Cryptic diversity within two widespread diadromous freshwater fishes (Teleostei: Galaxiidae)

Abstract

Identification of taxonomically cryptic species is essential for the effective conservation of biodiversity. Freshwater‐limited organisms tend to be genetically isolated by drainage boundaries, and thus may be expected to show substantial cryptic phylogenetic and taxonomic diversity. By comparison, populations of diadromous taxa, that migrate between freshwater and marine environments, are expected to show less genetic differentiation. Here we test for cryptic diversity in Australasian populations (both diadromous and non‐diadromous) of two widespread Southern Hemisphere fish species, Galaxias brevipinnis and Galaxias maculatus. Both mtDNA and nuclear markers reveal putative cryptic species within these taxa. The substantial diversity detected within G. brevipinnis may be explained by its strong climbing ability which allows it to form isolated inland populations. In island populations, G. brevipinnis similarly show deeper genetic divergence than those of G. maculatus, which may be explained by the greater abundance of G. maculatus larvae in the sea allowing more ongoing dispersal. Our study highlights that even widespread, ‘high‐dispersal’ species can harbour substantial cryptic diversity and therefore warrant increased taxonomic and conservation attention.

Full text

Ecology and Evolution. 2024;14:e11201.  
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1 of 16
https://doi.org/10.1002/ece3.11201
www.ecolevol.org
Received:4July2023
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Revised:3March2024
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Accepted:19March2024
DOI: 10.10 02/ece3.11201
RESEARCH ARTICLE
Cryptic diversity within two widespread diadromous
freshwater fishes (Teleostei: Galaxiidae)
Charlotte Jense1 | Mark Adams2,3 | Tarmo A. Raadik4 | Jonathan M. Waters5 |
David L. Morgan6 | Leon A. Barmuta1 | Scott A. Hardie1 | Bruce E. Deagle7 |
Christopher P. Burridge1
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,
providedtheoriginalworkisproperlycited.
©2024TheAuthors.Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.
1DisciplineofBiologicalSciences,School
ofNaturalSciences,Universit yof
Tasmania,Hobart,Tasmania,Australia
2EvolutionaryBiologyUnit,South
AustralianMuseum,Adelaide,South
Australia,Australia
3SchoolofBiologicalSciences,The
UniversityofAdelaide,Adelaide,South
Australia,Australia
4DepartmentofEnergy,Environmentand
ClimateAction,ArthurRylahInstitute
forEnvironmentalResearch,Heidelberg,
Victoria,Australia
5DepartmentofZoology,Universityof
Otago,Dunedin,NewZealand
6CentreforSustainableAquatic
Ecosystems,HarryButlerInstitute,
MurdochUniversity,Murdoch,Western
Australia,Australia
7AustralianNationalFishCollec tion,
CSIRONationalResearchCollections
Australia,Hobart,Tasmania,Australia
Correspondence
CharlotteJense,DisciplineofBiological
Sciences,SchoolofNaturalSciences,
UniversityofTasmania,LifeSciences
Building,BiologicalSciencesPrivateBag
55,Hobart,TAS7001,Australia.
Email:charlotte.jense@utas.edu.au
Funding information
UniversityofTasmania;Commonwealth
ScientificandIndustrialResearch
Organisation
Abstract
Identificationoftaxonomicallycrypticspeciesisessentialfortheeffectiveconserva-
tionofbiodiversity.Freshwater-limitedorganismstendtobe genetically isolatedby
drainageboundaries,andthusmaybeexpectedtoshowsubstantialcrypticphyloge-
neticandtaxonomicdiversity.By comparison, populationsofdiadromoustaxa,that
migrate bet ween freshwater and ma rine environment s, are expected to sh ow less
geneticdifferentiation.HerewetestforcrypticdiversityinAustralasianpopulations
(bothdiadromousandnon-diadromous)oftwowidespreadSouthernHemispherefish
species,Galaxias brevipinnisandGalaxias maculatus.BothmtDNAandnuclearmarkers
revealputativecryptic specieswithinthesetaxa.The substantialdiversity detected
withinG. brevipinnismaybeexplainedbyitsstrongclimbingabilitywhichallowsitto
formisolatedinlandpopulations.Inislandpopulations,G. brevipinnissimilarly show
deepergeneticdivergencethanthoseofG. maculatus,whichmaybeexplainedbythe
greaterabundanceofG. maculatuslarvaeintheseaallowingmoreongoingdispersal.
Ourstudyhighlightsthatevenwidespread,‘high-dispersal’speciescanharboursub-
stantialcrypticdiversity andthereforewarrantincreasedtaxonomicand conserva-
tionattention.
KEYWORDS
colonisation,delineation,Galaxias,geneflow,geographicalbarriers,imperilled,species
boundaries
TAXONOMY CLASSIFICATION
Biogeography,Conservationecology,Phylogenetics

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1 | INTRODUCTIO N
Amajorimpedimenttotheconservationofbiodiversityisthepres-
enceofcrypticspecies,whichistheoccurrenceofmultiplespecies
erroneouslyclassifiedasasinglespeciesduetoa lackof observed
or quantified genetic and/or morphologic differentiation (Delić
etal., 2017). Accurate species designation andidentificationisim-
portantforestimatingtheimpactofthreatsonpopulationsandbio-
diversity (Fiser et al., 2018)and the effectiveness of conservation
actions (Hoekzema & Sidlauskas, 2014). Spe cies' ranges and are a
of occurrence are generally overestimated when cr yptic species
arepresent,potentiallydiminishingtheirconservationstatus(Delić
etal.,2017).Despitetheiroften-closeevolutionaryaffinities,cryptic
species may also have different environmental needs and provide
differentecosystemservices(Fiseretal.,2018).Crypticspeciescan
contribute to the underestimation of anthropogenic extinctions,
whichimpedesourunderstandingoftheseprocessesandtheirpo-
tentialmitigation(Delićetal.,2017 ).
Identification of cryptic diversity has increased largely due
to the application of molecular systematic approaches to both
neglected and well-studied taxa (Brown et al., 20 07;Kochanova
etal.,2021;Pfenninger&Schwenk,2007;Rocaetal.,2001).For
example,ninelineageswerefoundintheGehyra nanaspeciescom-
plex of geckos withoverlapping distributions inseveral lineages
across the Australian Monsoonal Tropics (Moritz et al., 2018).
Widespread, polymorphic species appear particularly prone to
the presence of cryptic diversity (Adams et al., 20 14; Hammer
etal.,2013),andmanycrypticspecieshavebecomemorphologi-
callydiagnosable once theirexistence was revealedusingmolec-
ular genet ic or other data (H ammer et al., 2013; Raadik , 2014).
However, in some ins tances cr yptic spec ies remain mor phologi-
callyundiagnosable(Craigetal.,2009;Egge&Simons,2006; Eitel
etal.,2013;Konetal.,20 07),andsomeresearchersquestionthe
meritofformallydescribingcandidatespeciessolelyonmolecular
data(Strucketal.,2018).
MitochondrialDNA(mtDNA)isoftenusedtoresolvetaxonomic
uncertainties and determine populationstructure in many animals
because of its high rates of mutation and genetic drift (Ardren
etal.,2010;Konetal.,20 07;Lietal.,2006).However,hybridisation
andincompletelineagesortingcaneachresultinmtDNAgenetrees
being incongruent with their underlying species tree (Frankham
etal.,2002).Surveysofnucleargeneticmarkersinconjunctionwith
mtDNA are therefore desirable to provide multiple independent
perspectivesontaxonomic uncertainties (Grechko, 2013;Hammer
et al., 2013; Wan et al., 2004). Historically, allozymes were the
most wid ely used nuclear ma rkers for delinea ting species due to
theeaseofscreening~30–60independentloci(Adamsetal.,2014;
Hammeretal.,2007,2013).Althoughnowlargelyreplacedbyhigh-
throughputDNAsequencingofnuclearmarkers,allozymeshavestill
proveninsightfulfordelineatingspeciesandmajorphylogeographic
breakswithinspecies(Hammeretal.,2019;Unmacketal.,2017).
Molecular assessment of cryptic diversity is important for
freshwater biodiversity. Although freshwater environments only
compromise~0.3% of theEarth'ssurface,they harbour dispropor-
tionallyhighbiodiversity(e.g.,almost50%ofalldescribedfishspe-
cies;Reidetal.,2013). Despitethislimitedextentofhabitat,many
freshwaterfishspeciesremainundocumented,with~300newspe-
ciesdescribedannually(Dudgeonetal.,2006).Thishiddendiversity
isparticularlysignificant giventhat freshwater habitats areamong
the most imperilled in the world, with greater rates of population
decline and species extinction than terrestrial and marine taxa
(Reidetal., 2013).Fortypercentofknown freshwater fishspecies
areonthe IUCNRedList ofthreatened species (Reid et al.,2013),
with 20 41spe cies categori sed as vulnera ble, endange red or criti-
cally endangered in 2013 (Lintermans,2013). Keythreatening pro-
cesses comprise habitat degradation and fragmentation, invasive
species , climate change, overex ploitation and po llution (Dudgeon
etal.,2006;Jelksetal.,2008;Lintermansetal.,2020).
Freshwater-limited fish lineages often experience isolation
duetotheirhabitat constraints,leadingtogeneticdifferentiation.
However,cryptic diversity isalso a feature of diadromousfishes—
those thatmigrate between marine and freshwaters—despitetheir
typicallylargerpopulationsizesandhighergeneflow,which might
be expected to reduce diversification. Indeed, while diadromous
fishestypicallyexhibitcomparativelylowpopulationgeneticstruc-
turing(Allibone&Wallis,1993;Burridge&Waters,2020;DeWoody
&Avise,2000;Wardetal.,1994),diadromycanalsopotentiallyfacil-
itatelongdistancecolonisationandsubsequentfounderspeciation
(Burridge&Waters,2020).Notably,forsomefishspeciesdiadromy
can be fa cultative rat her than oblig ate (Closs et al., 2003; Feutr y
et al., 2013; Hicks et al., 2017). Diadromous species can also har-
bourlandlocked(freshwaterlimited)populationsthatexhibitgenetic
andmorphologicaldifferencesfromtheirdiadromouscounterparts
(Chapman et al.,20 06;Rojoetal.,2020;Tigano&Russello, 2022).
Speciation is also considered a common outcome from landlock-
ing(Lingetal.,2001; Ovenden&White,1990; Walliset al.,20 01).
Therefore,thepotentialforcrypticdiversitywithindiadromoustaxa
maybeunder-appreciated,andthusextinctionrisksunderestimated.
Galaxias brevipinnisand Galaxias maculatusarewidelydistrib-
uted diadr omous fishes tha t exhibit morphol ogical and life his-
tory variation throughout their range, including the presence of
landlockedpopulations.Galaxias brevipinnisisfoundintemperate
southeast Australia (including Tasmania) and throughout New
Zealandanditssub-Antarcticislands.Galaxias maculatusoccursin
South America, Australia andNewZealandaswellasneighbour-
ing island s such as the Falkla nds/Malvinas , Lord Howe, and th e
ChathamIslands.Galaxias maculatusoccupieslowelevationfresh-
water sys tems (Bice et al., 2019), whil e G. brevipinnishasgreat
climbing abilities and adults can penetrate farther inland (100's
kminland and elevations up to 1200 m)(Atlas of LivingAustralia
website, n.d.; Jung et al., 2009; McDowall, 2003; McDowall &
Suren, 1995). Bothspecieshave experiencedmajor declines due
tohabitat lossand degradationand are targeted by fisheriesfor
their ‘whitebait’ larvae (Bice et al., 2019; Raadik et al., 2019).
Introduced salmonids can also dramatically reduce the abun-
dance of G. brevipinnis through predation and displacement

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(Rowe et al., 20 02).Bothspecies may be experiencinglocalised
extirpationinlandlockedorisolatedpopulations(Biceetal.,2019;
Raadiketal.,2019).Therefore,itiscriticaltoclarifythepotential
presenceofcrypticdiversitywithinthesespecieswhichmaywar-
rantconservationactions.
Both G. brevipinnis and G. maculatus have been consi dered to
harbour cryptic species (Delgado et al., 2019; Jung et al., 2009;
Raadik, 2005; R aadik et al., 2019). Both speciesexhibitdeepmo-
leculardivergenceamong landmasses, suggestiveofcryptic diver-
sity (Watersetal., 2000,2010). DNA-basedphylogenieshavealso
rejectedmonophyly forAustralian andNew Zealand G. brevipinnis
(Campbelletal.,2022;Watersetal.,2010;Waters&Wallis,2001a),
and for all G. maculatus based on the placement of G. rostratus
(Burrid ge et al., 2012). Gene tic and morph ological di vergence has
alsobeenobservedamonglandlockedanddiadromouspopulations
in both spe cies (Campbel l et al., 2022; Carrea e t al., 2013; Dunn
et al., 2020; King et al., 2003; McDowall & Frankenberg, 1981;
Raadik, 2005; Rojo et al., 2020).However,despitetheseprevious
studiestherehasasofyetbeenlimitedinvestigationofregionalge-
neticvariationamongpopulationswithinAustraliaandNewZealand.
Thiscontrastsagainststudiesofnon-diadromousgalaxiids,inwhich
atleast 15 cryptic species have beenidentifiedwithinGalaxias oli-
duss.l.fromAustraliaand12crypticspeciesfromG. vulgariss.l.in
NewZealand, manyofwhichareextremelyrestrictedinrange and
highlythreatened(Campbelletal.,2022;Lintermans&Raadik,2019;
Raadik,2014 ,2019a,2019b).
1.1 | Aims
Theprimary aim of thisstudyistoaddresstheknowledgegap re-
gardinggeneticdivergenceamongpopulationsofG. brevipinnisand
G. maculatus within Aus tralia and New Ze aland. This in cludes the
first a ssessments fo r associated islan d populations in cluding Lord
Howe, Chath am, and New Zeala nd Subantarc tic islands . Both mi-
tochondrial DNAand nuclear allozymemarkerswere used for the
first timetoassessthe presence of candidates forcrypticspecies.
Inbothspecies,crypticdiversitymayrelateprimarilytomarinebar-
riers(e.g.,rangedisjunctionswithinlandmassesandamongislands).
However,wealso predictagreaterpropensity ofcryptic diversity
in G. brevipinnis dueto its greater climbing abilityand penetrance
inland(sensuRaadik,2005).
2 | METHODS
2.1 | Sample collection
Atotal of 259 G. brevipinnisindividuals were utilisedfor allozymes
(n = 149) and mtDNA (n = 149), including 14 GenBank sequences,
with 39 individuals in common across data sets. The G. macula-
tus samples comprised 117 individuals: allozymes n = 75, mtDNA
n = 71(this includes 17GenBanksequences),with28individualsin
commonacrossdatasets. WesampledthroughouttheirAustralian
ranges (52sitesforG. brevipinnis;50sitesforG. maculatus)and in-
cludedlocalitiesinNewZealand(30sitesforG. brevipinnis;foursites
forG. maculatus).AmongthesesiteswereNewZealandSubantarctic
Islands(CampbellIslandandAucklandIsland)forG. brevipinnis,Lord
HoweIslandforG. maculatus,andChathamIslandsforbothG. brevi-
pinnis and G. maculatus including PittIsland for G. brevipinnis (see
Figure 1; Tables S1and S2).Sampleswere preservedin95%etha-
nolorsnap-frozenusingliquidnitrogenandstoredattheAustralian
BiologicalTissuesCollection,basedattheSouthAustralianMuseum
orattheUniversityofTasmania.
2.2 | Allozyme laboratory procedures
Allozyme electrophoresis was undertaken on muscle homogen-
atesasdetailedpreviously(Adamsetal.,2 014;Morganetal.,2016;
Ovenden etal., 1993). Asearlier studieshaveshown thatG . brevi-
pinnis and G. maculatus are not close relat ives, each specie s was
screenedseparately.Outgroups comprised G. olidus (n = 4) for the
G. brevipinnisstudyandG. rostratus (n = 3)forG. maculatus.Thefol-
lowingenzymesandnon-enzymaticproteinsweresuccessfullysur-
veyedin one orboth species: ACON,ACP,ACYC, ADA, ADH,AK,
ALD,AP,CA, CK,DIA,ENOL,EST,FDP,FUM,G6PD, GAPD,GDA,
GLO, GOT, GP,GP I, GSR, IDH , LDH, MDH, ME, M PI, NDPK, NP,
PGAM, 6PGD, PGK, PGM, PK, PEPA, PEPB, PEPD, SOD, SORDH,
TPIandUGPP.Detailsofenzyme/locusabbreviations,enzymecom-
missionnumbers, electrophoretic conditions and stain recipes are
presentedinHammeretal.(20 07)andRichardsonetal.(1986).
2.3 | Analysis of allozyme data
Each allozyme data set was subjected to two types of analysis.
Initially,weemployedthemultivariateordinationtechniqueofprin-
cipal coordinates analysis(PCoA)in a stepwise manner toidentify
distinctgeneticlineagesandinstancesofputativegeneticadmixture
from fir st princip les, that is usin g individual s as the unit of anal y-
sisand withoutapriorireferencetolocality or mtDNAhaplotype.
Fulldetailsofthephilosophyandimplementationofthisprocedure
are prese nted in Adams et al . (2014). Thereafter,individualswere
assigned to their primary genetic lineage and furthergroupedinto
siteswithin eachprimarylineage—provided therewasnoevidence
ofgenetic heterogeneitywithinasite.Siteand/orlineage-specific
quantificationswerethen performed: (1)the numberofdiagnostic
differencesbetweenlineages(i.e.,fixeddifferences,allowingamaxi-
mumtoleranceof10%foranysharedalleleswhensummedtogether
fo ral o cus;seeA dams etal .(2014)fortherationaleunderpinningthis
approach),and(2)Nei'sunbiasedgeneticdistance(Nei'sD)between
sites. A neighbour-joining (NJ)tree wasalso constructed basedon
Nei's D. All methodological details for generating fixed difference
counts,Nei'sD,andNJtreesare providedinHammer etal.(20 07)
andAdamsetal.(2014).

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2.4 | DNA extraction and PCR
DNAwasextractedusingtwomethods:aChelexextractionmethod
with 200 μLof 5% Chelex and40 μg Proteinase Kwhere the sam-
ples were i ncubated at 56°C for 2 h or usi ng the Qiagen DNe asy
tissue kit according to the manufactures spin-column protocol
for animal tissue. DNA extracts were used for the amplification
of the mtDNA cytochrome b gene using the following primers:
Cytb-Glu 5′-GAAAAACCACCGTTGTTATTCA-3′andCytb-Thr5′-
CGACT TCCGGATTACAAGACT-3′ (Waters & Wallis, 2001a). PCR
was performed in 25 μL volumes containing 1x Readymix Buffer
(Sigma-AldrichCo.LLC),0.5 μMofeachprimerand2 μLofDNAtem-
plate.Thermocyclingcomprised95°Cfor3 minfollowedby34 cycles
of95°Cfor15 s, 55°Cor 52°C for15 sand 72°C for30 s. Galaxias
maculatussamplesfromwe stAus tra liaweredegraded,andas hor ter
fragmentof 399 bp was amplified usingprimers L147245′-CGAAG
CTTGATATG A A AA ACC ATCGT TG-3′andH151495′-AAACTGCAG
CCCCTCAGA ATGATATTTGTCCTCA-3′(Streelmanetal.,20 02)and
2.5 mMMgCl2.Thermocyclingwasinitiatedwith7 cyclesof95°Cfor
30s,45°Cfor30sand72°Cfor1 minpriortothe34 cyclesdescribed
abovebutwith a48°Cannealingtemperature.PCR productswere
sent to Macrogen (Seoul, S. Korea –h t t p : / / d n a . m a c r o g e n . c o m ) for
purifi cation and seque ncing using Cytb- Glu and L14724 for their
specificamplicons.
2.5 | Mitochondrial sequence analysis
Sequenceswereeditedandalignedtoareferencesequencewith
Geneious Prime version 2021.2 (h t t p s : / / w w w . g e n e i o u s . c o m ).
Individualcytochromebsequencesrangedfrom 367to 1145 bp,
andanym issingdat ainthealignmentwasco dedas‘ N’.J Mode lte st
FIGURE 1 MapdepictingthelocationofGalaxias brevipinnis(a,b)andGalaxias maculatus(c,d)samplesanalysedusingallozymesand
mitochondrialDNA(mtDNA).(a,c)samplelocationsinAustralia;(b,d)samplelocationsinNewZealand.(e)Overviewoflocationsof
samplesites.Coloursofthemapmatchesotherfigures.AmoredetailedmapofNth1andNth2canbefoundinFigure S1.SeeSupporting
Informationformoredetails.

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version 2.1 was used to identify the best-fit model of nucleo-
tide substitution from 88 candidates using the default settings
basedon thecorrectedakaikeinformationcriterion(Burnham &
Anderson,2004).Phylogenetic analyses were performedusing a
Bayesian inf erence strateg y implemented i n BEAST vers ion 2.6
(Bouckaertetal.,2019).BEAUti wasusedtocreateaninput file
forBEASTusingthebest-fit modelasdeterminedby jModelTest
2(Darribaetal.,2012;Guindon&Gascuel,2003),witharelaxed-
log-normal-clockmodelandaMCMCchainlengthof50,000,000
generations.TwotreepriorswereusedtocreateBayesianphylog-
enies: a Co alescent cons tant populat ion size tree prio r,suit able
for intra specific analy ses, and a Yule tree pr ior,w hich assumes
a consta nt rate of speciat ion and is often u sed for intersp ecific
analyses.Twoindependentruns wereconducted to testforsta-
tionarity and convergence of parameters, adequacy of burn-in,
andasufficienteffectivesamplesize(>200)usingTracerVersion
1.7(Rambautetal.,2018).Afteritwasestablishedeachruncon-
verged, LogCombinerwasused to combineoutputfrom the two
independentrunsusingaburn-inof10%.Tre eAnnotatorwasuse d
toprocessthetree,andFigtreeversion1.4(h t t p : / / t r e e . b i o . e d . a c . 
u k / s o f t w a r e / f i g t r e e / )forvisualisation.
Phylogenies were also inferred using maximum likelihood in
IQ-TREE2(Minhetal.,2020),with1000bootstrapreplicatesand
twoindependentrunsusingthebest-fitmodelasdeterminedby
jMode lTest2a saforemen tio ned.Additi ona lly,MrBaye swa sru nas
analternativeBayesianinterferencestrategybecauseitallowsfor
multifurcation(Huelsenbeck&Ronquist,2001).MrBayesversion
3.2.6wasrunasplugininGeneiousPrime,usingthesubstitution
modelmostsimilartothoseselectedbyjModelTest2.ForG. bre-
vipinnisaMCMCchainlengthof2,000,000generationswasused
subsamp ling every 500 ge nerations. For G. maculatus a MCMC
chain length of 1,100,000 generations was used subsampling
every 20 0 generatio ns. Stationar ity and conver gence of param-
eters, adequacyofburn-inand a sufficienteffective samplesize
(>200)werecheckedinGeneiousPrime.TreeAnnotatorwasused
toprocessthetreeusingaburn-inof10%,andFigtreeversion1.4
forvisualisation.
Toprovidecontexttothegeographicclusteringobservedinthe
mtDNA phylogeny, genetic distances were estimated among and
within geographically concordant clades.The p-distancehas been
showntobe more appropriateforquantifyinggenetic distancesin
barcodingstudiesthanKimura2-parameterdistance(Srivathsan&
Meier,2012) and was quantified inMEGA X (Stecher etal., 2020)
usingthedefaultsettings.BecausethewestAustralianG. maculatus
sequenceswere~400 bp,we also calculatedthe genetic distances
withthealignmentprunedto400 bp.
As previous phylogenetic analyses havesuggested that G. bre-
vipinnis from Australia and New Zealand may not be monophy-
letic (Burridge et al., 2012; Burridge & Waters, 2020; Campbell
et al., 2022; Waters e t al., 2010), we i ncluded mtDN A sequences
from near relatives for context, along with outgroup sequences
from: G. vulgaris s. l . group (OQ738806–O Q73880 8), G. johnstoni
(OQ738646)andG. auratus(JN232629.1).WealsoincludedG. occi-
dentalis(OQ738863)andG. rostratus(JN232631.1)asoutgroupsfor
G. maculatusandtoprovideacontrastintermsofinterspecificand
intraspecificdivergences.
3 | RESULTS
3.1 | Galaxias brevipinnis
3.1.1 | Allozymedataset
Thefinal datasetforG. brevipinniscomprised 149individuals(plus
fourG. olidus)genotypedfor57putativeallozymeloci.Apreliminary
PCoAonallG. brevipinnis (Figure S2)confirmedthegeneticdistinc-
tivenessoftheNewZealand and Australianlineagesandidentified
discrete ‘southern’ and ‘northern’ clusters among the Australian
sites. We fur ther explor ed this heterogen eity through a s eries of
follow-upPCoAs,eachtargetingvarioussubsetsofthefullallozyme
dataset.
Aninitial PCoAofall135Australianfish (Figure 2a)revealed
three pr imary cluster s, correspond ing to a ‘southern’ grou p for
mostsitesplustwo‘northern’clustersforthe12sitesrepresent-
ing the northern-most catchments where G. brevipinnis occurs
(sitedetailsinTable S1).APCoAofthebroadlydistributed‘south-
ern’cluster(Figure 2b)subsequently identifiedtwo distinctsub-
groups:onewidespreadonmainlandAustraliaandonerestricted
toTasmania,hereinreferredtoas‘WIDE’and‘TAS’,respectively.
The pres ence of two distinc tive lineages, he rein referred to as
‘Nth1’ and ‘Nth2’, was al so further c onfirmed for t he ‘northe rn’
sites (Figure 2c).Therewasnoevidenceofgeneticheterogeneity
betwee n the two NZ sites , located in the e ast and west of th e
South Island (analysis not shown). The five clusters (NZ, TAS,
WIDE,Nth1,Nth2)ultimatelyidentifiedbyPCoAwerefullydiag-
nosableby2–12fixeddifferences(Table 1).
The only additional substructure found via PCoA was within
Nth1(Figure S3),withindividualsclustering into oneofthree geo-
graphicallydefinedregions,namely(1)allsitesintheClarenceRiver
Basin, (2) al l but one site in the B ellinger River B asin, and (3) the
‘NeverNever’site,closetotheboundaryofthesetwoadjacentRiver
Basins(Figure S1).The geographicstructure within Nth1was sup-
portedby1–3fixeddifferencesamongthethreeclustersidentified
(Figure S3). A summar y of the allozy me profiles fo r the five main
groupsandthethreegeographicclusterswithinNth1arepresented
inTable S3.
Givennoevidenceofgeneticheterogeneitywithinanysite,the
genetic affinitiesamong individualsiteswere visualised usingaNJ
tree (Figure S4).Thegeneticgroupingsdepictedarelargelyconcor-
dantwiththoseidentifiedusingPCoA,withtwoexceptions:(1)The
TASsub-groupisnestedwithintheWidegroup, and (2)the‘Never
Never’siteisnestedwithintheClarencegroup,insteadofappearing
asdistinctentities.

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3.1.2 | Mitochondrialsequences
The mtDNA sequences were 618–1145 bp with an alignment
lengthof 1145 bp across 149 sequences from G. brevipinnis,three
from the G. vulgaris s. l. group,and single sequences fromG. aura-
tus and G. johnstoni.jModelTest2 selected Tamura-Nei+ Γ as the
optimal model of sequence evolution. Three geographically con-
cordantcladeswereevident fromallfourmtDNAphylogenies, de-
scribed here following previously defined allozyme descriptions:
(i) New Zealand, (ii) s outhern Austr alia (WIDE and TAS togeth er)
and(iii)northern-mostAustralia(Nth1andNth2together).TheNew
Zealandandnorthern-mostAustraliancladeswereeachsupported
withposteriorprobabilities>0.95in theBayesian phylogeniesand
withbootstrapvaluesof≥70%inthemaximumlikelihoodphylogeny
(Figure 3and Figures S5–S7).While the southern Australian clade
wassupportedwithaposteriorprobabilityof1.00intheCoalescent
Bayesianphylogenyandabootstrapsupportof88%inthemaximum
likelihoodphylogeny,itwas unsupportedintheother phylogenies.
ThephylogeniesdonotrecoverallG. brevipinnisasmonophyletic,as
NewZealandG. brevipinnisaresistertootherNewZealandGalaxias
withstrongsupportinallbuttheYuletreepriorphylogeny.Further
structuringwas observed withinNewZealand,with one claderep-
resenting both Auckland and Campbell Islands, and three sepa-
rate clad es representing S outh Island, No rth Island and Ch atham
Islands(includingPittIsland).Theseclades weresupported across
allphylogenieswith exception of the Chathams cladeforMrBayes
andthe Yule treeprior phylogeny.Relationshipsamongthese four
NewZealandcladesvariedacrossphylogeniesandmostlyreceived
lowsupport. Acrossall of G. brevipinnis,theminimumbetweendi-
vergence between clades mentioned above was 1.33% between
southernAustraliaandnorthern-mostAustralia,whilethemaximum
within-clade divergence was 2.51% for northern-most Australia
(Table 2).
3.2 | Galaxias maculatus
3.2.1 | Allozymedataset
Thefinal allozyme datasetforG. maculatuscomprised 52putative
loci for 75 indi viduals from ac ross eastern A ustralia plu s three G.
rostratus.As the raw dataclearly demonstratedthatthesetwosis-
terspecies were unequivocallydiagnosableatnumerousloci(eight
fixeddifferences;Table S4),theinitia lP CoA(Figure 4)wasrestricted
to G. maculatus. Two marginally distinctive clusters were evide nt
within G. maculatus, corresponding to (1) the L ake Hiawatha site
inNewSouthWales, Australia,and(2) all othersites.PCoA onthe
latterclusterdidnotrevealanyfurthergeneticdiscontinuities.The
twogroupsweredistinguishedbyasinglefixeddifferenceatGpand
major differencesinallelefrequency (Δp > 40%)atAcon,Got2 and
Pgm2 (Table S4).However,theNJtreedidnotdistinguishthesetwo
groups (Figure S8).
3.2.2 | Mitochondrialsequences
The mtDNA sequences were 367–1141 bp with an alignment of
1141 bp and comprising 71 G. maculatus sequences from across
Australia(includingwestAustralia),alongwithsequencesfromNew
Zealand,andSouthAmerica,plusoutgroupsequencesfromG. ros-
tratusand G. occidentalis.jModelTest2 selectedthebest-fitmodel
to be Tamura-Nei + I + Γ. Three mostly geographically concordant
FIGURE 2 Scatterplotsforthefirsttwodimensionsfromthe
PrincipalCoordinatesAnalyses(PCoA;dimension1onthex- a x i s 
anddimension2onthey-axis)oftheallozymedataforAustralian
Galaxias brevipinnis.Axesarescaledtoreflecttherelative
percentagecontributionofeachdimension(showninbrackets).(a)
initialPCoAofall135Australianfish;(b)PCoAofthe72individuals
comprisingthe‘southern’cluster;(c)PCoAofthe63individuals
referabletothetwo‘northern’clusters.
TAB LE 1 Pairwisemeasuresofdiagnosabilityandgenetic
divergenceamongthefivecandidatetaxaidentifiedfromthe
Galaxias brevipinnisallozymedatasetwithmaximumsamplesizes
foreachtaxoninbrackets.Lowerlefttriangle = numberoffixed
differences;upperrighttriangle = unbiasedNei'sD.
Lineage
NZ
(14)
Nth1
(45)
Nth2
(18)
WIDE
(68) TA S ( 4)
NZ —0.17 0.18 0 .14 0.28
Nth#1 7—0.07 0.07 0.20
Nth#2 7 2 —0.08 0.20
Wide 5 4 4 —0.14
TAS 12 9 9 4—

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clades were evident from all four phylogenies: South America,
New Zealand, and Australia (Figure 5 and Figures S9–S11). Each
of these gr oups received supp ort except for th e Australian cla de
in the Yule tree prior phylogeny (0.92 posterior probability).In all
four phylo genies, two se quences from t he Chatham Isla nds (New
Zealand) and the two from Lord Howe Island clustered closest to
easter n Australia n sequences , representi ng the disrupt ion to geo-
graphicconcordance. All phylogenies did notrecover G. maculatus
as monophyletic given the placement of G. rostratus as sister to
Australian and NewZealand,although topologicalsupport for this
waslowfromtheYuletreepriorphylogeny.Eachofthephylogenies
recognised west Australia as monophyletic with topological sup-
port.However,the placementofwestAustraliaassistertoalleast
Australianindividualswasonlyrecoveredandsupportedbythecoa-
lescentBayesianphylogeny(clusteringwithineastAustraliacannot
be refuted). The minimum divergence between the east and west
Australiansequenceswas2.56%(2.75%basedon400 bp)whilethe
maximumwithindivergencewas3.73%(3.85%basedon400 bp)for
SouthAmerica (Table 3).In contrast to allozymes,individuals from
LakeHiawathadidnotformtheirownclusterrelativetoothereast
Australiasequences.
4 | DISCUSSION
Freshwater-limited fishes have become well known for their
crypticdiversity (Adams et al., 2013,2014; Hammer et al.,2014;
Jerr y, 2008; Kir chner et al., 2021). However, such hidden diver-
sityislessexpectedfordiadromousfishes given theirgreaterdis-
persalabilities.Nevertheless,afewstudieshaverevealed cryptic
diversityindiadromousspecies(e.g.,Galván-Quesadaetal.,2016;
McMahan et al., 2013, 2021). Here, based on fixed differences
in allozymes and relationships and divergences estimated from
mtDNAsequences,wehaveidentifiedcrypticdiversitywithintwo
widespreadandwell-studieddiadromousfishesandsuggestcandi-
datesfordistincttaxa.
For both all ozymes and mtDN A cryptic dive rsity is sugge sted
within Australian G. brevipinnis, with two groups delineated: one
lineageinsouthernAustralia(TASandWIDE)andoneinthenorth
(Nth1 and Nth2). Allozymes further delineate the two northern-
mostgroups.Itisnotknown if thetwonorthern candidates(Nth1
and Nth2) are diadromous, nevertheless their catchments have
significant marine separation (>300 km) from other catchments
harbouring G. brevipinnis (Raadik,2005). Bot h candidate t axa also
display distinctive morphology from each other and from their
southern counterparts (Raadik, unpublished). Species distribution
modelsbasedonthelocationofotherG. brevipinnispopulationsand
including10environmental factors(e.g., averagerainfall,tempera-
ture,slopeandelevation),failedtopredicttheoccurrenceofthese
FIGURE 3 BayesianestimateofphylogenyforGalaxias
brevipinnisusingaCoalescentpriorinBEASTinferredfromthe
cytochromebregionofmitochondrialDNAandrootedusingG.
auratus(notshown).Numbersrepresentposteriorprobabilitiesand
thehorizontalbarsatnodesrepresentthe95%highestposterior
densityofthenodeheight.Thecoloursrepresentthecandidate
taxa(blue = TAS,pink = WIDE,purple = Nth1,orange = Nth2,
green = NZ).

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northern-most populatio ns (Growns & West, 2008). This suggest
the northern-most populations could belocally adapted ecotypes,
howevermoreextensivesamplingwouldbeneededtofullyresolve
thetaxonomy and reject phenotypic plasticity.Thenorthern-most
candidatetaxaalsooccupythesame catchmentandmayhavepar-
titionedtheavailablehabitatatthescaleofstreamreaches.Similar
geographicpatternshavebeenobservedbetweenNewZealandG.
‘southern’andG. gollumoides,with apparentpartitioningofhabitat
atafinescaleintherivernetworktheyinhabit(Crowetal.,2010).
The Tasmania (TAS) group is also somewhat distinctive from
the other s outhern mainl and populations ( WIDE), with fou r fixed
differences and their isolation by a marine distance of ~250 km.
However, it is neste d within these WI DE populations in t he allo-
zyme tree and lacks supportfrom mtDNA.The recognitionofthe
TAS group has been made based on previous mtDNA evidence
(Waters&Wallis,2001a).Galaxias coxii(Macleay1880)andG. wee-
doni(Johnston 1883) have precedence for mainland Australia and
Tasmania,respectively,butweresynonymisedwithG. brevipinnisby
McDowallandFrankenberg(1981).Itwouldbebeneficialforfuture
molecularandmorphologicalanalysestoemployincreasedsampling
of locations and loci to further assess the distinctiveness of the
Tasmanianpopulation(TASgroup).
Incomparisonwithallozymes,ourmtDNAanalyseswereunable
to disting uish the two nor thern-m ost (Nth1 and Nth2) Aust ralian
candidatetaxafromeachother.Thisoutcomemostlikelyreflectsthe
mtDNA genetree/speciestree discordancethat isoften observed
among closely relatedspecies (e.g., Hammer et al., 2014; Unmack
et al., 2019). Furthermore, minimum between-clade mtDNA dis-
tanceswerenotappreciablylargerthanmaximumwithin-cladedis-
tances(e.g.,1.33%amongAustraliancladesversus1.90%withinthe
southernAustralianclade).Likewise,inthecaseoftheG. oliduscom-
plex,15speciesweresuggestedbasedonfixedallozymeplusmor-
phological differences, butonly eight of these were monophyletic
formtDNA,andtwodidnotreceiveanytopologicalsupport(Adams
et al., 2014). Underestimation of species diversity from mtDNA
relative to a llozymes coul d reflect introg ressive hybridis ation and
mtDNAcapture(Moore,1995)orincompletelineagesorting(Avise
etal.,1986).Regardless,thelevelofamongcladecytochromeb di-
vergence in G. brevipinnisis comparablewith othercryptic species
complexe s (Bronaugh et al. , 2020;H oekzema & Sidlauskas, 2014;
Jirsova et al., 2019), including several freshwater-limited galaxiid
radiations (Adams et al., 2014; Chakona et al., 2013; Vanhaecke
etal.,2012;Waters&Wallis,2001b;Wishartetal.,2006).
WithrespecttoNewZealandG. brevipinnis,ourresultssupport
previous suggestions that G. brevipinnis contains cryptic diversit y.
Additionally,ourobservedmtDNArelationshipsamongNewZealand
andnearbyislandG. brevipinnispopulationsalsoraise the potential
significance of marinebarriers for cryptic diversity in this lineage.
Fourreciprocallymonophyleticcladesareevident:(i)Aucklandand
CampbellIsland,(ii)ChathamIslands,(iii)SouthIslandNewZealand
and (iv) Nor th Island New Ze aland. That not al l clades were sup-
portedcanbeexplainedbytheshallowdivergenceobservedamong
themanddoes not preclude completebutrecent geneticisolation.
While our s tudy lacked allozyme data for all these localities, we
predictfixeddifferencesgivenobservationsoftheirgreaterresolv-
ing power elsewhere in this (described above) and other studies
(Adams et al.,2014;Hammeretal., 2014).Furthermore,thediver-
gence among the subantarctic islands, Chatham Islands and the
South IslandNewZealandaresupportedbynuclearSNPevidence
(Darestanietal.,2023).However,non-diadromousSouthIslandLake
populat ions also exhibit nuclear SNP d istinction fro m each other
and diadromous populations (Darestani et al., 2023), which raises
thepossibilitythatdivergencesobservedduringthatstudymayre-
flect intraspecificspatialpopulation genetic structure, rather than
TAB LE 2 RangeofcytochromebDNAsequencep-distanceforGalaxias brevipinnisandnearrelativesamong(lowertriangle)andwithin
(diagonal)clades.
G. auratus New Zealand Southern Australia Northern- most Australia G. vulga ris group
G. auratus
NewZealand 0.152–0 .163 0.000–0.021
SouthernAustralia 0.154–0 .172 0.057–0.079 0.000–0.019
Northern-mostAustralia 0.166–0.171 0.061–0.084 0.013–0.043 0.000–0.025
G. vulgaris group 0.162–0.167 0.057–0.079 0.070–0.090 0.080–0.095 0.041–0.047
G. johnstoni 0.160 0.070–0.086 0.054–0.072 0.061–0.073 0.083–0.088
FIGURE 4 FirsttwodimensionsfortheinitialPrincipal
CoordinatesAnalysisofallozymevariationfrom75Galaxias
maculatus.Therelativecontributionofeachdimensionisshown
alongsideeachaxis.BasesymbolasforFigure 1;individualsfrom
theLakeHiawathasiteareoverlainwiththeletter‘H’.

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FIGURE 5 BayesianestimateofphylogenyforGalaxias maculatususingaCoalescentpriorinBEASTinferredfromcytochromebregion
ofmitochondrialDNA,rootedusingG . occidenta lis(notshown).Numbersrepresentposteriorprobabilitiesandthehorizontalbarsatnodes
representthe95%HighestPosteriorDensityofthenodeheight.Coloursrepresentthecandidatetaxa(red = Australia,green = NewZealand).

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supportforcrypticspecies.Nevertheless,werecommendfurther
geneticassessments(includingmorevigoroussamplingoftheNorth
Island)toassessthetaxonomicdistinctionoftheselineages.
Our analyses of G. maculatusalsorevealedcandidatesfor
cryptic diversity.Weconfirmthelargegeneticdivergencespre-
viously observed among South American, New Zealand and
AustralianG. maculatus(Pavuk,1997;Watersetal.,2000;Waters
& Burridge, 1999), for which different subspecies have been
suggested (Stokell, 1966). We also detected large divergence
(2.6%–6.6%)betweeneastandwestAustralia,spatiallyseparated
by~1500 km.This geneticdivergencecontrasts withgenetic ho-
mogeneitywithineastAustralianG. maculatusatcomparablespa-
tialscales(seealsoO'Dwyer etal., 2021),withonly somesubtle
divergenceevident in the landlockedHiawatha Lake population.
Althou gh we lacked allozy me data for sout h-west Aust ralian G.
maculatus, Pavuk (1997 ) observed that allozyme distinction of
this popu lation exceeded t hat between ea st Australia a nd New
Zealand, albeit based only a handful of allozyme loci, none of
whichdisplayedfixeddifferences.Afollow-upassessmentof the
taxonomicdistinctiveness of south-west AustralianG. maculatus
basedonadditionalnuclear loci is clearly desirable, despitenot
beingflaggedtoharbourcrypticdiversityduringconventionaltax-
onomictreatment(McDowall&Frankenberg,1981).
The divergence of the south-west Australian G. maculatus
population may reflect marine isolation, here representing the
complete absence ofrivers inthe intervening Euclabasin, span-
ning~1500 km (Unmack, 2001;Unmack et al.,2012). Thisregion
has also been implicated for divergences of terrestrial animals
and plants (Guay et al., 2010; Schmidt et al., 2014), although
other freshwater taxa appear to have surmounted it (Unmack
et al., 2011). Al ternatively, the re putedly land locked lifec ycle of
south-west Australian G. maculatus (Morgan et al., 2006) may
havepromotedgeneticdivergence, similar to that suggested for
south-west Australian Galaxias truttaceus (Morgan e t al., 2016),
andother populationsofG. maculatusinsouth-easternAustralia
(McDowall&Frankenberg,1981;Pollard,1971a,1971b)andSouth
Americaatmuchsmallerspatialscales(downto~2 km;McDowall
& Frankenberg, 1981; Pollar d, 1971a, 1971b; Rojo et al., 2020;
Zatt ara & Premoli, 2005). H owever, larv al gene flow from eas t
Australiawouldalsobeimpededbytheeast-flowingLeeuwincur-
rent,withoce ancurrentsalsoimplicatedforgeneticstr ucturingin
SouthAmericanG. maculatus(González-Wevaretal.,2015).
AcrosstheiroverlappingAustralianrange,wesuggestthreepu-
tativecrypticcandidatetaxawithinG. brevipinnis(southernAustralia
(TAS and WIDE together), Nth1, Nth2) and highlight largegenetic
divergencewithintheG. maculatusgroup.Thisisconsistentwithour
expectationsbasedonthegreaterclimbingability ofG. brevipinnis,
allowingittopenetratefartherinlandandformisolatedpopulations.
Incontrast,G. maculatusareunabletoovercome3 mslopedpassages
orotherinlandbarriersthatarereadilysurmountable byG. brevip-
innis(Doehringetal.,2012).Thisdifferenceinnumberofcandidate
cryptic taxa also mirrorsdifferences in the diversity of their close
relatives.Galaxias brevipinnishas12closerelativesinNewZealand,
representedbyG. vulgaris s.l. lineages (Campbelletal., 2022), plus
G. johnstoniandG. pedderensisinTasmania(Burridgeetal.,2012).In
contrast, over a much broader spatial scale onmainlandAustralia,
the only cl ose relatives of G. maculatus are G. occidentalis and G.
rostratus(Burridge et al., 2012). These observations match expec-
tations if the ancestors of the ‘maculatus’ and ‘brevipinnis’ groups
haddispersalabilitiessimilartoG. maculatusandG. brevipinnistoday,
respectively.
Differencesin marine dispersal ability couldalsoexplainthe
different levels of cryptic diversity within G. brevipinnis and G.
maculatus.While both species have similar lifecycles, with juve-
niles fro m diadromous pop ulations spendin g 4–6 months at sea
(Jung et al ., 2009), G. maculatus populations on oceanic islands
(Lord Howe, Chatham islands) provided genetic evidence for
dispers al across large ma rine barrier s, as shown by the t wo in-
dividualsfrom bothChathamand LordHowethat cluster within
Australiansamples. Watersetal.(2000)alsofound G. maculatus
TAB LE 3 CytochromebDNAsequencedivergenceestimates(p-distance)forGalaxias maculatusandnearrelativesamongidentified
(lowertriangle)andwithin(diagonal)clades.Divergenceestimatesfortheprunedalignmentareshowninparentheses.ThetwoChatham
islandsindividualsthatclusteredwithAustraliawerenotincludedhere,becauseitnotablychangedthedivergenceestimates.
G. occidentalis South America New Zealand East Australia West Australia
G. occidentalis
SouthAmerica 0.165–0.192 0.003–0.037
(0.167–0.185) (0.000–0.038)
NewZealand 0.179–0.201 0.112–0.154 0.000–0.015
(0.170–0 .176) (0.121–0.143) (0.000–0.013)
EastAustralia 0.162–0.197 0.122–0.172 0.047–0.103 0.000–0.026
(0.158–0.178) (0.129–0.172) (0.049–0.070) (0.000–0.026)
WestAustralia 0.158– 0.176 0.127–0.157 0.036–0.093 0.026–0.066 0.000–0.030
(0.156–0.173) (0.132–0.157 ) (0.038–0.093) (0.027–0.068) (0.000–0.030)
G. rostratus 0.199(0.178) 0.125– 0.148 0.070–0.097 0.077–0.094 0.085–0.093
(0.133–0.143) (0.074–0.083) (0.078–0.083) (0.088–0.093)

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individualsfromNewZealand withcloseraffiliationtoTasmania.
Incontrast,monophyly was observed forG. brevipinnisinsimilar
settings,suchasforTasmania,ChathamIslandsandNewZealand
sub-Antarcticislands(AucklandandCampbellIsland).Otolithsig-
natures also suggestthatthemajorityofdiadromousG. brevipin-
nisinNewZealand recruit to theirnatalstream,with larvae and
juveniles potentially orientating into nearshore river plumes to
limitdispersal (Augspurgeretal.,2021).Inmoreinsularsettings,
G. brevipinnisthatstraymayhavelimitedprobabilityofrecruiting
elsewhere.Additionally, even if they dorecruitelsewhere, they
maynotleavelong-term geneticsignatures(Waters etal., 2013).
Furthermore, G. maculatus appear to have greater larval abun-
dance at sea a s they dominate the w hitebait (larv ae) fishery in
NewZealand (McDowall, 1965;Yungnickel et al.,2020),andthis
confersgreatergeneflow.
5 | CONCLUSIONS AND TAXONOMIC
RECOMMENDATIONS
This study demonstrates additional cryptic diversity within two
widespreaddiadromousfishesoftheSouthernHemisphere.Based
onthesefindings, wesuggestthepresenceofseveralputativeun-
describedtaxa.We recommend futuresteps followtheintegrative
sp e cies deli n eati o nfra m ewo r kpro p osedbyUn macketal .(2021)and
include morphologicalanalyses such asthose described by Raadik
(2014). However, it shoul d be noted that a lack of mo rphologica l
distinctiondoes notnecessarilyprecludethe presenceofmultiple
species,northebenefitsoftheirrecognitionduringinvestigationsof
evolutionaryhistoryandecology(Delićetal.,2017 ).Thedifferentia-
tionweobservedwithinG. brevipinnisandG. maculatuscouldbea
resultof(geographic)isolation,habitatcomplexityorecologicaland
life history differences such as dispersal abilities andrecruitment.
Assessingthemigratorystatusofthetwonorthern-mostcandidate
taxaofG. brevipinnis,forexampleisessentialtounderstandpoten-
tial drive rs of diversifi cation. Thes e two north ern-mos t candidate
taxa of G. brevipinnis require more detailed stud y (morphological
andgenetic)butbasedoncurrentdata,G. brevipinnisinthat region
shouldberecognisedasaconservationunitseparatefromtheother
Australianpopulationsasaprecautionarymeasure.Whilewedonot
currentlysuggestsouth-westAustralia G. maculatusasacandidate
taxon,thelargegeneticdivergencewarrantsfurthergeneticassess-
mentand supportsits recognition as a separate conservationunit.
Therecognitionofsuchconservationunitswillhelpmaintainpoten-
tially important genetic diversity. While this study covers a broad
geographic range, finer geographic coverage may uncover other
regionallydistinctlineages.Indeed,withdiadromousfishesparticu-
larly vulnerabletohabitatlossand degradationinboth freshwater
andmarineenvironments, it is essentialthatsuch cryptic diversity
beidentifiedandconserved(Jungetal.,2009).
Cryptic diversity has beenpreviously suggestedin widespread
and vagil e taxa—those that ar e less affected by b arriers. This i n-
cludes both non-migratory and migratory bird species (Irwin
et al., 2011; Lohman et al., 2 010), planktonic marine copepods
(Halber t et al., 2013), an d marine and fre shwater bony and ca rti-
laginousfishes (D'Aloia et al., 2 017; Fahmi et al., 2021;Neilson &
Stepien,2009).For example,migratorypopulationsoftheWilson's
warbler (a bird) exhibit strong genetic differentiation, perhaps re-
flectingdifferencesinmigratorypatterns(Irwinetal.,2011).Similar
to diadromous fishes harbouring landlocked populations as a re-
sultofthelossoftheirmarinemigratoryphase,othertaxa,suchas
birds,experiencelossofmigrationresultinginresidentpopulations
that may livein sympatry but are reproductively isolated (Gómez-
Bahamónetal.,2020).Withenvironmentalchange,lossorchanges
inmigrationpathwaysacrosstaxaaretobeexpectedandcouldpro-
mote diversification (deZoeten &Pulido, 2020). Our resultshigh-
lightthatwidespreadandvagilespeciesshouldbeassessedtoavoid
erroneousrecognitionofspecies boundaries,theunderestimation
ofendemism(Lohmanet al., 2010),andinappropriatemanagement
andconservationpriorities.Suchassessmentsmaycorrectprevious
over-esti mation of species a bundance and ran ge. Genetic stu dies
such as ours can depict population structure andidentify popula-
tionsorconservationunitswithnovelgeneticdiversity tomaintain
andhighlightwhereecologicalworkandmanagementeffortsshould
befocussed.
AUTHOR CONTRIBUTIONS
Charlotte Jense:Conceptualization(equal);datacuration(equal);for-
malanalysis(lead);visualization(lead);writing–originaldraft(lead);
writing–reviewandediting(lead).Mark Adams:Conceptualization
(equal); data curation (equal); formal analysis (lead); visualization
(lead); writing –originaldraft(equal);writing– reviewand editing
(equal). Tarmo A. Raadik: Conceptualization (equal); data curation
(equal);writing–originaldraft (equal);writing–reviewandediting
(equal).Jonathan M. Waters:Datacuration(equal);writing–original
draft(equal);writing–reviewandediting(equal).David L. Morgan:
Data curation (equal); writing –originaldraft (equal);writing – re-
viewand editing(equal).Leon A. Barmuta:Writing–originaldraft
(equal); writing – reviewand editing (equal). Scott A. Hardie: Data
curation(equal);writing–originaldraft(equal);writing–reviewand
editing(equal).Bruce E. Deagle:Conceptualization(equal);datacu-
ration (equal); writing – original draft (equal);writing–review and
editing (equal). Christopher P. Burridge:Conceptualization (equal);
datacuration(equal);writing–originaldraft(equal);writing–review
andediting(equal).
ACKNOWLEDGEMENTS
This research was supported by the University of Tasmaniaand
byCommonwealthScientificandIndustrialResearchOrganization
(CSIRO). CJwasfunded by aCollegeofScience and Engineering
ResearchTrainingProgram(RTP)ScholarshipfromtheUniversity
ofTasmaniaand partiallybyaCSIROScienceLeader Fellowship
(R-91460) awarded to BED. Open access publishing facilitated
by Universi ty of Tasmania, as part of t he Wiley - Univers ity of
Tasmania agreement via the Council of Australian University
Librarians.

12 of 16
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JENSE et al.
The authors would like to acknowledge Travis Ingram, Grace
Fortune-Kellyand Tania Kingforprovidingadditional G. maculatus
sample s from the Chath am Islands. T he authors al so thank Grac e
SimandReikaTakeuchifortheirassistanceinthelab.
CONFLICT OF INTEREST STATEMENT
Theauthorsdeclarethattheyhavenoconflictofinterest.
DATA AVAIL AB ILI T Y STAT E MEN T
ThemtDNAsequencedata are availableonGenBankunderacces-
sionnumbersOQ738671–OQ738862.
ORCID
Charlotte Jense https://orcid.org/0000-0002-6902-2289
Mark Adams https://orcid.org/0000-0002-6010-7382
Bruce E. Deagle https://orcid.org/0000-0001-7651-3687
Christopher P. Burridge https://orcid.org/0000-0002-8185-6091
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SUPPORTING INFORMATION
Additional supporting information can be found online in the
SupportingInformationsectionattheendofthisarticle.
How to cite this article: Jense,C.,Adams,M.,Raadik,T.A.,
Waters,J.M.,Morgan,D.L.,Barmuta,L.A.,Hardie,S.A.,
Deagle,B.E.,&Burridge,C.P.(2024).Crypticdiversity
withintwowidespreaddiadromousfreshwaterfishes
(Teleostei:Galaxiidae).Ecology and Evolution,14,e11201.
https://doi.org/10.1002/ece3.11201