew
o (c) E. nee i (34.1 mm SL) and (d) E. niggie sp. no . (34.6 mm SL)
showing he p esence and absence o he da k ounded spo s on he
do sal su ace.
FIGURE 5 Alcohol p ese ed colou a ion o En e omius niggie
sp. no . (a) NPEJ21-B081 37.9 mm SL (SAIAB 236360) and (b) NPEJ-
B080, 41.1 mm SL, holo ype (SAIAB 236359).
FIGURE 6 Gene al body ea u es and li e colou a ion o
En e omius niggie sp. no . (a) Male du ing b eeding season, ield ID
NPEJ21-B081 37.9 mm SL (SAIAB360). (b) Male du ing non-b eeding
season, ield ID NPEJ21-B080, 41.1 mm SL, holo ype (SAIAB
236359).
1144 SCHEEPERS ET AL.
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TABLE 3 Mo phome ic and me is ic da a o En e omius niggie sp. no . and E. nee i.
Cha ac e
E. niggie sp. no . E. nee i
O he specimensHolo ype Pa a ypes Holo ype Pa a ypes
Numbe o specimens 1 14 1 8 7
S anda d leng h (SL) (mm) 41.1 33.6–44.4 32.2 25.0–37.0 24.4–40.0
Head leng h (HL) (mm) 10.9 8.4–10.4 8.5 6.9–9.3 6.5–9.9
Caudal peduncle leng h (CPL) (mm) 9.6 8.5–12.6 7.6 5.7–8.6 6.0–9.5
Pe cen age o SL (%)
HL 26.5 21.3–26.1 26.4 24.9–27.9 22.7–26.8
P edo sal leng h 52.4 48.9–53.8 49.7 51.5–54.8 49.9–53.3
Do sal- in base 15.4 12.0–17.2 15.2 10.8–14.5 11.9–15.6
Do sal- in heigh 25.2 22.4–28.1 NA 24.9–28.7 20.0–25.9
Pec o al- in leng h 19.9 17.6–21.0 NA NA 14.8–20.1
Pel ic- in leng h 19.0 17.0–19.5 NA NA 14.4–18.0
Pec o al- o pel ic- in leng h 24.9 18.7–28.3 23.6 18.8–24.1 21.0–26.7
Pel ic- o anal- in leng h 17.9 14.6–21.9 17.4 14.3–19.6 14.5–21.1
Anal- in base 8.3 7.5–10.8 6.8 6.4–7.7 5.7–8.7
Anal- in heigh 18.9 15.1–19.6 NA 16.2–22.0 14.1–18.4
Body dep h 26.9 23.2–28.0 27.6 27.5–30.0 24.8–29.2
Body wid h 15.8 11.8–15.1 NA NA 12.9–17.1
CPL 23.4 22.1–26.7 23.6 21.9–25.0 20.0–25.7
Pe cen age o HL (%)
Head dep h 80.9 77.0–88.0 75.3 74.1–77.5 74.9–91.7
O bi 32.1 30.8–37.7 36.5 34.4–38.4 29.8–40.8
In e -o bi 25.7 22.8–34.3 31.8 29.5–35.6 21.2–30.9
Snou leng h 26.6 20.8–30.8 24.7 21.8–26.9 21.6–25.1
Pos -o bi 43.3 30.5–45.5 45.9 42.7–47.4 37.8–46.5
An e io ba bell 23.8 19.8–36.4 31.8 20.3–37.1 12.0–34.2
Pos e io ba bell 40.0 38.1–51.1 38.8 37.1–49.4 14.9–58.7
Pe cen age o CPL (%)
Caudal peduncle dep h 54.3 42.4–66.7 61.8 52.3–64.1 51.6–63.3
Me is ics
Unb anched do sal- in ays iii iii iii iii iii
B anched do sal- in ays 8 8 8 8 8
Unb anched anal- in ays iii iii iii iii iii
B anched anal- in ays 5 5 5 5 5
Pec o al- in ays 13 12 (8–14) NA NA 11 (10–11)
Pel ic- in ays 8 8 (8–9) NA NA 8 (8–9)
La e al line scales (LL) 30 27 (27–30) 27 27 (26–28) 26 (24–28)
Scale ows be ween LL and do sal in 5 5 5 5 5
Scale ows be ween LL and pel ic in 3 3 3 3 3
Scale ows be ween LL and anal in 3 3 3 3 3
Ci cumpeduncula scales 12 12 11 12 (11–12) 12 (11–12)
P edo sal scale ows 12 11 (11–12) 11 12 12 (11–12)
To al e eb ae 32 33 (32–34) 32 32 32
P e-caudal e eb ae 18 18 18 18 18
Caudal e eb ae 14 15 (14–16) 14 14 14
P edo sal e eb ae 9 9 (8–10) 9 9 (8–9) 8 (8–9)
P e-anal e eb ae 19 19 (19–20) 19 19 19
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