39
E olu iona y ela ionships o Fish Lake Valley Tui Chub Sipha eles
obesus ssp. (Teleos ei, Cyp ini o mes, Leuciscidae) and a new
genus o leuciscid minnows om he Al o d Basin, wes e n
Uni ed S a es
Ma hew A. Campbell1,2 , Se a C. Pe y1, Khyana N. Yea wood1, G ace Au inge 1, Nick Buckmas e 3,
Amanda J. Finge 1
1 Genomic Va ia ion Labo a o y, Depa men o Animal Science, Uni e si y o Cali o nia, One Shields A enue, Da is CA, 95616, USA
2 Fishes and Ma ine In e eb a es, Uni e si y o Alaska Museum o he No h, 1962 Yukon D i e, Fai banks AK, 99775, USA
3 Cali o nia Depa men o Fish and Wildli e, Inland Dese s Region, He i age and Wild T ou P og am, Bishop, CA 93514, USA
Co esponding au ho s: Ma hew A. Campbell ([email p o ec ed]); Amanda J. Finge (aj inge @ucda is.edu)
Copy igh : © Ma hew A. Campbell e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
The Tui Chubs, Sipha eles spp., a e ound widely ac oss he G ea Basin and in some
adjacen egions. Nea ly all di e si y o Sipha eles has been consolida ed unde he
name S. bicolo and he e a e nume ous isola ed popula ions o Tui Chubs o unce ain
axonomic s anding and he e o e unclea conse a ion p io i y. The Fish Lake Valley Tui
Chub (FLVTC) has been ecognized in o mally as S. bicolo ssp. 4 wi h a limi ed na u al
dis ibu ion in Fish Lake Valley in sou hwes Ne ada. Conside ing ha a igo ous exam-
ina ion o he phylogene ic ela ionships o he FLVTC and o he Tui Chubs has no been
conduc ed, he FLVTC is placed in a axonomic amewo k by i s applying a species
delimi a ion me hod o S. bicolo sensu la o using mi ochond ial da a and hen con-
duc ing phylogene ic analyses o genome-wide SNP da a. Sipha eles bicolo is be e
cha ac e ized by se en species, all wi h exis ing names, which he e a e conside ed o
be alid Sipha eles species. Fu he mo e, he sepa a ion o Al o d Basin Sipha eles
om o he Sipha eles is appa en as a deeply di e gen lineage. As a esul , we p opose
Epizon Campbell & Finge , gen. no . o con ain hese ishes. The Fish Lake Valley Tui
Chub is ound o be he ea lies -b anching lineage o S. obesus in ou SNP da a se
and a e highly di e en ia ed om o he S. obesus. These indings a e conco dan wi h
geologic e idence ha indica es ha Fish Lake Valley became connec ed o he b oade
Lahon an Basin ~2 million yea s ago, wi h gene low possible un il ~0.5 million yea s
ago. Based on he geog aphic dis ibu ion and magni ude o gene ic di e gence, we ind
he ecogni ion o FLVTC as a subspecies o S. obesus is app op ia e.
Key wo ds: ASAP, Epizon, G ea Basin ishes, molecula phylogene ics, phylogene ic
ne wo ks, species delimi a ion me hods
Academic edi o : Ped o B agança
Recei ed:
28 Feb ua y 2025
Accep ed:
6 Oc obe 2025
Published:
21 No embe 2025
ZooBank: h ps://zoobank.
o g/5D51F97E-4F34-4E86-A214-
3339985E5632
Ci a ion: Campbell MA, Pe y
SC, Yea wood KN, Au inge G,
Buckmas e N, Finge AJ (2025)
E olu iona y ela ionships o Fish
Lake Valley Tui Chub Sipha eles
obesus ssp. (Teleos ei, Cyp ini o mes,
Leuciscidae) and a new genus o
leuciscid minnows om he Al o d
Basin, wes e n Uni ed S a es. ZooKeys
1261: 39–67. h ps://doi.o g/10.3897/
zookeys.1261.151636
ZooKeys 1261: 39–67 (2025)
DOI: 10.3897/zookeys.1261.51636
40
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
In oduc ion
Encompassing a as a ea o wes e n No h Ame ica, he G ea Basin is gene -
ally bo de ed by he Columbia Ri e o he no h, he Sie a Ne ada o he wes ,
and by he Rocky Moun ains o he eas (Hough on 1976). The name G ea
Basin e e s o he ac i is endo heic, and i is composed o la ge sub-basins,
he Lake Bonne ille Sys em, he Cen al Basins, he Dea h Valley Sys em, he
No hwes Lakes Basin, and he Lake Lahon an Sys em (Hough on 1976). The
ich hyo auna o he G ea Basin is axonomically di e se, bu no consis en ly
dis ibu ed ac oss all sub-basins and limi ed in species di e si y.
Aqua ic species di e si y in a biogeog aphic egion is la gely a unc ion o
his o ical p ocesses ha gene a e and p ese e species di e si y. Fo ishes
in he G ea Basin, wo o hese p ocesses may be dispe sal in o and subse-
quen specia ion wi hin he G ea Basin. P ocesses ha gene a e di e si y
a e opposed by p ocesses ha educe aqua ic species di e si y, such as
long pe iods o habi a educ ion, a idi y, and isola ion. Isola ion may also
con ibu e o species di e si y i popula ions a e no econnec ed o , i hey
do, ep oduc i e isola ion is in place. In he G ea Basin, he Pleis ocene
was cha ac e ized by d ama ic changes in habi a , wi h la ge plu ial lakes
appea ing and disappea ing (Mi lin and Whea 1979), al e na ing pe iods o
dispe sal and high habi a a ailabili y wi h pe iods o isola ion, di e en ia-
ion, and ex inc ion. These pe iods o high and low wa e a ailabili y ope a -
ed o e d ainage pa e ns ha ha e been shaped on million-yea ime scales
(e.g., Minckley 1986). Du ing he his o y o he G ea Basin, ecological and
habi a p e e ences o ishes also in luenced dispe sal and popula ion con-
nec i i y. High-g adien sys ems in oo hills and moun ains we e subjec o
a highe equency o in e -d ainage headwa e s eam cap u e in compa -
ison o lacus ine spillo e a ec ing low-g adien sys ems. These highe
g adien sys ems a e ypically occupied wi h moun ain sucke s (Pan os eus
spp.), salmonids (Onco hynchus spp. and P osopium spp.) and sculpins
(Co us spp.). The low g adien and low ele a ion habi a s mo e ypically
a e occupied by sucke s (Ca os omus spp. and Chasmis es spp.), minnows
(Leuciscidae), pup ishes (Cyp inodon spp.), and salmonids wi h his habi a
p e e ence (Smi h e al. 2002). The dis ibu ion and di e si y o G ea Basin
ish species esul om hese habi a associa ions and sha ed his o ical
p ocesses as well as chance. None heless, he e olu iona y his o y o all
G ea Basin ishes is e lec ed in hei gene ic s uc u ing and gene ic di e -
si y (e.g., Finge e al. 2022; Su e al. 2022; Campbell e al. 2023).
While species di e si y is gene a ed, main ained, and educed by na u al p o-
cesses, i s ecogni ion is hea ily in luenced by scien i ic e o (Lundbe g e
al. 2000). G ea Basin ishes emain incomple ely documen ed and cha ac e -
ized axonomically, and numbe ~55 ecognized species-le el axa. O e such
a la ge a ea, he G ea Basin con ains abou 8% (55/683) o ecognized ish
species di e si y in No h Ame ica (Jenkins e al. 2015). These a e om he
Gas e os eidae (n = 1), Co idae (n = 3–4), Cyp inodon idae (n = 5–7), Goodei-
dae (n = 4), Salmonidae (n ~ 10), Ca os omidae (n ~ 13) and Leuciscidae (n ~
16) (Smi h e al. 2002; Sigle and Sigle 2014; Unmack e al. 2014; Campbell e
al. 2023; Ha is e al. 2025).
41
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
The leuciscid minnow lineages ha e a long p esence in he G ea Basin os-
sil eco d despi e he challenges associa ed wi h p ese a ion and eco e y o
ossils om hese ishes, and se e al species a e s ill p esen in his sys em.
Fossil leuciscid minnows a e known om a leas he middle Miocene in he
G ea Basin (~16 mya) (Ca ende 1986). Th ee gene a ha e been pe sis en
and a e ound widely, hese a e Gila Bai d & Gi a d, 1853, he senio synonym
o Moapa Hubbs & Mille , 1948, Rhinich hys Agassiz, 1849, and Sipha eles
Cope, 1883. Sipha eles is known o be well- ep esen ed in he ossil eco d in
he Miocene and Pleis ocene o he Lahon an Basin and Moja e Ri e (Smi h
e al. 2002). The genus Sipha eles, despi e being widely dis ibu ed ac oss he
G ea Basin, con ains only h ee alid species (F icke e al. 2025). Sipha eles
bicolo (Gi a d, 1856) is dis ibu ed b oadly ac oss phylogeog aphic egions,
including he G ea Basin, in he Klama h Ri e d ainage, he Sac amen o Ri e
d ainage (Pi Ri e ), and he Columbia Ri e d ainage. The o he wo species
a e closely- ela ed and a e ound in he Al o d Basin: S. al o densis (Hubbs
& Mille , 1972) and S. bo axobius (Williams & Bond, 1980), and hey may
belong o a sepa a e genus al oge he (Schonhu h e al. 2012). Di e gence
ime es ima ion indica es ha he Al o d Basin leuciscids has been an inde-
penden lineage o ~10 million yea s (Rabosky e al. 2018). All o he G ea
Basin Sipha eles ha e been placed unde S. bicolo (Gi a d, 1856) and wi hin
S. bicolo he e a e a ious dis inc i e and isola ed ish popula ions, bu hey
a e o unce ain axonomic s anding and hus unclea conse a ion p io i y.
Molecula da ing places he age o he lineage o ish classi ied as S. bicolo
a ~ 12 million yea s (Kuma e al. 2017; Rabosky e al. 2018). In conjunc ion
wi h ossil e idence, he dis ibu ion and ages o hese ishes indica es ha
he e was ample ime and space o di e si ica ion wi hin Sipha eles beyond
he cu en ly ecognized h ee species. O he gene a wi h dis ibu ions span-
ning he G ea Basin o ha age exhibi dis inc species such as Rhinich hys
(Moyle e al. 2023), Pan os eus Cope, 1875 (Unmack e al. 2014), Ca os omus
Lesueu , 1817, and Chasmis es Jo dan, 1878 (e.g., Campbell e al. 2023). In
pa icula , he e a e geog aphically and gene ically s uc u ed popula ions
wi hin hese gene a dis ibu ed among Columbian, Sac amen o/Klama h and
Lahon an geog aphic egions ha may be ecognized as species.
Based on he age, wide ange, occu ence ac oss phylogeog aphic egions
known o endemism, and lowe ele a ion habi a p e e ence ha p omo es
ewe d ainage basin exchanges i is plausible ha S. bicolo con ains se e al
species and subspecies-le el axa. Lack o ecogni ion o hese axa is, in pa ,
because a igo ous and comp ehensi e phylogene ic examina ion o G ea
Basin Sipha eles has no been unde aken o es he species bounda ies o
hese di e se popula ions. An unde s anding o he di e si y wi hin S. bicolo
and he ela ionships be ween his lineage and o he s will in o m managemen
and conse a ion decisions in his a id landscape.
He e we conduc his analysis o G ea Basin Sipha eles and ocus on he Fish
Lake Valley Tui Chub (FLVTC) which has been ecognized in o mally as S. bicolo
ssp. 4. I occupies a limi ed na u al dis ibu ion in Fish Lake Valley in sou hwes
Ne ada whe e i is he only ex an na i e ish. Speci ically, we conduc a molecula
phylogene ic s udy wi h he aim o iden i ying he e olu iona y ela ionships o
FLVTC, cu en composi ion o he lineage, and i s axonomic s anding.
42
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
Ma e ials and me hods
Mi ochond ial phylogeny and species delimi a ion
We ob ained ep esen a i e sequences o Sipha eles om he NCBI GenBank
d awing om a mi ochond ial cy och ome b (cy b) PopSe 28190045 (Ha is
2000). Ou g oup sequences we e selec ed om 12 leuciscid minnows in
he La iniinae sub amily wi h Ch osomus e y h ogas e (Ra inesque, 1820)
used o oo ing (Schonhu h e al. 2012). Addi ional Sipha eles sequenc-
es wi h iden i ying in o ma ion ha could expand ep esen a ion we e
ob ained om GenBank h ough blas n sea ches agains he NCBI Nucle-
o ide da abase using cy b sequences om PopSe 28190045 (Al schul e
al. 1997). Du ing he collec ion o public sequence da a, we ob ained a
single po en ial ep esen a i e m DNA sequence om he Owens Valley
(AF370056.1). The Owens Valley is known o con ain dis inc i e endemic
Sipha eles lineages, bu also ou -o -basin gene ics om human media ed
mo emen s (Chen e al. 2007). The single publicly a ailable sequence was
ambiguous due o hese ac o s and we unde ook Sange sequencing o
cy b sequences om 12 indi iduals om he Owens Valley o known na i e
backg ounds o ep esen endemic Sipha eles mi ochond ial lineages. We
used wo p ime se s o ampli y wo agmen s o he cy b gene ha we e
combined in o a longe sequence. The p ime se s we e p e iously epo ed
by Za doya and Doad io (1998) and used o gene a e p e iously published
da a om Sipha eles (e.g., Schonhu h e al. 2012). The p ime s used we e
Glu-F 5’-GAAGAACCACCGTTGTTATTCAA-3’; Cy b-R 5’-TCTTTATATGAGAAR-
TANGGGTG-3’, Cy b-F 5’-CACGARACRGGRTCNAAYAA-3’ and Th -R 5’-CCTC-
CRATCTYCGGATTACA-3’ (Za doya and Doad io 1998).
Polyme ase Chain Reac ions (PCR) we e ca ied ou in a o al olume o
25 µL, using 5x Colo less GoTaq® Flexi Bu e (5 uL pe eac ion) wi h a inal
concen a ion o 200 µM o dNTP, 2 mM MgCl2, 0.2 µM o each p ime , 0.625
U o GoTaq® DNA polyme ase, and 15 ng o DNA ex ac . A e an ini ial de-
na u a ion s ep o 2 min a 95 °C, we an 40 PCR cycles consis ing o 1 min a
95 °C, 1 min a 45 °C, and 1 min and 45 s a 72 °C. These cycles we e ol-
lowed by a 7-min inal ex ension s ep a 72 °C. Fo wa d and e e se di ec ion
ch oma og ams we e aligned wi h Geneious (h ps://www.geneious.com),
and hen he wo p ime se con igs oge he we e mapped o a e e ence se-
quence om NCBI (Sipha eles bicolo ; accession: AY096010.1) wi h he ‘Map
o Re e ence’ unc ion wi hin Geneious.
A mul iple sequence alignmen (MSA) o cy b sequences was made wi h
MAFFT (Ka oh e al. 2002; Ka oh and Toh 2008; Ka oh and S andley 2013) and
Maximum-Likelihood (ML) phylogeny in e ed wi h IQ-TREE2 (Nguyen e al.
2014; Minh e al. 2020). Model selec ion was conduc ed by speci ying Mod-
elFinde Plus (-m MFP) (Kalyaanamoo hy e al. 2017) and suppo o nodes
e alua ed wi h 10,000 apid boo s aps (-bb 10000) (Hoang e al. 2018).
To delimi possible species wi hin S. bicolo , mi ochond ial sequences iden-
i ied as S. bicolo we e uploaded o he Assemble Species by Au oma ic Pa -
i ioning (ASAP) webse e (Puilland e e al. 2021, h ps://bioin o.mnhn. /abi/
public/asap/asapweb.h ml). Simple, unco ec ed dis ance was speci ied .
43
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
Indi idual and genomic sampling
Access o Sipha eles issues was possible h ough ecen ly collec ed speci-
mens o om issue a chi es. Ma e ial om ac oss he G ea Basin ange o
he genus (Fig. 2, Table 1), ep esen ing known di e gen lineages o Sipha eles
wi h emphasis on po en ial nea ela i es o Fish Lake Valley Tui Chub was
a ge ed (Fig. 1, Ha is 2000). Fin clips we e ei he d ied and s o ed in coin
en elopes o in 95% e hanol p io o ex ac ion o DNA. Sampling included
ep esen a i es o S. al o densis (n = 13, one loca ion) and a a ie y o po en-
ial species-le el lineages cu en ly classi ied as subspecies unde S. bicolo :
S. moha ensis Snyde , 1918 (n = 74, wo loca ions); S. isola us (Hubbs & Mille ,
1972) (n = 25, one loca ion); S. newa kensis (Hubbs & Mille , 1972) (n = 25,
wo loca ions); S. bicolo (n = 74, six loca ions); S. halassinus (Cope, 1883)
(n = 43, h ee loca ions); S. snyde i (Mille , 1973), including ‘ oikona’ lineage ish
(Chen e al. 2007) (n = 66, ou loca ions and n = 50, wo loca ions espec i ely).
Sipha eles obesus (Gi a d, 1856) we e conside ed po en ial nea ela i es o
FLVTC and nine loca ions we e sequenced (n = 223). One loca ion o S. obesus
is loca ed in Fish Lake Valley (McNe Ranch) and was conside ed ep esen a-
i e o he FLVTC lineage. Sequences o oo ing phylogene ic analyses we e
ob ained om Gila o cu ii (Eigenmann & Eigenmann, 1890) and Pogonich hys
mac olepido us (Ay es, 1854).
De e mina ion o Sipha eles lineages was in pa in o med by mi ochon-
d ial phylogene ic esul s (Suppl. ma e ial 1: ig S1), inclusion in p e ious
s udies, and by geog aphic loca ions. Sipha eles al o densis samples a e
om he Al o d Lake basin, and no om Bo ax Lake and can ep esen
S. al o densis. The loca ion, Thousand C eek Go ge was in es iga ed p e i-
ously in gene ic s udy o nume ous Al o d Lake basin Sipha eles popula ions
by Smi h e al. (2019) and is ep esen a i e o S. al o densis in ha s udy.
Sipha eles moha ensis is ex inc om i s ype locali y, was ansloca ed o
conse a ion, and we sampled he descendan s o his managemen ac ion
(Chen e al. 2013). Sipha eles isola us is also ex inc om i s ype locali y,
bu was sampled om nea by loca ions in Independence Valley. Sipha e-
les newa kensis is endemic o sp ings in he Newa k Valley wi h a es ic -
ed dis ibu ion, desc ibed om a sp ing on he wes e n side o he alley.
The NV12 sampling loca ion is loca ed ~5.5 km om he likely ype locali y
as indica ed by Hubbs and Mille (1972) as sp ings on an allu ial an nea
Diamond Peak, on he wes side o Newa k Valley (Ha is 2000). Sipha eles
bicolo is desc ibed om Uppe Klama h Lake, wi h phylogene ic a ini ies o
he ma e ial examined in his s udy indica ed as S. bicolo , wi h Rock C eek
in Ca low Valley sequenced o bo h mi ochond ial and nuclea da a ypes
(Suppl. ma e ial 1: ig S1). Sipha eles halassinus is desc ibed om Goose
Lake, in O egon and we did no ob ain ma e ial om ha loca ion o his
s udy. Howe e , we ob ained ma e ial om close geog aphically p oximi y
om he Wa ne Valley and he Pi Ri e (Goose Lake may be conside ed pa
o he Pi Ri e d ainage). The e a e m DNA lineages simila o Goose Lake
samples p esen in Cowhead Slough (Suppl. ma e ial 1: ig S1) as well. Sam-
ples o S. halassinus om Twen y Mile Slough and Big Sage Rese oi we e
44
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
p e iously analyzed by Chen e al. (2009) wi h associa ed museum speci-
mens OS 17847 and OS 17853 and a e ep esen a i e o S. halassinus in
ha s udy. Sipha eles snyde i and S. snyde i ‘ oikona’ issues we e ob ained
la gely om e uge popula ions main ained o his axon, and may be con-
side ed ep esen a i e o his lineage. The Mule Sp ing sampling loca ion
has been p e iously in es iga ed as a e uge popula ion o he ‘ oikona’ ge-
ne ic lineage, ounded by ansloca ion om Cabin Ba Ranch in 1990 (Chen
e al. 2007). Sipha eles obesus was desc ibed om he Humbold Ri e , and
we we e able o sample om his d ainage basin o his s udy, and he mi o-
chond ial phylogene ic analysis and geog aphic dis ibu ion a e conco dan
wi h ecognizing he sampling as being ep esen a i e o S. obesus.
Figu e 1. Maximum Likelihood phylogeny o mi ochond ial (m ) cy och ome b (cy b) da a ea u ing ‘Sipha eles bicolo ’
labeled sequences wi h esul s o species delimi a ion indica ed o six clus e s. Nodal suppo is indica ed by g ay
ci cles o boo s ap suppo (BS) > 90%, whi e ci cles o 90% > BS > 75% and no indica ed o BS < 75%.
Ch osomus e y h ogas e
P ychocheilus g andis
Epizon bo axobius
Epizon bo axobius
Epizon al o densis
Epizon al o densis
Epizon al o densis
Epizon bo axobius
Epizon al o densis
Epizon bo axobius
P ychocheilus lucius Gila o cu ii
Ac ocheilus alu aceus
Gila obus a
Gila coe ulea
Relic us soli a ius Hespe oleucus symme icus
E emich hys ac os
Mylopha odon conocephalus La inia exilicauda
S. obesus m lineage
S. bicolo m lineage
S. halassinus m lineage
S. newa kensis m lineage
S. isola us m lineage
S. moha ensis
m lineage
S. snyde i samples
45
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
Table 1. Taxonomic and geog aphic sampling. The la ge geog aphic a ea (Vicini y) is indica ed along wi h each sampling
loca ion. The sample size (n) used in analyses wi h he median ead coun a e il e ing is gi en as well as he o al numbe
o indi iduals sequenced (To al n). Fo he yea collec ed, i collec ed in di e en yea s, he numbe e ained om each
yea o analyses is gi en. The la i ude and longi ude and sou ce o samples a e also p o ided wi h any addi ional pe inen
in o ma ion. The axonomic sampling is om Pogonich hys mac olepido us (Ay es, 1854), Gila o cu ii (Eigenmann &
Eigenmann, 1890), Epizon al o densis (Hubbs and Mille , 1972) and Sipha eles Cope, 1883. Sipha eles is abb e ia ed S.
in he axon column wi h S. halassinus (Cope, 1883), S. bicolo (Gi a d, 1856), S. newa kensis (Hubbs & Mille , 1972),
S. isola us (Hubbs & Mille , 1972), S. snyde i (Mille , 1973), S. moha ensis Snyde , 1918, and S. obesus (Gi a d, 1856).
Taxon Vicini y Sampling loca ion Median
ead coun nTo al
n
Yea
collec ed La i ude, Longi ude Sou ce
Pogonich hys
mac olepido us
Unknown Unknown 2461487 912 CDFW
A chi es
Gila o cu ii Moja e Ri e D ainage Dese Disco e y Pond 2053059 2 2 2012
Epizon al o densis Sheldon Wildli e Re uge Thousand C eek Go ge 7589069 10 13 2023 41.8873, -118.9520
S. halassinus Pi Ri e Big Sage Rese oi 4944824 12 12 2005 41.5957, -120.6405
S. halassinus Valley Falls Honey C eek 2065216 815 2011 42.4262, -120.1009
S. halassinus Wa ne Sp ing Twen y Mile Slough 1439984 316 2004 42.1291, -119.8177
S. halassinus Modoc Coun y Cowhead Slough 2964294 12 20 2023 41.9193, -120.0322
S. bicolo Ca low Valley Rock C eek 1136559 3 6 2011 42.6854, -119.1888
S. bicolo Ha ney Coun y Kueny Canyon 3307963 11 12 2005 42.6818, -118.9958
S. bicolo Sheldon Wildli e Re uge Andy’s Place 2348208 15 16 n = 4, 2022
n = 11, 2023
41.7916, -119.3846
S. bicolo Sheldon Wildli e Re uge Bi ne Ranch 3167076 16 16 n = 4, 2022
n = 12, 2023
41.7368, -119.4686
S. bicolo Sheldon Wildli e Re uge Ho se Canyon 7898712 4 4 2022 41.7863, -119.3244
S. newa kensis Newa k Valley NN4 3110134 20 25 2022 39.7227, -115.6919
S. newa kensis Newa k Valley NV12 5185390 25 25 2022 39.6498, -115.7700
S. isola us Independence Valley Wa m Sp ings 3322395 14 25 2022 40.9545, -114.7490
S. snyde i Mammo h Moun ain So che Lake 1571551 11 23 n = 1, 1998
n = 10, 2022
37.6286, -119.0737
S. snyde i ‘ oikona’ Owens Ri e Co onwood Pond (Whi e MT
Resea ch Cen e )
1787193 21 25 2010 37.3606, -118.3276
S. snyde i ‘ oikona’ Owens Ri e Mule Sp ing 1410489 10 25 2010 37.1061, -118.3276
S. snyde i Owens Ri e NE Pond (Whi e Moun ain
Resea ch Cen e )
2154310 22 25 2017 37.3606, -118.3293
S. snyde i Owens Ri e SE Pond (Whi e Moun ain
Resea ch Cen e )
2307926 210 2022 37.3604, -118.3294
S. snyde i Owens Ri e SW Pond (Whi e Moun ain
Resea ch Cen e )
2532879 2 8 2022 37.3604, -118.3296
S. moha ensis Moja e Ri e Camp Cady 2230368 16 19 n = 6, 1997
n = 10, 2005
34.9451, -116.5993
S. moha ensis Moja e Ri e D ainage Tui Slough 1662314 35 55 2011 34.3483, -117.2411
S. obesus Ca son Ri e , Ca son Dese Li le Soda Lake 2696470 22 25 2006 39.5147, -118.8833
S. obesus Fish Lake Valley Lida Pond 2590847 928 2022 37.4571, -117.4959
S. obesus Fish Lake Valley McNe Ranch 2008366 625 2021 37.8443, -118.0086
S. obesus Ho C eek Valley Twin Sp ings Slough 3127174 38 49 n = 16, 2011
n = 22, 2022
38.1969, -116.1656
S. obesus Humbold Ri e Sys em Uppe Humbold Ri e 2920540 25 25 2022 41.1564, -115.0271
S. obesus Lassen Coun y Eagle Lake 6658527 20 20 2023 40.6015, -120.7589
S. obesus Li le Fish Lake Valley Li le Fish Lake 1754730 612 2011 38.6181, -116.4710
S. obesus Rail oad Valley Flowing Wells 1647665 15 23 n = 14, 2011
n = 1, 2022
38.4081, -115.7003
S. obesus Walke Lake Rose C eek Rese oi 3571471 14 16 2023 38.5899, -118.6329
46
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
Genomic DNA was ex ac ed om he chub in clips using he QIAGEN
DNeasy Blood and Tissue Ki acco ding o he manu ac u e ’s p o ocol. Be-
cause he in clips used in his p ojec could be o subs an ial age (25+ yea s,
Table 1), we adjus ed he elu ion s ep o inc ease DNA yields by incuba ing
molecula g ade wa e a 56 °C o 10 min. DNA was elu ed in 25 μL o wa med
molecula g ade wa e hen cen i uged o 4 min a 5788xg. This s ep was e-
pea ed in o de o p oduce a o al o 50 μL o inal DNA p oduc .
Lib a y p epa a ion ollowed he Bes RAD p o ocol (Ali e al. 2016) wi h he
Sb I es ic ion enzyme and indi idual ba codes liga ed o sequences. Lib a ies
we e pooled and sequenced ac oss wo lanes on an No aSeqX wi h pai ed-end
Figu e 2. Sampling map o Sipha eles spp. sequenced in his s udy wi h RADseq included in analyses (n = 427). The ou g oup
axa o A oyo Chub (Gila o cu ii, n = 2) and Sac amen o Spli ail (Pogonich hys mac olepido us, n = 9) a e no shown.
Speci ic collec ion locali ies o S. obesus a e labeled. The inse indica es he ex en o he main map wi hin No h Ame ica.
Eagle Lake
Flowing Wells
Lida Pond
Li le Fish Lake
Li le Soda Lake
McNe Ranch
Rose C eek Rese oi
Twin Sp ings Slough
Uppe Humbold Ri e
35.0
37.5
40.0
42.5
−122 −120 −118 −116
Longi ude
La i ude
Epizon al o densis
S. halassinus
S. newa kensis
S. isola us
S. moha ensis
S. obesus
Sample Size
10
20
30
S. snyde i
400 km
N
S. bicolo
S. bicolo
47
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
150 bp 25B sequencing chemis y. Sequence da a we e demul iplexed o he
pla e le el hen combined om di e en lanes o indi idual demul iplexing.
Pai ed eads om indi iduals we e aligned o he G. o cu ii e e ence genome
(GCA_026230005.1) wi h he Bu ows-Wheele aligne and he MEM algo i hm
(bwa mem) (Li and Du bin 2010). Alignmen s we e so ed, il e ed o p op-
e pai ing, PCR duplica es emo ed, and numbe o aligned eads calcula ed
wi h SAM ools (Li e al. 2009). Indi iduals wi h mo e han 1 million il e ed and
aligned eads we e e ained o analyses.
Genome-wide phylogene ic analyses
A se o SNP geno ypes was gene a ed by i s calling SNPs wi h Analysis o
Nex Gene a ion Sequence Da a (ANGSD) (Ko neliussen e al. 2014). Quali y
con ol o SNPs was en o ced by equi ing SNPs o be p esen in 95% o indi-
iduals, minimum mapping and base quali y alues o 20 (-minMapQ 20, -minQ
20), a signi icance alue o 1.0 x 10-6 (-SNP_p al 1e-6), a pos e io p obabili y
alue o 0.95 (-pos Cu o 0.95) and a minimum Mino Allele F equency o 0.01
(-minMa 0.01). A SAM ools geno ype calling model was used and a PLINK- o -
ma ed ile gene a ed. Subsequen ly, he PLINK- o ma ed ile was con e ed o
a VCF- o ma ed ile wi h PLINK (Pu cell e al. 2007) and he VCF- o ma ed ile
was il e ed o a MAF o 0.05 and ‘p uned’ wi h BCF ools o ob ain unlinked SNPs
(Danecek e al. 2021). The speci ic op ions supplied o p uning wi h BCF ools
we e +p une -m 0.20 -w 10000. A Mul iple Sequence Alignmen (MSA) o SNPs
in PHYLIP o ma was made wi h he c 2phylip.py sc ip (h ps://gi hub.com/
edga domo iz/ c 2phylip/blob/mas e / c 2phylip.py) om he p uned VCF
ile. Subsequen con e sion o he PHYLIP- o ma ed ile o NEXUS o ma was
done wi h unc ions o he ape package in R (Pa adis 2010).
A conca ena ed Maximum-Likelihood (ML) phylogene ic ee was gene a ed
a he indi idual le el wi h IQ-TREE2 (Minh e al. 2020). A Gene al Time Re e sible
(GTR) model o nucleo ide e olu ion wi h asce ainmen bias co ec ion (+ASC)
was speci ied. Suppo o nodes was e alua ed wi h he ul a as boo s apping
algo i hm (-bb 1000 -nm 2000 -bco 0.9) (Hoang e al. 2018). A species- ee ha
inco po a es he mul ispecies coalescen (explici ly modeling incomple e lineage
so ing) was cons uc ed wi h SVDQua e s in PAUP* (Swo o d 2003; Chi man
and Kuba ko 2014, 2015). Indi iduals we e pooled a he collec ion loca ion le el
o his analysis, wi h 10 million andom qua e s e alua ed. Nodal suppo was
e alua ed wi h 1,000 boo s ap eplica es. A hi d analysis, an implici phyloge-
ne ic ne wo k was unde aken o isualize possible disco dance mo e ully as a
esul o hyb idiza ion and incomple e lineage so ing wi h Spli sT ee (Huson and
B yan 2005). Unco ec ed dis ances we e used wi h he Neighbo -Ne algo i hm.
Resul s
Mi ochond ial phylogeny and species delimi a ion
We analyzed an alignmen o 127 sequences wi h sequence accessions and
me ada a p o ided in Suppl. ma e ial 2. Mi ochond ial sequences a e om
12 leuciscid minnow species no classi ied in Sipha eles (n = 12 sequences)
and 115 o he sequences a e indica ed o be om di e en Sipha eles
54
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
1980). A e age numbe o do sal- in ays o 7, anal ays 7, pel ic ays 8, and
caudal- in ays 19. Epizon al o densis commonly has 13 pec o al- in ays and
E. bo axobius 14 (Williams and Bond 1980).
Epizon is endemic o he Al o d Basin o Eas e n O egon o he No hwes
Lakes sub egion, o he G ea Basin (Hough on 1976). The Al o d Basin is a
no h-sou h o ien ed alley bounded by S eens Moun ain o he wes and he
Owyhee Pla eau o he eas , wi h o ma ion o he alley du ing he Miocene
(O and O 2012). I appea s ha he ances al lineage o Epizon was iso-
la ed du ing his ime. Du ing he Pleis ocene, he plu ial Lake Al o d was
~19 km wide and 113 km long du ing maximal ex en (Reheis 1999). Subse-
quen d ying isola ed ishes in limi ed sui able habi a s in a cyclical ashion,
esul ing in E. al o densis being mo e b oadly dis ibu ed wi hin he basin o
o me Lake Al o d and E. bo axobius es ic ed o he he mal sp ing- ed Bo-
ax Lake and p oxima e habi a s. Wi hin Bo ax Lake, ish li e ypically o a yea
and a e o a smalle size (33–45 mm SL) whe eas in he di e se habi a s else-
whe e in Al o d Basin E. al o densis ish li e longe and g ow la ge , 4–5 yea s
and ≤ 120 mm SL (Williams and Bond 1983; Sigle and Sigle 2014). A sample
o 50 ish om G idley Sp ings we e epo ed o be 27–91 mm SL wi h a peak
a 30-38 mm SL and ish dis ibu ed in o la ge sizes ≤ 91 mm SL (Williams
and Bond 1983). As such, E. bo axobius had been no ed p io o o mal ax-
onomic desc ip ion as a dwa o m o E. al o densis. Bo ax Lake is ed by
sp ings wi h 35–40 °C ou lows esul ing in a lake o ypically 29–32 °C em-
pe a u es, wi h E. bo axobius a oiding empe a u es abo e 34 °C and a loss
o equilib ium a 34.5 °C (Williams and Bond 1983). Epizon al o densis occu-
pies a a ie y o habi a s in sp ings, c eeks, ponds, and ese oi s, in se en
o eigh his o ically occupied d ainages (Schee e e al. 2015). This axon is
ound in cold, cool, and wa m wa e s, bu no abo e 31.1 °C (Williams and
Bond 1983). Di e en ia ion o he wo species o Epizon may be d i en by
adap a ion o he unique cha ac e is ics o he he mal-sp ing discha ge
wi h possible sympa ic specia ion o E. al o densis and E. bo axobius (e.g.,
Smi h e al. 2019). In addi ion o he cha ac e s o Williams and Bond (1980),
nuclea gene ic da a sepa a es he wo species (Smi h e al. 2019). Remple
Posi ion Epizon gen. no . Sipha eles T ansi ion o T ans e sion
876 A C T
885 G A Ti
897 T A o G T
901 T C Ti
909 C G T
912 G A Ti
918 T C Ti
933 A G Ti
957 C T Ti
999 A T T
1002 T C Ti
1017 G A Ti
1023 A G Ti
1026 T C Ti
1059 A C T
1068 T C Ti
1122 C T Ti
55
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
(2013) epo s ha la ge eye diame e , longe snou leng h and longe head
leng h di e en ia ing E. al o densis and E. bo axobius a e appa en in ea ly
li e s ages and can be used o di e en ia e la ae.
Nup ial ube cles a e p esen in males wi h sexual dimo phism epo ed in
E. bo axobius based on ela i e leng h o ins, which a e all longe in males
han emales (Williams and Bond 1980). This cha ac e is ic is also appa en
in E. al o densis (Sigle and Sigle 2014). Gi en he cons an empe a u es o
Bo ax Lake, yea - ound spawning occu s wi h E. bo axobius. Epizon al o densis
in he mally luc ua ing habi a s, spawns only once a yea (Williams and Bond
1983). Epizon a e oppo unis ic omni o es wi h a die closely ela ed o he p o-
duc ion o hei habi a s and a iable wi h seasons (e.g., Williams and Williams
1980). Top die a y i ems a e mic oc us aceans, chi onomids and dia oms
(Williams and Bond 1983).
E ymology. Romanized G eek e sion o επιζών, meaning su i o in e e -
ence o he pe sis ence o his genus in he di e se and challenging dese
habi a s i has ound i sel . Gende masculine.
Discussion
Composi ion o Sipha eles
Analyses p esen ed he e (Figs 1, 3–5) and by o he s (Schonhu h e al. 2012,
2014, 2018; Rabosky e al. 2018;) indica e ha he Al o d Basin leuciscids
a e subs an ially di e ged om o he membe s o Sipha eles. Upon desc ip-
ion, he a ini ies o Gila al o densis Hubbs & Mille , 1972 we e o he hen
subgenus Sipha eles. The ecogni ion o Sipha eles as a genus by Simons
and Mayden (1998) did no include ep esen a ion o he Al o d Basin leu-
ciscids in hei molecula da a se ; howe e , Al o d Basin leuciscids we e
included in he ele a ion o Sipha eles a ha poin . Subsequen molecu-
la phylogene ic analyses o Al o d Basin leuciscids lack clea esolu ion
o hei placemen , nei he being a sis e lineage o Sipha eles no being
mo e-closely ela ed o o he ishes con incingly indica ed (e.g., Schonhu h
e al. 2012, 2014, 2018). We ad oca e ha he Al o d Basin leuciscids
should be placed in a sepa a e genus, Epizon gen. no ., desc ibed in he e-
sul s sec ion o his manusc ip . We choose o ecognize wo alid species
o Epizon al hough mi ochond ial ba code da a does no esol e hese wo
species phylogene ically (Fig. 1). P e ious in es iga ion o E. al o densis
and E. bo axobius wi h mic osa elli e and genome-wide SNP da a, howe e ,
ind suppo o a phylogene ic di ision o hese wo axa ha p eda es he
end o Pleis ocene (Smi h e al. 2019). The implica ion is ha hese wo
lineages may ha e specia ed sympa ically wi hin he Al o d Basin. These
lines o e idence as well as ana omical di e gence and adap a ion o ho -
sp ings habi a o E. bo axobius lead us o conclude in suppo o i s alidi y.
Monophyly o a less inclusi e Sipha eles is suppo ed ac oss di e en mo-
lecula da a ypes – mi ochond ial loci, nuclea loci, and genome-wide SNP
da a (e.g., Schonhu h e al. 2012, 2014, 2018) and Figs 1, 3, 4 in his s udy.
Mi ochond ial, genome-wide SNP da a and mic osa elli e da a indica e ha
his less inclusi e Sipha eles may be di ided in o se e al allopa ic uni s ep e-
sen ing dis inc clades (e.g., Figs 1–3; Ha is 2000; Remple 2013). Sipha eles
56
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
as de ined he e, con ains se en species: S. isola us, S. newa kensis, S. bicolo ,
S. halassinus, S. moha ensis, S. snyde i and S. obesus.
A majo phylogene ic g ouping p esen wi hin Sipha eles is o S. isola us
and S. newa kensis, ound in Independence Valley and Newa k Valley, Ne a-
da espec i ely. These lineages a e ound in he Cen al Basins egion o he
G ea Basin ollowing Hough on (1976). Wi hin hese species, he subspecies
S. isola us euchila (Hubbs & Mille , 1972) om Fish C eek Valley as well as
he nominal subspecies S. isola us isola us om Independence Valley may be
easonably ecognized. Fu he s udies speci ically on his ques ion, including
mo e comp ehensi e ana omical in es iga ion a e needed o e alua e he axo-
nomic s a us o S. isola us isola us and S. isola us euchila.
Ano he majo phylogene ic g ouping is o S. bicolo and S. halassinus. O e -
all, hese lineages a e ound dis ibu ed in he Klama h and Pi i e basins,
he No hwes Lakes sec ion o he G ea Basin (Hough on 1976), and he Co-
lumbia Ri e basin mo e gene ally (e.g., Lubinski and Scholz 2021). Sipha eles
bicolo con ains se e al subspecies including he named S. bicolo columbianus
(Synde , 1908) and S. bicolo eu ysoma (Williams & Bond, 1981). Unlike Ha is
(2000), sequences om he ‘Sil e Lake’ sampling loca ion a e nes ed wi hin
S. bicolo (Suppl. ma e ial 1: ig S1) and because o ha we do no conclude
i ep esen s an undesc ibed dis inc species as indica ed by Ha is (2000).
We also ind Sipha eles halassinus o be alid wi h he subspecies Gila bicolo
accaceps Bills & Bond, 1980 placed in molecula phylogene ic analysis as a
subspecies o S. halassinus. Tha is, S. halassinus accaceps (Bills & Bond,
1980), ound in he Cow Head Basin.
A hi d g ouping o Sipha eles, including S. moha ensis, S. snyde i and S. obesus
is also ound in ou genome-wide analyses. Geog aphically, hese lineages
a e ound in he Dea h Valley Sys em, Cen al Valleys, Lake Lahon an Sys em
and No hwes Lakes G ea Basin sub- egions o Hough on (1976). Sipha eles
moha ensis a e ound na u ally in he Moja e Ri e basin while S. snyde i is
ound in he Owens Valley. As p e iously indica ed by mic osa elli e analysis,
he ‘ oikona’ lineage is dis inc i e and es ic ed o he Owens Valley (Chen e al.
2007). Wi h ou b oad geog aphic and lineage sampling we iden i y ha he ‘ oi-
kona’ Tui Chub has close a ini ies o o he Tui Chubs om he Owens Valley and
may be conside ed a subspecies o S. snyde i, bu wi hou a o mal axonomic
name ye . We ep esen ed S. obesus wi h nume ous sampling loca ions and ad-
d ess he composi ion o his lineage in a sepa a e ollowing sec ion.
The h ee main lineages o his g ouping, S. moha ensis, S. snyde i and
S. obesus exhibi subs an ial mi onuclea disco dance (Figs 1, 3). The mi o-
chond ial da a om S. moha ensis indica es i is he ea lies b anching lineage
o Sipha eles as de ined he e, howe e he nuclea da a places S. moha ensis
as clea ly mos closely ela ed o S. snyde i and S. obesus. Geologic e idence
indica es ha he Owens Valley was he likely sou ce o Moja e Ri e Sipha eles
(Sol z and Naiman 1978) and ha he e we e connec ions ia he Ama gosa
Ri e o he Moja e Ri e whe e S. moha ensis was na u ally dis ibu ed. Con-
nec ions om he Owens Valley o Dea h Valley did occu up un il he end o
he Pleis ocene, bu how much gene low occu ed is unclea . No Sipha eles a e
known om ecen imes in he Ama gosa Ri e o o he in e media e sys ems
be ween he Moja e Ri e and Owens Ri e , and i p esen , would be insigh ul.
In Fig. 1 he b anching o de o m DNA lineages may no e lec he ue species
57
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
his o y and mo e complex scena ios a e no necessa y o in oke. The mi o-
nuclea disco dance obse ed be ween S. snyde i and S. obesus m DNA and
genome-wide SNP da a, howe e , may equi e a mo e complex scena io.
P e iously an Owens Ri e ish was sequenced o mi ochond ial cy b
(AF370056.1), and he h ee No heas Pond sampling loca ion ish we se-
quenced sha ed an iden ical haplo ype. The ou o he indi iduals success ully
sequenced om S. snyde i including oikona lineage ish, only di e ed by 2–4
mu a ional s eps om each o he , bu we e no all iden ical. Because hese
sequenced indi iduals come om e ugial popula ions ha a e managed o
conse a ion pu poses, he e we e conce ns abou he le el o po en ial hy-
b idiza ion in indi iduals used in ansloca ions in o he Owens Valley. How-
e e , hese S. snyde i ish did no show e idence o ecen hyb idiza ion in he
nuclea genome (Fig. 5). While we obse e deep di e gences in he nuclea
da a-based analyses, he mi ochond ial da a om he Owens Valley is nes ed
wi hin a b oadly dis ibu ed clade o S. obesus ish. The e a e h ee esul an
hypo heses ha can be explo ed. The i s is ha e y ecen in og ession has
occu ed leading o he obse ed pa e ns. This is unlikely gi en ha he e is mi-
ochond ial di e si y ac oss he Owens Valley ish sequenced and ha all ish
sequenced success ully o cy b ha e an obesus-like m DNA lineage. The sec-
ond hypo hesis may be ha he e was his o ic gene low be ween he Lahon an
Basin and he p ehis o ic Lake Russell, ep esen ed oday by Mono Lake. The e
is no cu en paleo-hyd ological in o ma ion suppo ing a connec ion be ween
he Lahon an and Mono Basins a e he 3.2 mya closu e o he no he n ou -
le o Lake Russell. The p esence o ac i e no mal and ans ensional aul ing
along he no he n edge o he Mono Basin makes he p obabili y o d ainage
cap u e a likely mechanism o he in oduc ion o he S. obesus mi ochond ial
lineage in o Lake Russell. Subsequen ou low e en s om Lake Russell would
ha e led o he exchange o mi ochond ial DNA lineages be ween he Lahon an
Basin and he Owens Valley, wi h selec ion d i ing he in og ession o m DNA.
Such e en s ha e been obse ed in se e al ish gene a leading o examples o
mi onuclea disco dance (Moyle and Campbell 2022; Campbell e al. 2024).
Finally, i is also possible ha he depic ed pa e n canno be ully in e p e ed
gi en he ew sequences gene a ed. Ou e o o gene a e and sequence cy b
amplicons om S. snyde i was no b oadly success ul. We ini ially a ge ed 12
indi iduals bu gene a ed only se en sequences. Addi ional sequencing o mi-
ochond ial da a om S. snyde i and dedica ed in es iga ion o S. moha ensis,
S. snyde i and S. obesus could be unde aken o cla i y he sou ce o his mi o-
nuclea disco dance.
Composi ion o Sipha eles obesus
Sipha eles obesus is ound ac oss he Lake Lahon an Sys em wi h some loca-
ions in he Cen al Basins and m DNA sequences om a egion o he No h-
wes Lakes a e placed wi h S. obesus hough no sequenced om he nuclea
genome in his s udy. The e a e h ee main lineages p esen wi hin S. obesus
in genomic sequence da a examined in his s udy. The i s di ision is a Fish
Lake Valley lineage ep esen ed by wo sampling loca ions (Lida Pond and
McNe Ranch), wi h Rail oad and Li le Fish Lake alleys o ming a second
lineage, and he emainde o S. obesus sampling loca ions (Walke , Lahon an
58
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
d ainages, e c.) o ming a hi d lineage. The m DNA da a analyzed also con-
ained a dis inc i e clade om he Summe Lake Basin in O egon, pa o plu i-
al Lake Chewaucan (Suppl. ma e ial 1: ig S1). This biogeog aphic egion was
no sampled in his s udy o genomic sequencing bu may be easoned o be
ano he dis inc i e gene ic lineage wi hin S. obesus. This inding p esen s a dis-
junc dis ibu ion o S. obesus ha may ep esen his o ical p ocesses such as
aul -block opog aphy o clima ic change esul ing in a ica ian e en (Ha is
2000). Al e na i ely, he m DNA may ail o accu a ely po ay e olu iona y ela-
ionships (e.g., Zink and Ba owclough 2008; Edwa ds and Bensch 2009).
The Fish Lake Valley lineage is well-suppo ed in phylogene ic analysis, wi h
maximal suppo o monophyly in conca ena ed ML and species- ee analyses
(Figs 3, 4). Fu he mo e, i is di ided in o wo disc e e uni s comp ising he
sepa a e sampling loca ions (Fig. 3, Suppl. ma e ial 1: ig S2). Based on he
esul s o he analyses p esen ed he e, i is unlikely ha he Lida Pond loca ion
ou side Fish Lake Valley was ounded by human-media ed mo emen om he
McNe Ranch sampling loca ion. The Lida Pond ish appea o ha e unique
gene ic di e si y wi hin FLVTC no ep esen ed in McNe Ranch and ha may
now be los om he na i e ange. Resolu ion o FLVTC as he ea lies -b anch-
ing lineage o S. obesus is indica ed by ou species- ee analysis, hough his
esul ecei es low (BS = 51%) suppo (Fig. 4). Simila ly in Relic Dace Relic us
soli a ius Hubbs & Mille , 1972, he e is clea e idence o sepa a ion o popula-
ions o his ish in Goshu e, S ep oe, and Sp ing alleys in eas e n Ne ada om
he wes e n popula ions o his ish. Suppo o monophyly o eas e n alleys
is high, bu suppo o b anching ela ionships wi hin he eas e n clade a e only
mode a e (BS = 75–78%) in a species ee analysis. Rela i ely apid spli ing o
popula ions, bo lenecking and gene ic d i can ac o educe he numbe o
si es ha exis o suppo b anching pa e ns, p oducing a lowe signal o noise
ela ionship in hese dese ishes (Finge e al. 2022).
Geog aphic e idence suppo s a mig a ion pa hway om he Lahon an Basin
in o Fish Lake Valley ha was dis up ed ~2 million yea s ago by ac ion along
he Hun oon Valley aul sys em (Reheis e al. 2002). A e his ime, a plu ial
lake exis ed un il ~ 0.5 million yea s wi h pe iodic ou lows ha may ha e al-
lowed gene low (Reheis e al. 1991). The phylogene ic placemen o FLVTC as
closely ela ed o Lahon an lineages wi h subs an ial di e gence is conco dan
wi h his geog aphical in o ma ion.
The Rail oad Valley and Li le Fish Lake Valley lineage also is highly suppo ed
(e.g., Fig. 4); howe e , he sepa a ion o Rail oad Valley sampling loca ions a
an indi idual le el does no occu (Suppl. ma e ial 1: ig S2). This indica es a
lack o gene ic s uc u ing be ween Rail oad Valley sampling loca ions. Finally,
a hi d lineage o b oadly Lahon an Basin S. obesus is p esen ac oss analyses
composed o sampling loca ions ha exhibi gene ic s uc u ing (e.g., Figs 3–5).
The eshwa e biodi e si y c isis: a pe spec i e om he G ea Basin
and adjacen a eas
The G ea Basin is a ela i ely accessible a ea in a de eloped coun y, wi h a
limi ed ich hyo auna. As a esul , leuciscid minnows ha e ecei ed subs an-
ial ana omical analysis leading o a ious classi ica ion schemes discussed in
de ail by Simons and Mayden (1998). Impo an ly, wo k by Uyeno (1961) lead
59
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
o he consolida ion o nume ous ishes unde he genus Gila, wi h a eali y,
as in e p e ed h ough molecula phylogene ics, ha is much mo e complex
(e.g., Simons and Mayden 1998; Schonhu h e al. 2012). Indeed, he ana omi-
cal di e en ia ion o species wi hin Sipha eles may be challenging as well, and
Sipha eles moha ensis, was no ed by Mille (1973: 8) o be a subspecies as
Gila bicolo moha ensis, accompanied by he s a emen “I ha e no been able
o disco e cha ac e s ha will sepa a e i speci ically om all popula ions o
Gila bicolo in he Lahon an basin”. The g ea amoun o ana omical a ia ion
ac oss he ange o a ish species in he G ea Basin, such as all Sipha eles
obesus in he Lahon an Basin, is likely a esul o ecological plas ici y and he
occupa ion o a di e si y o habi a s and ecologies. Habi a s in he G ea Basin
include ho sp ings, cold sp ings, lakes, and i e s, all wi h co-occu ing ood
i ems and o he ishes ha also a y o e such la ge geog aphic a eas.
Sipha eles moha ensis is clea ly di e en ia ed om o he Sipha eles a mi o-
chond ial and nuclea loci wi h an ancien mo emen in o he Moja e Ri e and
ex inc ion om o he habi a s ha may ha e been occupied. Molecula phyloge-
ne ic analyses as p esen ed he e a e use ul o iden i y a eas whe e axonomic
e inemen is possible. Ou wo k suppo s he ecogni ion o se en species p e-
iously classi ied unde one, as alid. As species a e a cu ency in biodi e si y e-
sea ch, lack o ecogni ion o species-le el di e si y is de imen al o success ul
conse a ion ac ions (B own and Lomolino 1998). Simila s udies ha e imp o ed
axonomy a he species-le el wi hin Pan os eus Cope, 1875 and Rhinich hys
Agassiz, 1849 in he G ea Basin and adjacen egions (Unmack e al. 2014; Su
e al. 2022; Moyle e al. 2023). Molecula analyses ha e also indica ed ha in
wes e n No h Ame ica adjacen o and in he G ea Basin P osopium williamsoni
(Gi a d, 1856) and Ca os omus a dens Jo dan & Gilbe , 1881 con ain di e gen
and un ecognized species lineages (Mille 2003; Mock e al. 2006).
Fu he in es iga ions a e wa an ed o examine hese ishes o he p es-
ence o no o ana omical cha ac e is ics o o he ai s in suppo o species
desc ip ions. A a highe le el, molecula phylogene ic s udies o G ea Basin
ishes such as his one p o ide addi ional axonomic imp o emen s a he ge-
nus-le el. Genomic s udies examining o he axa such as he Ca os omidae
may also be impo an o unde s anding he gene ic ela ionships o ha
g oup (e.g., Campbell e al. 2023; Ha is e al. 2025). Wi h due conside a ion
and applica ion o in eg a i e app oaches (O oni e al. 2025), he con ibu ion
o he G ea Basin o No h Ame ican ich hyo auna is likely o con inue o in-
c ease, mo e accu a ely in o ming biodi e si y conse a ion in he egion.
In esponse o he wo ldwide biodi e si y c isis, in species ich sys ems con-
se a ionis s ad oca e he conse a ion o ‘ ype locali y ho spo s’ whe e he e
a e ype locali ies o mul iple and dis inc axonomic g oups, such as plan o
bu e ly species, o example. Fo eshwa e ish, such loca ions migh be in
he species- ich Amazon basin (e.g., Aze edo-San os and O oni 2025). How-
e e , his conse a ion s a egy alls sho in he G ea Basin, whe e he discon-
nec ed inland wa e s a e o en no species ich, and he e o e ype locali ies
wi h only a single species wa an p o ec ion. Indeed, some endemic G ea Ba-
sin ishes like he Relic Dace a e he only na u ally-dis ibu ed ishes ound in
hei habi a s e en i o e a a he la ge a ea. In o he G ea Basin ishes, he
ype locali y may ep esen he only ex an popula ion such as he Wall Canyon
Sucke Ca os omus mu i allis Ha is, Ma kle & Campbell, 2025.
60
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
While some G ea Basin ish species like Relic Dace and he Wall Canyon Suck-
e s ill exis in hei ype locali ies, many o he s do no , o en due o he in oduc-
ion o non-na i e ishes (Cuche ousse and Olden 2011) and human al e a ions o
habi a s (O oni e al. 2023). Fo example, S. isola us is no longe ound in i s ype
locali y and S. moha ensis was ex i pa ed om i s o iginal dis ibu ion because o
non-na i e ish in oduc ions (Ha is 2000). Ano he example is he ’ oikona’ Tui
Chub which was i s documen ed in 1987 om a single loca ion and ish exam-
ined in his s udy descend om 24 ish ansloca ed o a i icial e uge pounds in
1989 (Chen e al. 2007). Conse a ion ac ions aimed a he ype locali y o a hypo-
he ical ‘ oikona’ Tui Chub axon would no p o ide b oad conse a ion bene i s o
eshwa e ishes in he G ea Basin. Howe e , in eg a ion ac oss aqua ic axa (e.g.,
including sp ing snails Gas opoda: So beoconcha: Hyd obiidae) may be an e ec-
i e conse a ion s a egy ha iden i ies aqua ic ype locali y ho spo s ha may be
emphasized wi hin conse a ion amewo ks in he G ea Basin.
Conclusions
We ind he ishes p e iously classi ied unde Sipha eles o me i axonomic e-
inemen . A he highes le el, a deep gene ic di e gence be ween he Al o d Basin
leuciscids and o he ishes is p esen and we p opose Epizon gen. no . o he Al-
o d Basin leuciscids. Fu he mo e, wha has been consolida ed unde S. bicolo is
be e cha ac e ized as se en species-le el en i ies, all wi h exis ing names. Wi h-
in S. obesus we ind ha Fish Lake Valley Tui Chub is phylogene ically he ea li-
es -b anching lineage and is highly gene ically di e en ia ed om o he S. obesus
lineages. I also occupies a limi ed geog aphic a ea, sepa a e om all o he Tui
Chubs. Based on he le el o gene ic di e en ia ion and geog aphic dis ibu ion
o Fish Lake Valley Tui Chub, i me i s ecogni ion as a subspecies. Ou sea ch
h ough he li e a u e did no ind an a ailable name, he e o e o mal axonomic
desc ip ion would be necessa y o p o ide an o icial name o his subspecies.
Acknowledgemen s
We would like o ecognize he sampling e o s ha made his possible. Samples
we e p o ided by Cali o nia Depa men o Fish and Wildli e Fishe ies B anch
Cen al Valley Tissue A chi e and Ne ada Depa men o Wildli e, Cali o nia
Depa men o Fish and Wildli e, O egon Depa men o Fish and Wildli e, and US
Fish and Wildli e Se ice (USFWS) pe sonnel. Ka e ina S asinopoulou assis ed
wi h he e ymology and spelling o Epizon gen. no . Pic u es o he holo ypes
o Epizon al o densis and Epizon bo axobius a e cou esy o he Uni e si y o
Michigan Museum o Zoology Di ision o Fishes.
Addi ional in o ma ion
Con lic o in e es
The au ho s ha e decla ed ha no compe ing in e es s exis .
E hical s a emen
Ma e ial collec ed in suppo o his s udy was ob ained unde ins i u ional and e hical
guidelines o NDOW, CDFW, ODFW, and USFWS by pe sonnel o hose agencies.
61
ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636
Ma hew A. Campbell e al.: E olu iona y ela ionships o Fish Lake Valley Tui Chub
Use o AI
No use o AI was epo ed.
Funding
The au ho s would like o acknowledge unding o his p ojec h ough he USFWS
g an #F22AC00854-00. The sequencing was ca ied ou by he DNA Technologies and
Exp ession Analysis Co e a he UC Da is Genome Cen e , suppo ed by NIH Sha ed
Ins umen a ion G an 1S10OD010786-01.
Au ho con ibu ions
Concep ualiza ion: MAC. Da a cu a ion: KNY, SCP, MAC, GA. Fo mal analysis: MAC, GA.
Funding acquisi ion: AJF, MAC. Me hodology: AJF, MAC. P ojec adminis a ion: AJF.
Resou ces: NB, AJF. Supe ision: AJF. Visualiza ion: MAC. W i ing - o iginal d a : AJF,
MAC. W i ing - e iew and edi ing: NB, SCP, KNY, AJF, GA.
Au ho ORCIDs
Ma hew A. Campbell h ps://o cid.o g/0000-0002-5826-0329
G ace Au inge h ps://o cid.o g/0000-0002-7639-3766
Amanda J. Finge h ps://o cid.o g/0000-0003-3850-3685
Da a a ailabili y
Demul iplexed NGS sequence da a has been deposi ed o he NCBI Sequence Read A -
chi e unde BioP ojec PRJNA1327467. Sange sequenced mi ochond ial sequences
a e a ailable om GenBank (h ps://www.ncbi.nlm.nih.go /genbank/) wi h accessions
epo ed in Suppl. ma e ial 2. T ee iles used in gene a ion o igu es and ASAP esul s
a e p o ided in Suppl. ma e ial 3. Indi idual me ada a is p o ided as a Suppl. ma e ial 4.
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