Ga eways o he unde wo ld: molecula phylogene ic
analyses iden i y pa e ns o g oundwa e auna and
wo new species o hypogean Hyalella Smi h, 1874
(Amphipoda, Hyalellidae) om
he no he n Moja e Dese
And ew G. Cannizza o1, Co ey J. Lange2, Da id J. Be g3
1Depa men o Biology, Miami Uni e si y, Ox o d, OH 45056, USA 2US Bu eau o Land Managemen ,
Sou he n Ne ada Dis ic O ice, Las Vegas, NV 89130, USA 3Depa men o Biology, Miami Uni e si y,
Hamil on, OH 45011, USA
Co esponding au ho : And ew G. Cannizza o ([email p o ec ed])
Academic edi o : Denis Copilas-Ciocianu|Recei ed 14 Augus 2025|Accep ed 10 Oc obe 2025|Published 21 No embe 2025
h ps://zoobank.o g/730DE74C-1DF1-4E65-B6A9-7B1104D72A01
Ci a ion: Cannizza o AG, Lange CJ, Be g DJ (2025) Ga eways o he unde wo ld: molecula phylogene ic analyses
iden i y pa e ns o g oundwa e auna and wo new species o hypogean Hyalella Smi h, 1874 (Amphipoda, Hyalellidae)
om he no he n Moja e Dese . Sub e anean Biology 54: 35–68. h ps://doi.o g/10.3897/sub biol.54.168846
Abs ac
In No h Ame ica, he genus Hyalella (C us acea: Amphipoda) is p ima ily epigean, wi h only wo de-
sc ibed species ound in g oundwa e . In con as , S ygob omus is an amphipod genus occupying sub e -
anean habi a s h oughou mos o he Nea c ic. Using bo h molecula and mo phological ai s, we
desc ibe wo new species o s ygobi ic amphipods, Hyalella plu onia sp. no . and Hyalella keepuikan un
sp. no . om g oundwa e habi a s in Nye Coun y, Ne ada, USA. These species o m a monophyle ic
g oup ha also con ains he simila H. c e ae om Ash Meadows Na ional Wildli e Re uge. Membe s
o his “H. c e ae” complex a e simila in e ms o o e all mo phology bu can be dis inguished h ough
examina ion o axonomically ele an cha ac e s wi hin he genus such as he hi d u opod, gna hopods,
and pe eopod 7. The desc ip ion o hese species highligh s he unexpec ed di e si y o s ygobi ic Hya-
lella wi hin he no he n Moja e Dese , whe e ou such species comp ise wo independen lineages.
S ygob omus is absen om hese loca ions. This di e si y o Hyalella is hypo hesized o be due in pa o
he e olu iona y his o y he genus and he ecological/clima ic condi ions o he egion i sel , which may
exclude o he mo e wide- anging Nea c ic s ygobi es like S ygob omus.
Keywo ds
De ils Hole, Hyalella mue a, Hyalella c e ae, Ne ada
Sub e anean Biology 54: 35–68 (2025)
doi: 10.3897/sub biol.54.168846
h ps://sub biol.penso .ne
Copy igh And ew G. Cannizza o 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.
RESEARCH ARTICLE
Sub e anean
Biology Published by
The In e na ional Socie y
o Sub e anean Biology
A pee - e iewed open-access jou nal
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
36
In oduc ion
While en i onmen s such as ponds, lakes, and s eams a e mos commonly en isioned
when conside ing eshwa e on Ea h, hese habi a s make up less han one pe cen o
he Ea h’s eshwa e ; nea ly 70% is con ained in glacie s, pe ma os , and ice caps, and
he emaining ~30% exis s as g oundwa e (Gleick 1993). Simila ends ollow he auna
associa ed wi h eshwa e s: common su ace inhabi an s o amilia , accessible, sys ems
come o mind (spo ish, insec s and insec la ae such as he “EPT axa”, e c.) when
conside ing “ eshwa e auna”, bu a signi ican numbe o axa occupy g oundwa e
sys ems as well. Many o he la e —including e eb a es such as salamande s and ishes,
and in e eb a es including Mollusca, A h opoda, Annelida, Pla yhelmin hes, and o he
phyla—ha e been eco ded in hypogean en i onmen s; in ac , hese axa o en display
ema kably high species ichness (S och 1995; Danielpol e al. 2000; Mau ice and Bloom-
ield 2012). Such di e si y wi hin hese en i onmen s is o en p omo ed by hei na u e,
as p edic ed by he “adap i e zone model” (S och 1995). This model highligh s he high
po en ial o coloniza ion o occu in hypogean sys ems which can hen lead o specia ion
and o en adia ion; his may, in pa , explain he high species di e si y obse ed among
c us acean axa in se e al g oundwa e sys ems (S och 1995; Danielpol e al. 2000).
In he Nea c ic, many axa ha e been eco ded om hypogean sys ems, bu pe haps
some o he highes such biodi e si y can be obse ed wi hin he C us acea B ünnich,
1772, especially he abundan and species- ich membe s o he pe aca id o de Am-
phipoda La eille, 1816 (S och 1995). To da e, o e 200 species om 19 gene a and 8
amilies o amphipods ha e been iden i ied om g oundwa e habi a s in he Nea c ic
(Ho on e al. 2025). A majo i y o hese species a e con ained wi hin amilies/gene a
which could be conside ed g oundwa e -obliga es, such as he genus S ygob omus Cope,
1872 and mos gene a o he amily Hadziidae S. Ka aman, 1943 which do no pos-
sess su ace ep esen a i es (Holsinge 1986; Ho on e al. 2025). These axa ypically
display highly s ygomo phic acies and o en ep esen olde , o en elic ual, lineages
(Holsinge 1986). Con e sely, a smalle subse o axa possesses bo h epigean and hy-
pogean ep esen a i es; such a dis ibu ion is likely due o mo e ecen in oduc ions o
g oundwa e . Examples o such axa a e ound in he gene a Gamma us Fab icius, 1775
and Hyalella Smi h, 1874 (Holsinge 1986; Cannizza o e al. 2023; Ho on e al. 2025).
Hyalella is a p ima ily epigean genus endemic o he New Wo ld, wi h o e 100 spe-
cies cu en ly desc ibed (Ho on e al. 2025). The cen e o di e si y o he genus is in
he Neo opical ealm, whe e an o e whelming majo i y (~80%) o species occu (De
Los Ríos e al. 2012; Ho on e al. 2025). He e, species ha e been eco ded om a wide
ange o epigean (sp ings, swamps, lakes, s eams, e c.) and hypogean (ca es, sp ings,
hypo heic habi a s) habi a s. While a numbe o Hyalella spp. ha e been eco ded om
a a ie y o g oundwa e habi a s in he Neo opics, hypogean Hyalella a e only known
om sp ing habi a s in he G ea Basin o Cali o nia and Ne ada wi hin he Nea c ic.
Two species ha e ecen ly been desc ibed: Hyalella mue a Baldinge , Shepa d & The o ,
2000 om Inyo Coun y, Cali o nia and Hyalella c e ae Cannizza o, Lange, & Be g,
2023 om Nye Coun y, Ne ada. The p esence o Hyalella in some Nea c ic g oundwa-
Two new hypogean Hyalella om Ne ada 37
e s i sel is cu ious, as hese habi a s a e mos ly occupied by membe s o he genus S y-
gob omus (Holsinge 1986). While S ygob omus spp. a e p esen in he G ea Basin, hey
a e no sympa ic wi h Hyalella spp. and a e absen h oughou he Moja e Basin and
Range and mos o he Sono an Dese . Fu he mo e, molecula phylogene ic analyses
o H. mue a and H. c e ae sugges while simila mo phologically and sepa a ed by an
o e land dis ance o ~45 km, he wo species a e no sis e axa and belong o di e en
lineages wi hin he genus, likely colonizing g oundwa e habi a s independen ly (Can-
nizza o e al. 2023). The p esence o mul iple Hyalella lineages in g oundwa e wi hin
he egion and he absence o S ygob omus spp. sugges ha Hyalella we e able o exploi
g oundwa e habi a s he e ha a e no occupied by o he amphipod axa (Cannizza o e
al. 2023). As a esul , i is likely ha wi hin he Moja e Basin and Range Hyalella spp.
would ollow di e si ica ion ends simila o hose o o he g oundwa e axa such as
S ygob omus, whe e di e en locali ies would con ain isola ed, endemic species.
Collec ions om Nye Coun y, Ne ada, USA ha e e ealed he p esence o addi-
ional popula ions o s ygobi ic Hyalella (Fig. 1). He e we examine hese popula ions
and simila species using bo h mo phological analyses and molecula gene ic compa i-
sons o h ee genes commonly assessed o amphipod molecula phylogene ics: nuclea
18S ibosomal DNA (Cannizza o e al. 2023; Cannizza o e al. 2025b), nuclea 28S
ibosomal DNA (Wi e al. 2006; Hou e al. 2007; Flo e al. 2010; Cannizza o e al.
2023; Cannizza o e al. 2025a, 2025b), and mi ochond ial 16S DNA (Macdonald e
Figu e 1. Upda ed dis ibu ions o hypogean Hyalella spp. in Nye Coun y, Ne ada and Inyo Coun y,
Cali o nia, USA.
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
38
al. 2005; Hou e al. 2007; Cannizza o e al. 2023; Cannizza o e al. 2025a, 2025b).
The mi ochond ial cy och ome c oxidase subuni I (COI) gene which is also o en as-
sessed was excluded om hese analyses due o issues wi h pseudogenes.
Ma e ials and me hods
Collec ion o specimens
Indi iduals we e collec ed li e, using hand ne s o small s aine s, om bo h an un-
named sp ing, ~0.7 km no h o he Highway 95 and Oleo Rd in e sec ion nea Bea -
y, and De ils Hole, Nye Coun y, Ne ada (Fig. 1). Collec ed specimens we e p ese ed
in 95% e hanol and s o ed a -20 °C. Specimens examined he e we e deposi ed a he
Na ional Museum o Na u al His o y, Smi hsonian Ins i u ion (USNM).
Mo phological analyses
To aid in mo phological analysis, ou specimens o each popula ion we e clea ed and
s ained p io o dissec ion. These indi iduals we e diges ed in a mix u e o 400 μL o
Molecula G ade Wa e , 360 μL o 2× Diges ion Bu e (Zymo Resea ch, I ine, CA,
USA), and 40 μL o P o einase K; samples we e incuba ed in his mix u e a 37 °C o
2 hou s and hen a oom empe a u e o e nigh . Once su icien ly diges ed, specimens
we e ans e ed o a solu ion o Lignin Pink o 2–3 hou s; a e specimens we e su i-
cien ly s ained, hey we e ans e ed o glyce in o dissec ion using a Nikon SMZ-8000
s e eomic oscope. The appendages we e moun ed on empo a y glyce in slides o acili-
a e examina ion using a Nikon Alphapho -2 YS2 compound mic oscope wi h a ached
d awing ube. Pla es we e p epa ed using Adobe Illus a o CC®. Body leng h measu e-
men s we e aken by measu ing he dis ance om he os um o he base o he pleo-
elson, ollowing he con ou o he body using ImageJ so wa e (Ab àmo e al. 2004).
The e m “de ining angle” e e s o he pos e io ma gin o he palm and he dis al-mos
poin o he pos e io ma gin o he p opodus, he a ea whe e he ip o he dac ylus
closes on he p opodus. Nomencla u e o he e ms “se a(e)” and “ obus se a(e)” ol-
lows published guidelines (Wa ling 1989), as does he nomencla u e o se al ypes (Zim-
me e al. 2009). The e m “se a(e)” e e s o “simple se a(e)” unless o he wise speci ied.
DNA ex ac ion and sequencing
Genomic DNA (gDNA) was ex ac ed om he diges ed ma e ial p epa ed du ing he
specimen clea ing p ocess. Ex ac ions we e pe o med using Tissue & Insec DNA
MiniP ep ki s (Zymo Resea ch, I ine, CA, USA), u ilizing a modi ied p o ocol (Can-
nizza o e . al., 2020). Ex ac ed DNA was s o ed a -20 °C and quan i ied using a
Qubi luo ome e (In i ogen, Wal ham, MA, USA).
Polyme ase chain eac ions (PCR) using p ime pai s 18SF/18S1000F and
18S700F/18S1250R (Englisch and Koenemann 2001) ampli ied ~1000 base pai s (bp)
Two new hypogean Hyalella om Ne ada 39
Table 1. GenBank accession numbe s gene a ed as a pa o his s udy.
O iginal
ca alog #
Museum
#
Species Locali y Coll
Da e
Collec o (s) La i ude,
Longi ude
18S
Accession #
28S
Accession #
16S
Accession #
AGC-1143.1 USNM
1694150
Hyalella
plu onia
sp. no .
Sp ing, ~11 km
NNE o Bea y,
Nye Coun y,
NV, USA
Dec-14-
2023
Co ey J. Lange 37.00512,
-116.72684
PX560105 PX560112 PX560116
AGC-1143.2 USNM
1694151
Hyalella
plu onia
sp. no .
Sp ing, ~11 km
NNE o Bea y,
Nye Coun y,
NV, USA
Dec-14-
2023
Co ey J. Lange 37.00512,
-116.72684
PX560106 PX560113 PX560117
AGC-1124.1 USNM
1694155
Hyalella
keepuikan un
sp. no .
De ils Hole II,
Nye Coun y,
NV, USA
Jan-08-
2020
Amb e
Chaudoin,
Randall Paylo
36.42714,
-116.29117
PX560103 PX560110 PX560114
AGC-1125.1 USNM
1694154
Hyalella
keepuikan un
sp. no .
De ils Hole II,
Nye Coun y,
NV, USA
Feb-07-
2017
Je ey
Golds ein, Olin
Feue bache
36.42714,
-116.29117
PX560104 PX560111 PX560115
o nuclea 18S DNA; 28S_le 2 and 28S_des1 (Ve o nik e al. 2005) ampli ied a ~730-
bp segmen o he nuclea 28S DNA gene; and he p ime pai s 16S F (Macdonald e
al. 2005) and 16SBR (Palumbi e al. 1991) ampli ied ~350 bp o he mi ochond ial 16S
DNA gene. To al PCR olumes o 35 μl con ained 30–70 ng o ex ac ed gDNA. PCR
mas e mix con ained 17.5 μl GoTaq Mas e mix (P omega Co po a ion, Madison, WI,
USA), 1 μl o each 10 μM p ime , and 13.5 μl o molecula g ade wa e . PCR was pe -
o med on a Bio-Rad T100 he mal cycle (Bio-Rad Labo a o ies, He cules, CA, USA).
A nega i e con ol lacking only gDNA was included o all se s o PCR eac ions o ule
ou con amina ion. The ollowing he mal cycle p o ocols we e ollowed: 18S: 95 °C
o 5 min, ollowed by 35 cycles o 95 °C o 30 s, 68.5 °C o 30 s, and 72 °C o 2 min,
ending wi h a 10 min inal ex ension a 72 °C; 28S: 95 °C o 5 minu es, ollowed by 37
cycles o 94 °C o 45 seconds, 45–53 °C o 30 seconds, and 72 °C o 50 seconds, end-
ing wi h a 5-minu e inal ex ension a 72 °C; 16S: 95 °C o 2 minu es, ollowed by 37
cycles o 95 °C o 40 seconds, 42–45 °C o 40 seconds, and 72 °C o 1 minu e, ending
wi h a 8-minu e inal ex ension a 72 °C. PCR p oduc s we e p epa ed o sequencing
using Exo-CIP Rapid PCR Cleanup (New England Biolabs, Ipswich, MA, USA). Sange
sequencing o pu i ied PCR p oduc s was ca ied ou a Eu o ins Genomics (Louis ille,
KY, USA) using indus y-s anda d Sange sequencing me hodology. All sequences gen-
e a ed as a pa o his s udy we e submi ed o GenBank (Table 1).
Phylogene ic analyses
Pai wise sequence alignmen was conduc ed using MAFFT (Ka oh e al. 2002; Ka oh
and S andley 2013) in GENEIOUS PRIME (www.geneious.com) and checked u -
he by eye. Mul iple sequence alignmen s we e pe o med sepa a ely o each gene,
accomplished unde he same pa ame e s as he pai wise alignmen s. All ma ke s
we e conca ena ed in o a inal alignmen using SEQUENCE MATRIX .1.8 (Vaid-
ya e al. 2011). We econs uc ed phylogene ic ela ionships wi h maximum likeli-
hood (ML) analyses using IQTREE .2.3.6 (Minh e al. 2020). The IQTREE sea ch
was un using he MODELFINDER algo i hm o selec bes - i ing subs i u ion
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
40
models o indi idual pa i ions, which we e analyzed unde an edge-linked model.
Fo bo h he 18S and 28S DNA loci, a TIM model wi h equal/empi ical base e-
quencies, and a F eeRa e model wi h 2 ca ego ies (TIM+F+R2) was selec ed; o
he 16S DNA, an HKY model wi h equal/empi ical base equencies, and a gam-
ma dis ibu ion wi h ou ca ego ies (HKY+F+G4) was selec ed. S a is ical suppo
was es ima ed using 1,000 ul a as boo s ap eplica es (Minh e al. 2013) and he
Shimoda ia-Hasegawa app oxima e likelihood a io es (Shimoda ia and Hasegawa
1999; Guindon e al. 2010). Accession numbe s o all indi iduals included in he
phylogene ic analyses a e p o ided in Suppl. ma e ial 1.
Resul s
Molecula phylogene ic analyses
Phylogene ic ees econs uc ed he e eco e ed indi iduals o Hyalella om De -
ils Hole II and he unnamed sp ing nea Bea y as a monophyle ic g oup which
also included H. c e ae (Fig. 2). These popula ions di e ed om H. c e ae a le els
simila o hose obse ed in o he amphipod axa (~98–99% simila i y in nuclea
genes and ~90% in he 16S DNA), especially in he 18S DNA, which has dem-
ons a ed g ea u ili y in iden i ying species in se e al amphipod axa (Whi e 2011;
Sisco and Sawicki 2023; Cannizza o e al. 2025b).
Mo phological analyses
Indi iduals o Hyalella examined om he unnamed sp ing nea Bea y and De ils
Hole II di e ed om he o he s ygomo phic Hyalella species in he Nea c ic (H. mue -
a and H. c e ae) in se e al key mo phological cha ac e is ics including he shape o he
gna hopods, p opo ions and se a ion o he basis o pe eopod 6, and he a mamen o
he elson and u opod 3 (Table 2). Based on ou mo phological and molecula esul s,
we desc ibe each o hese popula ions as a new species.
Table 2. Diagnos ic mo phological cha ac e is ics among s ygobi ic Hyalella in he Nea c ic. Abb e ia-
ions: GN = gna hopod; CPL = ca pus pos e io lobe.
Cha ac e H. c e ae H. mue a H. plu onia sp. no . H. keepuikan un
sp. no .
Maxilliped inne pla e apical obus se ae 3 3 2 4
Gn2 (♂) p opodus dac ylus e ical alignmen well beyond CPL beyond CPL beyond CPL well beyond CPL
Gn2 (♂) palma ma gin an e io no ch dis inc , pos e io angle dis inc , pos e io angle dis inc , angled weak
Pe eopod 7 basis pos e io ma gin se ae 8 8 6 3
Pe eopod 7 basis pos e io ma gin dis al-mos se ae obus small/simple small/simple small/simple
Pe eopod basis p ojec ion pas ischium 1.5× 1.5× 2.0× 1.1×
Pe eopod basis s. ischium-dac ylus 33% 33% 33% 25%
U opod 3 amus apex se ae longes obus longes simple longes simple longes obus
Telson apical se ae 4 4 2 4
Gn1/2 (♀) palm conca e s aigh /con ex s aigh s aigh
Two new hypogean Hyalella om Ne ada 41
Sys ema ics
O de Amphipoda La eille, 1816
Subo de Sen icauda a Low y & Mye s, 2013
In ao de Tali ida Ra inesque, 1815
Pa o de Tali idi a Ra inesque, 1815
Supe amily Hyaloidea Bulyče a, 1957
Family Hyalellidae Bulyče a, 1957
Genus Hyalella Smi h, 1874
Figu e 2. Maximum-likelihood phylogeny econs uc ed om he conca ena ed da ase (18S DNA,
28S DNA, 16S DNA) using IQTREE. Node suppo o wo me hodologies is indica ed by shaded
ci cles placed on nodes; weakly suppo ed nodes (< 0.40) no ma ked. Membe s o he H. c e ae complex
a e highligh ed by colo ed g adien s. A Hyalella plu onia sp. no . emale, 3.43 mm, unnamed sp ing,
~0.7 km no h o Hwy 95 and Oleo Rd, Nye Coun y, Ne ada, USA B Hyalella keepuikan un sp. no .
male, 3.15 mm, De ils Hole II, Nye Coun y, Ne ada, USA. UFBS, ul a as boo s ap, SHalRT, Shimo-
dai a-Hasegawa app oxima e likelihood a io es . Whi e scale ba ep esen s 1 mm.
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
42
Hyalella plu onia Cannizza o, Lange & Be g, sp. no .
h ps://zoobank.o g/D855A0A5-1777-4CEF-8658-5A513EC4728E
Figs 3A, B, 4–10
Ma e ial examined. • Holo ype, male 4.20 mm: Unnamed sp ing, ~0.7 km no h o High-
way 95 and Oleo Rd in e sec ion, Nye Coun y, Ne ada, USA (37.005118, -116.726843);
collec o : Co ey J. Lange, 14 Decembe 2023; USNM 1694150. • Allo ype, emale 3.25
mm: same locali y and collec ion da a as holo ype; USNM 1694151. • Pa a ype males,
n = 2, same locali y and collec ion da a as holo ype; USNM 1694153. • Pa a ype e-
males, n = 2, same locali y and collec ion da a as holo ype; USNM 1694152.
Type locali y. Unnamed sp ing, ~0.7 km no h o Highway 95 and Oleo Road
in e sec ion (on wes side o highway), Oasis Valley, Nye Coun y, Ne ada, USA
(37.005118, -116.726843).
E ymology. The speci ic epi he plu onia is he eminine o m o he La in Plu o
and he Ancien G eek Πλούτων (Plu on), he god o he Unde wo ld. Plu o’s Ga es
o plou onion we e sh ines e ec ed o Plu o; one o he mos amous is he sh ine a
Hie apolis, commonly called he “Ga eway o he Unde wo ld.” This epi he is gi en
bo h in e e ence o he hypogean habi a o he species and o play o he nickname
o he nea by own o Bea y: “Ga eway o Dea h Valley.”
Figu e 3. Habi us pho og aphs A Hyalella plu onia sp. no . holo ype male, unnamed sp ing, ~0.7 km
no h o Hwy 95 and Oleo Rd, Nye Coun y, Ne ada, USA (USNM 1694150), 4.20 mm B Hyalella
plu onia sp. no ., allo ype emale, unnamed sp ing, ~0.7 km no h o Hwy 95 and Oleo Rd, Nye Coun y,
Ne ada, USA (USNM 1694151), 3.25 mm C Hyalella keepuikan un sp. no ., holo ype male, De ils Hole
II, Nye Coun y, Ne ada, USA (USNM 1694154), 3.48 mm D Hyalella keepuikan un sp. no ., allo ype
emale, De ils Hole II, Nye Coun y, Ne ada, USA (USNM 1694155), 3.62 mm.
Two new hypogean Hyalella om Ne ada 43
Diagnosis. Small-sized, s ygomo phic species dis inguished om congene s—
excep membe s o he H. mue a and H. c e ae complex—by he combina ion o
he ollowing cha ac e s: eyes absen ; an enna 1 longe han an enna 2; pleoni es
lacking do sopos e io ca inae; maxilla 1 inne pla e wi h 2 apical pappose se-
ae; male u opod 1 inne amus lacking long cu ed se ae. Di e en ia ed om
H. mue a and o he membe s o he H. c e ae complex by he combina ion o
he ollowing cha ac e s: maxilliped inne pla e wi h 2 apical obus se ae; male
gna hopod 2 palma ma gin wi h dis inc an e io no ch; pe eopod 7 basis p o-
jec ing > 1.5 × he leng h o he ischium, wi h > 5 pos e io se ae, all simple,
leng h > 25% emainde o he pe eopod segmen s; u opod 3 amus wi h simple
se ae; elson wi h 2 long apical se ae.
Desc ip ion. Holo ype male (Figs 3A, 4–8): 4.20 mm in leng h. Eyes and in egu-
men a y pigmen absen (Fig. 3A).
An ennae. An enna 1 (Fig. 4A): App oxima ely 1.2 × leng h o an enna 2; pedun-
cle segmen 1 lacking pos e odis al obus se ae; p ima y lagellum wi h 12 segmen s,
aes he ascs on dis al segmen s, aes he ascs sho e han espec i e segmen s, unpai ed.
An enna 2 (Fig. 4B): app oxima ely 30% body leng h, gland cone dis inc ; peduncula
segmen 5 1.2 × leng h o segmen 4; lagellum wi h 10 segmen s.
Mou hpa s. Mandibles: le mandible (Fig. 4C): inciso 9-den a e, lacinia mobilis
5-den a e, accesso y se al ow wi h 2 plumose se ae; mola p ocess well-de eloped, cy-
lind ical, i u a i e, lacking se a. Righ mandible (Fig. 4D): inciso 5-den a e, lacinia
mobilis bi u ca e, lobes wi h nume ous ine den a ions; accesso y se ae ow wi h 2
plumose se ae; mola simila in o m o le mandible.
Uppe lip (Fig. 5A): ounded, apical ma gin o lab um wi h nume ous se ules.
Lowe lip (Fig. 5B): inne lobes indis inc , ou e ma gin o inne and ou e lobes
co e ed in se ules; ace o lip co e ed in pubescen se ules.
Maxilla 1 (Fig. 5C): inne pla e sho e han ou e pla e, wi h 2 apical pappose
se ae, and ine pubescen se ules co e ing he en i e pla e; ou e pla e wi h 9 apical
se a e se ae, pubescen se ules spa se and dec easing la e ally and p oximally; palp
one-segmen ed, es igial, 20% leng h o ou e pla e, ape ing dis ally, wi h pubescen
se ules, palp eme ges a sligh ly o e hal he leng h o he ou e pla e.
Maxilla 2 (Fig. 5D): bo h inne and ou e pla es co e ed in pubescen se ules;
ou e pla e subequal in leng h o inne pla e, wi h nume ous apical se ae; inne pla e
na owing sligh ly dis ally wi h nume ous apical se ae and 2 pappose acial se ae.
Maxilliped (Fig. 5E): inne pla e sho e han ou e pla e wi h 2 una med cuspi-
da e se ae along apical ma gin, su ace o pla e co e ed in pubescen se ules; ou e pla e
wi h nume ous se ae, lacking pubescen se ules; palp 4–segmen ed, second and hi d
segmen s wi h nume ous ma ginal, and subma ginal se ae; dac ylus su ace lacking
comb-scales, nail no elonga e.
Gna hopods. Gna hopod 1 (Fig. 6A): coxal pla e wi h 5 sho apical se ae; ba-
sis na owing p oximally, dis opos e io su ace o segmen lacking comb-scales; is-
chium wi h a pos e io se a; me us wi h 3 pos e io se ae, dis opos e io su ace o
segmen wi h spa se comb-scales; ca pus subequal in leng h o p opodus, wi h clus e
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
50
Figu e 8. Hyalella plu onia sp. no . holo ype male, unnamed sp ing, ~0.7 km no h o Hwy 95 and Oleo
Rd, Nye Coun y, Ne ada, USA (USNM 1694150), 4.20 mm A epime a B pleopod 1 (coupling spine
enla ged) C u opod 1 D u opod 2 E u opod 3 F elson. Scale ba s: 0.25 mm (A, B); 0.1 mm (C–E).
Ne ada Depa men o T anspo a ion (NDOT) and lies 45 m om he edge o Hwy
95. The Fede al Highway Adminis a ion (FHA) and NDOT a e planning o expand
and edesigna e Hwy 95 o In e s a e 11 h ough his a ea, which will in ol e upda ing
he wo-lane highway o a sepa a ed ou -lane in e s a e. Addi ionally, g oundwa e
Two new hypogean Hyalella om Ne ada 51
Figu e 9. Hyalella plu onia sp. no . allo ype emale, unnamed sp ing, ~0.7 km no h o Hwy 95 and
Oleo Rd, Nye Coun y, Ne ada, USA (USNM 1694151), 3.25 mm A gna hopod 1 (palma ma gin and
dac ylus enla ged) B gna hopod 2 (palma ma gin and dac ylus enla ged). Scale ba : 0.25 mm.
wi hd awal o he own o Bea y, and p oposed and cu en gold and li hium mines
in he immedia e a ea, pose a subs an ial isk o he con inued exis ence o he ype
locali y sp ing and H. plu onia sp. no . The ype locali y sp ing occu s nea he edge o
p i a e p ope y whe e a couple o addi ional sp ings occu on he wes side o Hwy 95;
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
52
Figu e 10. Hyalella plu onia sp. no . allo ype emale, unnamed sp ing, ~0.7 km no h o Hwy 95 and
Oleo Rd, Nye Coun y, Ne ada, USA (USNM 1694151), 3.25 mm A u opod 1 B u opod 2 C u opod 3
D elson. Scale ba s: 0.1 mm.
howe e , su eys we e no conduc ed on his p i a e p ope y. The second au ho has
su eyed o e 20 addi ional sp ings in Oasis Valley, on bo h p i a e and public land,
bu no o he popula ions o s ygobi ic hyalellid amphipods ha e been ound; only nu-
me ous popula ions o epigean hyalellid amphipods ha e been ound. Hyalella plu onia
sp. no . co-occu s wi h he endemic Oasis Valley sp ingsnail (Py gulopsis mic ococcus)
and Ama gosa oad (Anaxy us nelsoni), which we e ound du ing a su ey o he sp ing.
Two new hypogean Hyalella om Ne ada 53
Hyalella keepuikan un Cannizza o, Lange & Be g, sp. no .
h ps://zoobank.o g/E2FC2F71-5446-4BF2-9FE6-10B80600D83A
Figs 11–17
Ma e ial examined. • Holo ype, male 3.48 mm: De ils Hole II, Nye Coun y, Ne ada,
USA (36.42714, -116.29117); collec o s: Je ey Golds ein / Olin Feue bache , 08
Feb ua y 2017; USNM 1694154. • Allo ype, emale 3.62 mm: De ils Hole II, Nye
Coun y, Ne ada, USA (36.42714, -116.29117); collec o s: Je ey Golds ein / Olin
Feue bache , 08 Janua y 2020; USNM 1694155. • Pa a ype, male, same locali y and
collec ion in o ma ion as holo ype; USNM 1694157. • Pa a ype male, same locali y
and collec ion in o ma ion as allo ype; USNM 1694156.
Type locali y. De ils Hole II, Dea h Valley Na ional Pa k, Nye Coun y, Ne ada,
USA (36.42714, -116.29117).
E ymology. The speci ic epi he keepuikan un means “blind” in Timbisha Sho-
shone (li e ally “no eye ha ing”, kee(no/nega i e)pui(eye)ka ün(possession o ha ing))
is gi en in e e ence o he lack o eyes and he loca ion o De ils Hole II wi hin Dea h
Valley, he homeland o he Timbisha Shoshone T ibe. This name was selec ed in con-
sul a ion wi h he Timbisha Shoshone T ibal His o ic P ese a ion O ice.
Diagnosis. Small sized, s ygomo phic species dis inguished om congene s—
excep membe s o he H. mue a and H. c e ae complex—by he combina ion o
he ollowing cha ac e s: eyes absen ; an enna 1 longe han an enna 2; pleoni es
lacking do sopos e io ca inae; maxilla 1 inne pla e wi h 2 apical pappose se-
ae; male u opod 1 inne amus lacking long cu ed se ae. Di e en ia ed om
H. mue a and o he membe s o he H. c e ae complex by he combina ion o
he ollowing cha ac e s: maxilliped inne pla e wi h 4 apical obus se ae; male
gna hopod 2 palma ma gin wi h weak an e io no ch; pe eopod 7 basis p ojec -
ing < 1.2 × he leng h o he ischium, wi h < 5 pos e io se ae, all simple, leng h
25% emainde o he pe eopod segmen s; u opod 3 amus wi h obus se ae;
elson wi h 4 apical se ae.
Desc ip ion. Holo ype male (Figs 3C, 11–15): 3.48 mm in leng h. Eyes and in-
egumen a y pigmen absen (Fig. 3C).
An ennae. An enna 1 (Fig. 11A): app oxima ely 1.4 × leng h o an enna 2; pe-
duncle segmen 1 wi h pos e odis al obus se a; p ima y lagellum wi h 9 segmen s,
Table 3. Mo phological a ia ion obse ed among indi iduals o H. plu onia sp. no . and H keepui-
kan un sp. no .
Cha ac e Va iabili y (H. plu onia sp. no .) Va iabili y (H. keepuikan un sp. no .)
Body leng h ~2–4 mm ~2–4 mm
An enna 1 lagella segmen s 9–12 9–10
An enna 2 lagella segmen s 9–11 8–9
An enna 2 leng h o body leng h 1.1–1.3× 1.1–1.5×
Pe eopod 7 basis pos . se ae 6–8 4–5
Pe eopod 7 basis pos lobe s ischium leng h 1.8–2.3× 1.1–1.3×
U opod 1/2 peduncle inne se ae obus o simple obus o simple
U opod 3 amus se al leng h sho / long sho / long
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
54
aes he ascs on dis al segmen s, aes he ascs sho e han espec i e segmen s, an epenul-
ima e 3 segmen s wi h pai ed aes he ascs.
An enna 2 (Fig. 11B): app oxima ely 25% body leng h, gland cone dis inc ; pe-
duncula segmen 5 subequal in leng h o segmen 4; lagellum wi h 8 segmen s.
Mou hpa s. Mandibles: le mandible (Fig. 11C): inciso 8-den a e, lacinia mobi-
lis 5-den a e, accesso y se al ow wi h 3 plumose se ae; mola p ocess well-de eloped,
cylind ical i u a i e, wi h plumose se a.
Figu e 11. Hyalella keepuikan un sp. no ., holo ype male, De ils Hole II, Nye Coun y, Ne ada, USA
(USNM 1694154), 3.48 mm A an enna 1 (single aes he asc enla ged) B an enna 2 C le mandible
D igh mandible (lacinia mobilis enla ged). Scale ba s: 0.25 mm (A, B); 0.1 mm (C, D).
Two new hypogean Hyalella om Ne ada 55
Righ mandible (Fig. 11D): inciso 7-den a e, lacinia mobilis i id, lobes wi h
nume ous ine den a ions; accesso y se ae ow wi h 2 plumose se ae; mola simila in
o m o le mandible.
Uppe lip (Fig. 12A): ounded, apical ma gin o lab um wi h nume ous se ules.
Lowe lip (Fig. 12B): inne lobes indis inc , ou e ma gin o inne and ou e lobes
co e ed in se ules; ace o lip co e ed in pubescen se ules.
Figu e 12. Hyalella keepuikan un sp. no ., holo ype male, De ils Hole II, Nye Coun y, Ne ada, USA
(USNM 1694154), 3.48 mm A uppe lip B lowe lip C maxilla 1 (ou e pla e se a e se ae enla ged)
D maxilla 2 E maxilliped (apical ma gin o inne pla e enla ged). Scale ba s: 0.1 mm.
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
56
Maxilla 1 (Fig. 12C): inne pla e sho e han ou e pla e, wi h 2 apical pappose
se ae, and ine pubescen se ules co e ing he en i e pla e; ou e pla e wi h 9 apical
se a e se ae, pubescen se ules spa se dec easing la e ally and p oximally; palp one-
segmen ed, es igial, 20% leng h o ou e pla e, ape ing s ongly dis ally, wi h spa se
pubescen se ules, palp eme ges a app oxima ely 70% he leng h o he ou e pla e.
Maxilla 2 (Fig. 12D): bo h inne and ou e pla es co e ed in pubescen se ules;
ou e pla e subequal in leng h o inne pla e, wi h nume ous apical se ae; inne pla e
na owing sligh ly dis ally wi h nume ous apical se ae and 2 pappose acial se ae.
Maxilliped (Fig. 12E): inne pla e sho e han ou e pla e wi h 4 una med cus-
pida e se ae along apical ma gin, su ace o pla e co e ed in pubescen se ules; ou e
pla e wi h nume ous se ae, lacking pubescen se ules; palp 4-segmen ed, second and
hi d segmen s wi h nume ous ma ginal, subma ginal se ae; dac ylus su ace lacking
comb-scales, nail no elonga e.
Gna hopods. Gna hopod 1 (Fig. 13A): coxal pla e wi h 4 sho apical se ae;
basis na owing p oximally, dis opos e io su ace o segmen lacking comb-scales;
ischium wi h a pos e io se a; me us wi h 3 pos e io se ae, dis opos e io su ace o
segmen lacking comb-scales; ca pus subequal in leng h o p opodus, wi h clus e
o pos e io se ula e se ae; p opodus 1.3 × longe han b oad wi h 5 inne medial
se ula e se ae, an e odis al co ne wi h u o se ae, spa se comb-scales p esen
along pos e io ma gin; palm s aigh , wi h 5–9 se ae on inne /ou e ma gins; de-
ining angle s aigh wi h 2 obus se ae; dac ylus wi h plumose se a on ou e ma -
gin and 3 inne se ae, su ace lacking comb-scales.
Gna hopod 2 (Fig. 13B): coxal pla e wi h 3 sho apical se ae; basis na ow-
ing weakly p oximally; ischium wi h a pos e io se a; me us wi h 3 pos e io se-
ae, dis opos e io su ace o segmen lacking comb-scales; ca pus 40% leng h o
p opodus, pos e io ma gin loba e, 33% b ead h o segmen , lacking comb-scales;
p opodus enla ged, powe ul, 1.1 × longe han b oad, wi h u o an e odis al se-
ae, pos e io se ae absen ; palm s aigh , i egula wi h weak an e io no ch, palm
wi h 9 inne and 11 ou e obus se ae; de ining angle weak, p ojec ing sligh ly
pas ca pus pos e io lobe, wi h 2 obus se ae a anged a ound no ch whe e ip o
dac ylus es s, comb-scales p esen apically; dac ylus wi h plumose se a on ou e
ma gin placed p oximally and 3 inne se ae.
Pe eopods. Pe eopod 3 (Fig. 14A): coxal pla e wi h 5 sho apical se ae, pla e no
exca a ed pos e op oximally; basis much longe han coxal pla e lacking pos e io ma -
ginal se ae; me us subequal in leng h o ca pus; ca pus 80% leng h o p opodus; dac-
ylus 50% leng h o p opodus, wi h plumose se a placed p oximally on ou e ma gin
and 2 se ae inse ed adjacen o base o nail.
Pe eopod 4 (Fig. 14B): coxal pla e wi h 4 sho apical se ae, pla e exca a ed pos-
e op oximally; basis much longe han coxal pla e lacking pos e io ma ginal se ae;
me us subequal o ca pus in leng h; ca pus subequal in leng h o p opodus; dac ylus
50% leng h o p opodus, se a ion as in pe eopod 3.
Pe eopod 5 (Fig. 14C): coxal pla e la ge, biloba e, an e io /pos e io lobes bo h
wi h a se a; basis pos e io ma gin weakly con ex, wi h 4 sho se ae, pos e odis al co -
ne con ex, an e io ma gin wi h 4 obus se ae; me us subequal in leng h o ca pus;
Two new hypogean Hyalella om Ne ada 57
ca pus 70% leng h o p opodus, dac ylus 40% leng h o p opodus, wi h plumose se a
placed p oximally on pos e io ma gin and 2 se ae inse ed adjacen o base o nail.
Pe eopod 6 (Fig. 14D): coxal pla e la ge, biloba e, wi h p oduced pos e io lobe; ba-
sis pos e io ma gin s aigh o con ex, wi h 3 sho se ae, pos e odis al co ne s aigh ,
an e io ma gin wi h 2 obus se ae; me us subequal in leng h o ca pus; ca pus 85%
leng h o p opodus; dac ylus 40% leng h o p opodus, se a ion as in pe eopod 5.
Figu e 13. Hyalella keepuikan un sp. no ., holo ype male, De ils Hole II, Nye Coun y, Ne ada, USA
(USNM 1694154), 3.48 mm A gna hopod 1 (palma ma gin and dac ylus enla ged) B gna hopod 2
(palma ma gin and dac ylus enla ged). Scale ba : 0.25 mm.
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
58
Figu e 14. Hyalella keepuikan un sp. no ., holo ype male, De ils Hole II, Nye Coun y, Ne ada, USA
(USNM 1694154), 3.48 mm A pe eopod 3 B pe eopod 4 C pe eopod 5 D pe eopod 6 E pe eopod 7.
Scale ba : 0.25 mm.
Pe eopod 7 (Fig. 14E): coxal pla e diminu i e, lobes indis inc ; basis pos e io
ma gin weakly con ex, p ojec ing 1.1 × pas he ischium, wi h 4 sho se ae ex ending
o s a o pos e odis al co ne , obus se ae absen , pos e odis al co ne ounded, an-
e io ma gin wi h 2 obus se ae, basis leng h 25% emainde o pe eopod segmen s;
Two new hypogean Hyalella om Ne ada 59
me us 70% leng h o ca pus; ca pus 90% leng h o p opodus; dac ylus 50% leng h o
p opodus, se a ion like pe eopods 5 and 6.
Gills. Coxal gills p esen on somi es 2–7, s e nal gills p esen on somi es 3–6 (Figs
13B, 14A-E).
Pleon. Pleosome somi es lacking do sopos e io ca inae. Epime a (Fig. 15A): i s
epime on en al ma gin una med, dis opos e io co ne no p oduced, pos e io
Figu e 15. Hyalella keepuikan un sp. no ., holo ype male, De ils Hole II, Nye Coun y, Ne ada, USA
(USNM 1694154), 3.48 mm A epime a B pleopod 1 (coupling spine enla ged) C u opod 1 D u opod 2
E u opod 3 F elson. Scale ba s: 0.25 mm (A, B); 0.1 mm (C–E).
And ew G. Cannizza o e al. / Sub e anean Biology 54: 35–68 (2025)
66
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Supplemen a y ma e ial 1
GenBank/BOLD accession numbe s o sequences u ilized in his s udy
Au ho s: And ew G. Cannizza o, Co ey J. Lange, Da id J. Be g
Da a ype: xlsx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License
(h p://openda acommons.o g/licenses/odbl/1.0/). The Open Da abase License
(ODbL) is a license ag eemen in ended o allow use s o eely sha e, modi y, and
use his Da ase while main aining his same eedom o o he s, p o ided ha he
o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/sub biol.54.168846.suppl1
