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\begin{document}

\title{Phylogeography of a widespread Australian freshwater fish, western carp
gudgeon (Eleotridae: Hypseleotris klunzingeri): cryptic species, hybrid
zones and strong intra-specific divergences}



\author[1]{Peter J. Unmack}%
\author[2]{Benjamin Cook}%
\author[3]{Jerald Johnson}%
\author[4]{Michael Hammer}%
\author[5]{Mark Adams}%
\affil[1]{University of Canberra}%
\affil[2]{Griffith University Australian Rivers Institute}%
\affil[3]{Brigham Young University}%
\affil[4]{Museum \& Art Gallery of the Northern Territory}%
\affil[5]{The University of Adelaide}%


\vspace{-1em}



  
  \date{March 14, 2025}


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\selectlanguage{english}
\begin{abstract}
Despite belonging to the most abundant and widespread genus of
freshwater fishes in the region, the carp gudgeons of eastern Australia
(genus Hypseleotris) have proved taxonomically and ecologically
problematic to science since the 19th century. Several molecular studies
and a recent taxonomic revision have now shed light on the complex
biology and evolutionary history that underlies this group. These
studies have demonstrated that carp gudgeons include a sexual/unisexual
complex (five sexual species plus an assortment of hemiclonal lineages),
many members of which also co-occur with an independent sexual relative,
the western carp gudgeon (H. klunzingeri). Here we fill yet another
knowledge gap for this important group by presenting a detailed
molecular phylogeographic assessment of the western carp gudgeon across
its entire and extensive geographic range. We use a suite of nuclear
genetic markers (SNPs and allozymes) plus a matrilineal genealogy (cytb)
to demonstrate that H. klunzingeri s.l. also displays considerable
taxonomic and phylogeographic complexity. All molecular datasets concur
in recognizing the presence of multiple candidate species, two instances
of historic between-species admixture, and the existence of a natural
hybrid zone between two of the three candidate species found in the
Murray Darling Basin. We also discuss the major phylogeographic patterns
evident within each taxon. Together these analyses provide a robust
molecular, taxonomic, and distributional framework to underpin future
morphological and ecological investigations on this prominent member of
regional freshwater ecosystems in eastern Australia.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Phylogeographic assessments of freshwater fishes have commonly revealed
cryptic species (e.g., Baumsteiger, Kinziger \& Aquilar, 2012; Adams et
al., 2014; Pinacho-Pinacho et al., 2018), thereby improving knowledge of
species richness and diversity in aquatic ecosystems globally (Seehausen
\& Wagner, 2014). In Australia, for example, the number of recognised
(but not necessarily described) species increased by 39 between 2002 and
2013 (Allen, Midgley \& Allen, 2002; Unmack, 2013), with many of the
newly-defined species revealed by molecular phylogenetic and
phylogeographic evidence (Adams et al. 2023). Furthermore,
phylogeographic studies have indicated that historical, geological,
and/or climatic processes can be determinants of contemporary patterns
of biodiversity and distribution across riverine landscapes (e.g.,
Buckley et al., 2021; Shelley et al., 2020; Waters, Burridge \& Craw,
2020). Consequently, the phylogeographic assessment of freshwater fishes
provides foundational diversity and biogeographic knowledge upon which
valid ecological and environmental management studies are dependent
(Page et al., 2017).

In this study, we present a molecular phylogeographic assessment of the
western carp gudgeon (Eleotridae: \emph{Hypseleotris klunzingeri} )
throughout its entire Australian range. This small-bodied freshwater
fish (approximate maximum size is 60~mm TL) is one of the most abundant
and widely-distributed species in eastern Australia (Unmack, 2000;
Pusey, Kennard \& Arthington, 2004). Here it occurs as a disjunct
northern population in the Burdekin Basin in central-northern
Queensland, then in coastal drainages from just north of the Fitzroy
Basin to the Clarence Basin, as three disjunct southern populations in
the Macleay, Hunter and Shoalhaven basins in central New South Wales,
inland throughout the Murray-Darling Basin (MDB), and finally west to
the Bulloo River and Cooper Creek in the Lake Eyre Basin (Fig. 1;
Table1). \emph{Hypseleotris klunzingeri} is often found in sympatry with
other carp gudgeons, all belonging to a species complex comprising a
suite of other sexual and hemi-clonal congeners, but is reproductively
isolated from them (Bertozzi, Adams \& Walker, 2000; Schmidt et al.,
2011; Unmack et al., 2019).

As reviewed by Pusey et al. (2004), \emph{H. klunzingeri} occurs in a
wide range of lotic and lentic habitats, with aquatic plants and
leaf-litter beds, and slow-flowing water (i.e. \textless{}0.2~m/s) the
preferred (but not exclusively used) habitat features. The species is
primarily benthic, with a diet dominated by microcrustaceans and
macroinvertebrates (e.g., chironomids, ephemeropterans and
trichotperans). \emph{Hypseleotris klunzingeri} has a wide range of
water quality tolerances but is only known from upper estuarine areas
downstream of tidal barrages that prevent upstream movement, indicating
that estuarine habitats are not naturally used by the species. Spawning
can occur most of the year (excluding winter), but peaks in spring and
early summer in response to increasing water temperature and daylight
length, and possibly flow events, with flow events reported to catalyse
the onset of mass migration of \emph{H. klunzingeri} (Pusey et al.,
2004).

As with many other Australian freshwater fishes, the taxonomy and
distribution of \emph{H. klunzingeri} has historically been equivocal,
having first been mistaken by Klunzinger (1872, 1880) as \emph{H.
cyprinoides} (which does not occur in Australia), then described
as\emph{Carrassiops klunzingeri} by Ogilby (1898), to be later
reassigned to the genus \emph{Hypseleotris} . Its common name of western
carp gudgeon relates to the early thought that the taxon primarily
occurred west of the Great Dividing Range compared with the more
easterly\emph{H. galii} (e.g., Anderson, Lake \& Mackay, 1971), whereas
today we know that representatives of both lineages naturally occur both
east and west of the Great Dividing Range (Thacker, Geiger \& Unmack,
2022a). While the current nomenclature suggests a robust taxonomic
status, matrilineal phylogeographic data presented by Thacker et al.
(2007) indicated several divergent lineages within \emph{H. klunzingeri}
that to date have not been closely re-assessed. Furthermore, their
phylogeographic analyses suggest the low elevation divide between the
coastal Burnett River and the Condamine River in the northern MDB is the
pathway by which \emph{H. klunzingeri} was exchanged between these
basins (see also Unmack, 2013).

The purpose of this study is to present a thorough molecular
phylogeographic assessment of \emph{H. klunzingeri} , using
comprehensive sampling and an expanded suite of molecular datasets.
Regarding the latter, our choice of two independent sets of co-dominant
nuclear markers (single nucleotide polymorphisms {[}SNPs{]} and allozyme
loci), plus matrilineal sequence data (the mtDNA gene Cytochrome
\emph{b}{[}cyt\emph{b} {]}), ensures that this study is ideally placed
to help resolve the above-described taxonomic uncertainty and
re-evaluate biogeographic patterns in this prominent Australian
freshwater fish.

\textbf{Materials \& Methods}

\textbf{Sample collection}

All sampling was approved by the University of Canberra Committee for
Ethics in Animal Experimentation (approval codes CEAE 13-06, 15-06 and
20180442) and undertaken under the following state wildlife collecting
licences: New South Wales P07/0007-5.0 and P18/0027-1.1; Queensland
168221, 191126 and 212524; South Australia S115 and ME9902959; Victoria
RP1146 and RP1344. Fish were ethically euthanized using either AQUI-STM
or dilute clove oil, snap frozen in liquid nitrogen, and deposited in
the SA Museum's Australian Biological Tissues Collection.

We sampled throughout range of \emph{H. klunzingeri} for a total of 120
sites (Fig. 1, Table 1). All river names used herein are
cross-referenced to site numbers in Table 1. Each molecular dataset has
a different number of populations and individuals but overlap between
marker types was high. The SNP dataset consisted of 106 sites and 204
individuals, allozymes had 80 sites and 233 individuals and
cyt\emph{b}included 101 sites and 293 individuals (Table 1). The
cyt\emph{b}dataset includes all \emph{H. klunzingeri} sequences from
Thacker et al. (2007). Two versions of the cyt\emph{b} dataset were
constructed, one for the whole gene with 267 individuals, and a second
one for the first 601 base pairs to accommodate an additional 26
individuals from four locations (marked in Table 1 with an asterisk)
that provided additional phylogeographic insights within the MDB using
these larger sample sizes. For ease of presentation, individuals are
identified throughout by their final taxon assignment, namely pure taxa
KN, KE, KS, and KW (reflecting their various compass orientations), plus
assorted hybrid/admixed combinations thereof (KE\textsuperscript{m} = KE
from the MDB, KW\textsuperscript{m} = KW from the MDB, and KSxKE =
hybrids between KS and KE).

\textbf{SNP genotyping}

DNA was extracted by Diversity Arrays Technologies (DArT Pty Ltd,
Canberra, Australia, www.diversityarrays.com) using a NucleoMag 96
Tissue Kit (Macherey-Nagel, D\selectlanguage{ngerman}üren, Germany\emph{)} coupled with
NucleoMag SEP to allow automated separation of high-quality DNA on a
Freedom Evo robotic liquid handler (TERAN Pty Ltd, Port Melbourne,
Australia).

Sequencing for SNP genotyping was done using DArTseq\selectlanguage{english} (DArT Pty Ltd)
which uses a combination of complexity reduction using restriction
enzymes, implicit fragment size selection and next generation sequencing
(Sansaloni et al., 2011), as described in detail by Kilian et al.
(2012). The technique is similar to double-digest restriction associated
DNA sequencing (ddRAD) (Peterson et al., 2012), but has the advantages
of accepting lower quantities of DNA, greater tolerance of lower quality
DNA and higher call rates (Sansaloni et al., 2011). The restriction
enzyme combination of PstI (recognition sequence 5\selectlanguage{english}'-CTGCA\textbar{}G-3\selectlanguage{english}')
and SphI (5'-GCATG\textbar{}C-3') was used for the double digestion.

The PstI-compatible adapter included the Illumina flowcell attachment
sequence, a sequencing primer sequence, a barcode region of variable
length (see Elshire et al., 2011) and the PstI-compatible overhang
sequence. The reverse adapter contained flowcell attachment sequence and
SphI-compatible overhang sequence. Only fragments generated by the
PstI-SphI double digest were effectively amplified by polymerase chain
reaction (PCR) (Georges et al., 2018).

Sequences generated from each lane were processed using proprietary DArT
analytical pipelines as outlined by Georges et al. (2018) to yield
repeatable SNP markers. In addition, DArT processes approximately one
third of the samples twice from DNA to allelic calls as technical
replicates and scoring consistency (repeatability) was used as the main
selection criteria for high quality/low error rate markers.

\textbf{SNP filtering}

After receiving the SNP data from DArT Pty Ltd, the SNP data and
associated metadata were read into a genlight object as implemented in R
package adegenet (Jombart, 2008) to facilitate subsequent processing
with R package dartR (Gruber et al., 2018). We created two different
datasets based upon different filtering of the initial 19903 polymorphic
SNP loci, one for the phylogenetic analysis (`phylo' dataset), and the
other for the PCoA and fixed difference analyses (`PCoA' dataset). The
phylo dataset was initially filtered to remove any
obviously-introgressed individuals within the MDB (identified using the
PCoA dataset), as reticulation events are not compatible with
bifurcating trees. The next step retained only loci for which
repeatability was greater than 0.99 and all loci with a callrate above
0.6. The PCoA dataset included all individuals and was first filtered
for repeatability to include values greater than 0.99. The second
filtering step removed all secondary loci (loci found within the same
sequenced fragment) with the locus retained having the higher
polymorphism information content (PIC) value. Finally, loci with a
callrate above 0.9 were retained. The additional filtering steps were
undertaken on the PCoA dataset for the two analyses that are sensitive
to the presence of too many missing values and/or tightly-linked loci
(ordination and the calculation of fixed differences). The data
remaining after these primary filtering steps for both datasets are
regarded as highly reliable. The PCoA dataset was used for each of the
additional (stepwise) PCoA analyses based on a subset of individuals
being compared, with additional filtering applied to remove any loci
that become monomorphic in such subsets.

\textbf{SNP analyses}

Genetic similarity among individuals and populations was visualized
using ordination (Principal Coordinates Analysis {[}PCoA{]}; Gower,
1966), using individuals as entities and loci as attributes and
implemented by the gl.pcoa and gl.pcoa.plot functions of dartR. For our
phylogenetic analysis we constructed SNP genotypes for each individual
by concatenating only the variable bases from each SNP locus into a
single partition. A few loci had the SNP removed with the adaptor,
because of chance matching of the adaptor sequence to the terminal
region containing the SNP. These loci were removed prior to
concatenation. Heterozygous SNP positions were represented by the
standard ambiguity codes. We generated a phylogenetic tree using Maximum
likelihood (ML) applied to concatenated sequences. ML analyses were
conducted using RAxML 8.2.12 (Stamatakis 2014) on the CIPRES cluster
(Miller, Pfeiffer \& Schwartz, 2010) using the model GTRCAT and
searching for the best-scoring ML tree using the model GTRGAMMA in a
single program run, with bootstrapping set to finish based on the
autoMRE majority rule criterion. The tree was imported to Mega 7.0.18
(Kumar, Stecher \& Tamura, 2016), formatted and mid-point rooted. To
assist with identifying potential introgressed individuals,
heterozygosity was calculated in R using the command ``het \textless{}-
rowMeans(as.matrix(gl)==1, na.rm=T)'' followed by ``write.csv (het,
file=''het.csv'')''.

The diagnosability of lineages and candidate species was assessed by
calculating the number of pairwise fixed differences (both absolute and
allowing a 5\% tolerance for shared alleles at each locus) and the
associated probabilities that such values could arise through sampling
error alone (dartR command gl.fixed.diff; parameter tloc = 0 or tloc =
0.05; see Unmack et al., 2022 for rationale and methods involved).

\textbf{Allozyme genotyping and analyses}

Our allozyme dataset comprised the same 54 putative allozyme loci as
employed by Unmack et al. (2019) and was generated according to the
principles and procedures presented in Richardson, Baverstock \& Adams
(1986) and Hammer et al. (2007). We used PCoA, coupled with assessments
of diagnosability (fixed differences, allowing a 10\% cumulative
tolerance for shared alleles at a locus as advocated by Adams et al.,
2014 for allozyme markers) and admixture (intermediate positioning
between parental taxa for PCoA, higher levels of heterozygosity, and
lack of fixed differences at otherwise diagnostic loci), to explore the
broader taxonomic and phylogeographic patterns evident in this dataset.
The rationale and methods for these analyses follow Adams et al. (2014)
and Unmack et al. (2022).

\textbf{MtDNA genotyping and analyses}

The mitochondrial cyt\emph{b} gene was sequenced following the PCR
protocols in Hammer et al. (2014), except that samples were amplified
with the following primer pairs: Glu18 TAACCAGGACTAATGRCTTGAA with
Hd.alt GGRTTGTTGGAGCCTGTTTCAT or Hd.Hyps GGGTTGTTGGAGCCSGTTTCGT and
midg.496 GGCGGCTTTTCRGTAGATAA with Eleo.Thr.40 GATTTTAACCTCCTGCGTCCG.
Sequences were edited using Chromas 2.6.5 (Technelysium, Tewantin,
Queensland, Australia) and imported into BioEdit 7.2.5 (Hall 1999).
Sequences were aligned by eye and checked via amino acid coding in MEGA
to test for unexpected frame shift errors or stop codons. Data were
analysed phylogenetically using RAxML as per for the SNP analysis but
using the model GTRGAMMA.

\textbf{Results}

\textbf{Identification of primary taxa and lineages}

Ordination of SNPs

An initial PCoA of the stringently-filtered `PCoA' dataset revealed
three primary clusters, ultimately referable to the taxon groupings KN,
KE/KE\textsuperscript{m}, and KS/KW/KW\textsuperscript{m}, plus a series
of 16 admixed individuals which linked the latter two clusters in the
first dimension (Fig. 2a). Further targeted PCoAs (Fig. 2b, Fig. S1),
supported by all other molecular datasets, consistently demonstrated
that these admixed individuals, representing populations from the Bogan
and Castlereagh Rivers (Darling River tributaries), the lower Darling
River, and Murray River populations downstream from the Darling River
junction, mark the presence of at least one hybrid zone between pure KS
and pure KE. These additional PCoAs also revealed that pure KS
individuals were readily diagnosable from those occurring in the
north-western inland rivers (Fig. 1) and further revealed that this
latter taxon itself comprised two allied but distinctive lineages, pure
KW in the Cooper and Bulloo systems, and KW\textsuperscript{m} in two
MDB rivers, the Warrego and Paroo (Figs. 2b, 2c). Although only subtly
supported by PCoA (Figs. 2, S1), it became evident across all other
analyses that the KE individuals in the upper MDB (Condamine system down
to the mid-Darling River) displayed modest levels of introgression with
KS. In consequence, we also assigned a unique taxon identifier
(KE\textsuperscript{m}) to individuals from these sites.

Strong support for the presence of four primary taxa (KN, KE, KS, and
KW) plus three KS-admixed groupings in the MDB (KE\textsuperscript{m},
KSxKE, KW\textsuperscript{m}) is also evident in the fixed difference
counts (Table 2). These data clearly demonstrate that the primary taxa
are all readily diagnosable at multiple loci (range 38--432 absolute
fixed differences; 64--525 near-fixed differences) whereas KSxKE
displays no fixed differences from either parental taxon (as expected
for recently-admixed populations) and both KE\textsuperscript{m} and
KW\textsuperscript{m} display fewer fixed differences from KS than do
their pure parental taxa (as typically found for historic
admixture/introgression). Additional support is presented by the
observed heterozygosity counts for each group (Table 2), which show all
pure taxa display comparatively low levels of heterozygosity (range
0.0098--0.0218) when compared to KE\textsuperscript{m} (almost double
that of pure KE), KW\textsuperscript{m} (more than double that of pure
KW) and most notably KSxKE (more than fourfold higher than either
parent).

Phylogenetic analysis of SNPs

Our initial `phylo' dataset comprised all individuals except for the 16
hybrid KSxKE fish. Surprisingly, the presence of the nine admixed
KE\textsuperscript{m} individuals caused considerable distortion of the
relationships among populations and taxa in the resultant ML tree (Fig.
3a), reflecting the reality that reticulate evolution often leads to
data that do not exhibit tree-like behaviour (Unmack et al., 2022).
Removal of these KE\textsuperscript{m} individuals followed by
refiltering resulted in a final `phylo' dataset of 179 individuals for
7419 polymorphic SNP loci.

ML recovered one tree with a -ln score of -72522.995738 and the rapid
bootstrap search finished at 450 replicates (Fig. 3b; full tree in Fig.
S2). Support across most of the deeper nodes of the tree was strong,
with once again four principal lineages recognised, namely KN and KE (as
sister clades), with both being sister to clades KS and KW. Taxon KN
represents northern coastal populations from the Burdekin, Fitzroy, and
the coastal Boyne rivers (note there are two Boyne rivers in our study,
the second being a tributary to the Burnett River). Apart from its
presence in the Condamine and Darling (as lineage
KE\textsuperscript{m}), taxon KE consists of populations from eastern
coastal rivers from Baffle Creek south to the Clarence River, plus a
recently introduced population in the Barron River in far north
Queensland (site 1). Taxon KS contains individuals from three coastal
New South Wales rivers (Macleay, Hunter and Shoalhaven) along with
non-introgressed populations from the MDB, except those in the Warrego
and Paroo rivers (lineage KW\textsuperscript{m}) which, along with
Bulloo River and Cooper Creek populations, are referable to taxon KW.
Outside of the MDB, most individuals tend to group closely with others
from the same or adjacent river and there is obvious phylogeographic
structure at the regional level in all taxa except KS.

Allozyme analyses

The results of our allozyme analyses closely mirrored those obtained for
the SNPs dataset, both in terms of primary genetic lineages and
identifying pure versus admixed populations. A series of PCoA analyses
of the allozyme data (Fig. 4) displayed a near-identical association
between individuals as depicted for the SNPs (Fig. 2), and together
supported the presence of the same seven primary groupings, namely KN,
pure KE, KE\textsuperscript{m} (close to pure KE but slightly displaced
towards KS), KSxKE, pure KS, KW\textsuperscript{m}, and pure KW. The
same assignment of sites into primary groupings is shown in an unrooted
NJ tree (Fig. 5). These findings are further validated by the fixed
difference and observed heterozygosity counts for each grouping (Table
3), which show the same patterns of diagnosability and comparative
levels of heterozygosity as found for the SNPs. Together our two nuclear
datasets fully support the presence of four primary taxa in the western
carp gudgeon, namely KN, KE+ (KE+KE\textsuperscript{m}), KS, and KW+
(KW+KW\textsuperscript{m}), all readily diagnosable by numerous SNP and
allozyme loci (Table 4).

MtDNA analyses

The whole and half cyt\emph{b} datasets consisted of 1141 and 601 base
pairs for 267 and 293 individuals respectively. Maximum Likelihood (ML)
was run on the two cyt\emph{b} datasets with RAxML producing trees with
likelihood scores of -3145.296216 and -1595.307571, and the rapid
bootstrap search for both analyses finishing at 650 and 500 replicates
respectively (simplified tree for whole cyt\emph{b} in Fig. 6; detailed
trees for both whole and half cyt\emph{b} in Figs. S3 and S4
respectively).

For the full cyt\emph{b} dataset, the deeper phylogenetic relationships
were moderately well-supported and largely similar to those found for
SNPs. The KN clade was sister to the KE clade and both were sister to a
composite KS/KW clade, comprising three distinctive sub-lineages
referable to taxon KS plus well-supported, sister sub-lineages for KW
and KW\textsuperscript{m} (Figs. 6a, S3). Consistent with their hybrid
status, KSxKE individuals displayed either KS-derived or KE-derived
haplotypes (ratio 14:15, Fig. S4). For the non-hybrid taxa, an
individual's mtDNA clade membership was concordant with their
SNP/allozyme primary lineage identification in all instances apart from
six KS fish, all from three northern MDB rivers (sites 65, 66, and 68;
Gwydir, Namoi, and Macquarie Rivers).

Mapping the distribution of the major cyt\emph{b} lineages (Fig. 6)
clearly demonstrates that KE-derived haplotypes have intruded into the
KSxKE hybrid zones as identified using our nuclear datasets (Fig. 1 and
shaded in Fig. 6) plus are present in the pure KS populations from some
upper MDB rivers. It also reveals that haplotypes from two rare
KS-lineages, otherwise characteristic of the Border Rivers in the upper
MDB (sites 59, 62, and 63) have also spread across the drainage divide
into the upper Clarence River (site 41) and far downstream into the
KSxKE hybrid zone in the lower Murray.

\textbf{Origin and dynamics of KSxKE hybrid zones}

Our molecular data provide several additional perspectives on the KSxKE
hybrid zones. Consistent with geographic expectations, stepwise PCoA of
the SNPs dataset consistently identified the Condamine (ie. taxon
KE\textsuperscript{m}) as the most likely source population for the KE+
parent (Figs. 3b, S1). Second, individuals displayed varying degrees of
admixture between their two parental taxa (Fig. S1), indicating that
F\textsubscript{1} hybrids must be sufficiently fertile to at least
produce F\textsubscript{2} and/or backcross offspring. Finally, there
was no obvious correlation between the distance from the
KE\textsuperscript{m} source and the extent of introgression for
KE-derived alleles (Figs. 1, S1).

\textbf{Within-taxon phylogeographic structure}

Both our SNP and mtDNA datasets contain sufficient genetic insights to
explore phylogeographic trends within the four primary taxa. In addition
to the well-supported dichotomy between KW and KW\textsuperscript{m},
the SNPs identified a shallow split in KW between Bulloo and Cooper
populations (sites 111--114 versus 115--120; Fig. 3), They also revealed
additional albeit more complicated phylogeographic structure for the
other three taxa (Figs. 3, 4), herein further explored using
taxon-specific PCoAs (Fig. 7).

For KN, there was a primary dichotomy between the three northern and
four southern sites which reflects latitude rather than river basin
membership (Fig. 7a). Most notably, the Burdekin KN were closely allied
with those in the Fitzroy River (Figs. 3, 7) despite being somewhat of a
northern outlier (Fig. 1).

A more complex pattern is evident within KE+. All relevant PCoAs (Figs.
2b, 2d, 4c, S1) consistently found that Condamine fish
(KE\textsuperscript{m}) were genetically most similar to those in the
Burnett, while two geographic outliers (sites 1 and 16) and a number of
regional population clusters were present for pure KE in the RAxML tree
(Fig. 3b). A KE-specific PCoA revealed a similar pattern of diversity,
including the site 1 and 16 outliers (discussed separately), but was
also able to detect a primary phylogeographic split between sites from
the Maroochy River northwards and west to the Burnett (sites 10--29,
excluding site 16) versus sites south of and including the Pine and
Brisbane Rivers (sites 28--48). The northern outlier (site 1, Barron
River) clearly clusters with sites 19--24 (Burrum and Mary Rivers),
supporting its likely status as an introduced population from that part
of KE's range. Intriguingly, both individuals from site 16 (Burnett
River) are anomalously placed, one intermediate between the two primary
clusters (and showing elevated heterozygosity levels) and the other
clustering with the southern phylogroup. This same pattern is displayed
in the allozyme data (PCoA not shown) and in the mtDNA tree (Fig. S3),
with one individual from site 16 clustering with Brisbane River
haplotypes (southern phylogroup) and the other with Burnett River
haplotypes (northern phylogroup).

With respect to KS, most sites are relatively homogeneous, with only
modest structure relating to geographic outlying populations in two of
the Border Rivers (Severn and McIntyre Rivers; sites 60, 61), the
Macleay River (site 49), and the Shoalhaven River (site 52), the latter
clustering with one of the sites in the adjacent drainage divide (site
83, Murrumbidgee River) and therefore inferring a source population.
Although the mtDNA data are also relatively homogeneous for KS, the
Border Rivers harbored two distinctive cyt\emph{b} lineages at high
frequency that were absent elsewhere in pure KS (Fig. 6).

\textbf{Discussion} Building on the work of Thacker et al. (2007), we
present three comprehensive molecular datasets that together identify
four primary taxa (KN, KE+, KS, and KW+) plus several examples of
historic (KE\textsuperscript{m}, KW\textsuperscript{m}) and relatively
recent admixture (KSxKE) within the parent species \emph{H. klunzingeri}
. All primary taxa, lineages and admixed zones are fully diagnosable by
numerous independent genetic markers and our intensive sampling provides
an ideal starting point for future field surveys to plug apparent
distributional gaps, ecological assessments of the \emph{H.
klunzingeri}complex, or formal taxonomic revision. Regarding the latter,
allocating the nominal form of \emph{H. klunzingeri} s.s. (Ogilby, 1898)
to a specific taxon may prove problematic, given the type locality (the
Murray River in South Australia) is apparently, at least the time of
collecting for genetic evaluation, a hybrid zone (KSxKE).

\textbf{Candidate species}

The genetic resolution of candidate species has often relied on
identifying lineages using either gene genealogies (e.g., mtDNA or nDNA
gene trees) or multi-locus population trees (for allozymes or genomic
data). However, as such tree-only approaches detect genetic structure
rather than candidate species \emph{per se}(Sukumaran \& Knowles, 2017;
Unmack et al., 2022), lineages delineated in this manner need not
directly equate to biological or evolutionary species but may instead
reflect major phylogeographic breaks within a species or a composite of
two or more species plus admixed individuals.

While there is no simple formula for deciding whether two genetically
distinctive allopatric populations are conspecific or represent
different species, we have recently advocated a six-step approach to
assist in this task (Unmack et al., 2022). These steps are: identify
lineages, hybrids, and introgressed populations using a combination of
ordination of individuals (step 1) plus phylogenetic methods (step 2),
followed by pairwise assessments of lineage diagnosability (step 3),
comparative geographic distribution (step 4), and sampling intensity
(step 5), and concluding with a review of any other biological
information that might indicate that lineages are not conspecific (step
6). Unfortunately, observations relevant to this final step are largely
unavailable in the literature, since many ecological studies
of\emph{Hypseleotris} in eastern Australia have not attempted to
reliably distinguish \emph{H. klunzingeri} from a suite of congeneric
and often co-occurring taxa (e.g., Meredith, Matveev \& Mayes, 2003;
Lintermans, 2007), now known to comprise a complex of sexual species and
`unisexual' (hybridogenetic) lineages (Unmack et al., 2019; Thacker et
al., 2022b). We hope that a recent taxonomic revision by Thacker et al.
(2022a) for this hemiclonal species complex, which includes five sexual
species and multiple unisexual combinations, will help establish a more
robust taxonomic framework for identifying individuals to their correct
sexual group and hence facilitate the documentation of comparative
biological information for all sexual forms of \emph{Hypseleotris} ,
including those referrable to the \emph{H. klunzingeri} complex.

Table 4 summarizes the outcomes of applying steps 3--5 to the primary
taxa identified for \emph{H. klunzingeri} using steps 1 and 2. As shown,
there is strong evidence that KN, KE+, and KS are all valid candidate
species, being unequivocally or effectively diagnosable from each other
at hundreds of unlinked genes and displaying distributional patterns
that are inconsistent with being phylogeographic lineages within a
single species (Table 4, Fig. S5). Given their comparatively low number
of diagnostic differences, the decision as to whether the allopatric
taxa KS and KW+ are conspecific or represent distinct evolutionary
species remains the only taxonomic question not fully resolved by our
stand-alone genetic datasets. However, as the number of molecular
characters that diagnose KS from KW+ greatly exceeds the nine
partially-diagnostic morphological characters that delineate other
co-occurring species of \emph{Hypseleotris} (Thacker et al., 2022a), we
have concluded that KW+ `probably' represents a fourth candidate
species. A full resolution of its taxonomic status will require
additional targeted assessments of any morphological and other
biological differences between KS and KW+, and must include exemplars of
pure KS, pure KW, and KW\textsuperscript{m}. The scenario of sister
Cooper versus MDB candidate taxa is also evident in another co-occurring
freshwater fish (Australian smelt, \emph{Retropinna} spp.; Unmack et
al., 2022).

\textbf{Broad patterns within and between candidate species}

Taxon KN has an unusual distribution and genetic pattern. The southern
portion of the Fitzroy River Basin is quite different to the northern
portion, a pattern not replicated in any species examined so far. The
species is unknown from coastal basins between the Fitzroy and Burdekin
Basins except for the small Herbert Creek catchment (tissues were not
available for this study). This absence appears to be real, with
moderately-intensive sampling only finding its congener \emph{H.
bucephala} at many sites. Within the Burdekin Basin, KN has a narrow
distribution, being found primarily in Lake Dalyrmple and in the
Burdekin River upstream of the dam until the vicinity of Charters
Towers. It is absent from the arid Belyando River, the main southern
tributary to Lake Dalyrymple and containing habitats that are otherwise
commonly inhabited by \emph{H. klunzingeri} . Again, \emph{H.
bucephala}is present at many sites across the entire Burdekin Basin.
Either KN was once widespread in coastal basins north to the Burdekin
Basin, or it crossed over the drainage divide between the Fitzroy and
Burdekin basins, such as via the Rugby portal (Georges et al. 2018) with
subsequent extirpation of intervening populations. It is also not fully
possible to rule out that KN have been translocated to the Burdekin
Basin from the northern Fitzroy Basin as fish stocking contaminants.

The separation between taxa KN and KE corresponds to the Boyne River and
Baffle Creek catchment boundary. This separation is also found between
two rainbowfish species (\emph{Melanotaenia splendida} and \emph{M.
duboulayi} ) as well as two hardyhead species (\emph{Craterocephalus
fulvus} and \emph{C. stercusmuscarum} ), plus is represented by a major
disjunction in \emph{Hypseleotris acropinna} . It also represents the
northern most distribution points of two other species (\emph{Retropinna
semoni} and \emph{Philypnodon macrostomus} ) which do not occur north of
Baffle Creek (Unmack 2001). Taxon KE is continuously distributed and
common in most streams from Baffle Creek south to the Clarence River.
Populations are primarily structured by river basin, with a deeper
divergence between Baffle Creek, Burnett River, the Burrum system south
to the Maroochy River and populations from the Caboolture River south to
the Clarence River. Two populations represented geographic outliers. The
first is a clearly introduced population in the Barron Basin (site 1),
with a likely origin from the Burrum Basin. The second is the presence
of fish in Barkers Creek (site 16, Burnett system) that display both
nuclear and matrilinear evidence of admixture between two otherwise
distinctive ``northern'' and ``southern'' phylogroups. It is unclear
from our data if this represents a natural occurrence or a past
translocation event.

Complex geographic and genetic patterns are found for \emph{H.
klunzingeri} populations within the MDB. Befitting its extensive
geographic coverage and low-relief topography, the basin harbors the
pure taxa KS, KE+, and KW+, the latter two showing evidence of historic
admixture with KS (as lineages KE\textsuperscript{m} and
KW\textsuperscript{m}), plus a relatively-recent and possibly ongoing
hybrid zone (KSxKE) between upstream KE+ and its downstream congener KS.
Within KS there is no strong pattern of phylogeographic structure. These
populations are primarily found in the Murray subcatchment upstream of
the Darling River confluence, along with the Macquarie, Gwydir, Namoi
and Macintyre subcatchments. Taxon KE+ is restricted to the
Condamine-Balonne subcatchment. These KE\textsuperscript{m} lineage
individuals have obvious genetic affinities to populations from the
Burnett River, a common pattern for those MDB species which are also
found in coastal river basins in southeastern Queensland (Unmack 2013).
Fish with the KSxKE genetic profile are present in the Darling River
south into the lower Murray River, plus in the Castlereagh and Bogan
subcatchments. These are likely a result of KE\textsuperscript{m} fish
from the Balonne River dispersing further downstream, but also managing
to push upstream into nearby tributaries like the Bogan and Castlereagh.
Contemporary patterns in the Darling River are likely to vary over time
as drought eliminates populations (due to excess water extraction), with
recolonisation either coming via floodwaters from either the Balonne
(introducing more KE\textsuperscript{m} fish) or Macintyre/Barwon rivers
(taxon KS fish), carrying different genotypes into the lower Darling
River. The western portion of the Murray-Darling Basin in the Warrego
and Paroo subcatchments has admixed populations between KS and KW,
represented by KW\textsuperscript{m}. These populations share a long
drainage basin boundary with both Bulloo River and Cooper Creek (which
contains pure KW). One other fish species has crossed from the Lake Eyre
Basin rivers into the Paroo and Warrego subcatchments,\emph{Melanotaenia
splendida} (Lintermans 2007), while the turtle\emph{Emydura macquarii}
has crossed from the Bulloo River into Paroo (Georges et al. 2018). The
most likely spot for faunal exchange is via the Bindegolly portal
(Georges et al. 2018).

There have been four invasions into east coast river basins of KS from
the MDB. There first is located in the upper Maryvale River in the upper
Clarence Basin (site 41) which has a mitochondrial haplotype identical
to those adjacent in the Border Rivers subscatchment (upper Macintyre
River), along with a similar relationship based on SNPs (Figs. S2, S3).
The second invasion occurred in the upper Macleay River in Salisbury
Waters (site 49), which is adjacent to the Gwydir subcatchment. The fish
from Salisbury Waters are most similar to those from the Border Rivers
subcatchment in the upper Macintyre River for SNPs, while for
mitochondrial DNA from the upper Macintyre and the Gwydir subcatchments
were similar. The third transfer occurred with the Hunter Basin. This
population has long been considered likely native as they are known to
be widespread, although patchy in occurrence, and several other fishes
are shared with the Hunter, but not in surrounding coastal basins
(e.g.,\emph{Craterocephalus amniculus} , \emph{Mogurnda adspersa} ), or
they are also present in some additional surrounding basins (e.g.,
\emph{Tandanus tandanus} ). Hunter Basin KS (sites 50, 51) had a clear
genetic affinity with fish from the Murrumbidgee subcatchment rather
than the adjacent Macquarie and Namoi subcatchments. The fourth coastal
basin population was found in the Shoalhaven Basin (site 52). This
population was genetically closest to fish primarily from the adjacent
Murrumbidgee subcatchment.

It is tempting to speculate on whether these four geographic outliers
represent native populations or human-mediated introductions. While many
such introductions likely remain undocumented, over 50 Australian
freshwater fishes are already known or presumed to have been either
deliberately or accidentally introduced into catchments outside their
native range (Lintermans 2004). If these four coastal KS populations
were native, we would expect each to show the greatest genetic
similarity to its adjacent MDB population. This is the case for three of
these comparisons, with the Hunter Basin being the exception. In
addition, we might expect native occurrences to have broader
distributions within coastal basins provided they have had a large
period of time to disperse. Instead, all but the Hunter appear to only
harbor localised populations which have not dispersed far. Introductions
could come from nearby populations, thus mimicking a native pattern, or
they could be from distant populations, if accidentally introduced along
with deliberately-stocked, hatchery-reared sportfish. Unfortunately,
each of these basins lacks early historical records, a common situation
for Australia's freshwater fishes. At this stage we consider these four
populations are likely introduced, although we acknowledge the evidence
is equivocal.

The presence of three out of the four candidate species in the MDB is an
unusual distributional pattern. The majority of species of native fish
known to occur in the MDB do not share the Basin with widely-distributed
and truly-sibling congeners, the exceptions being \emph{Craterocephalus
fluviatilis} (Murray Hardyhead) and \emph{C. amniculus} (Darling
Hardyhead) and several \emph{Galaxias} species in the mountain galaxiid
complex which are closely related (Lintermans 2007; Raadik 2014). Some
introgression has been recorded in both fish groups (Adams et al. 2011;
Adams et al. 2014). There are also three congeneric species groups
present in the MDB which are known to produce hybrids from the
genera\emph{Maccullochella} (Douglas et al. 1995) and
\emph{Philypnodon}(Hammer et al. 2019). In other groups there are
examples of introgression such as in the genus \emph{Melanotaenia}
between three species (Unmack et al., unpublished data), along with an
admixture zone in the genus \emph{Retropinna} (Hammer et al. 2007;
Unmack et al. 2022). In addition, there is the hemiclonal complex of
\emph{Hypseleotris} carp gudgeons which have hybrid origins (Unmack et
al. 2019). Given that even distantly-related fish species are known to
readily hybridize (Vespoor \& Hammar 1991), the MDB provides
considerable opportunities for mixing gene pools from different
colonisations and reinvasions of the basin from surrounding river basins
over evolutionary time frames across a range of species with different
levels of genetic divergence. It is also likely that opportunities for
hybridisation have increased as natural habitats in the MDB have been
anthropogenically altered or degraded (Scribner, Page \& Bartron, 2001;
Lintermans, 2007).

\textbf{Cryptic biodiversity in Australian freshwater fishes}

The resolution of cryptic species diversity within \emph{H.
klunzingeri}accords with the trend of finding new candidate species in
Australian freshwater fish guided by molecular data (e.g., Adams et al.,
2014; Hammer et al., 2019; Unmack et al., 2022). Such data can not only
reveal the presence of cryptic species but can also uncover nuanced
evidence for admixture and introgression that was often not detectable
prior to the advent of detailed genomic datasets and the coupling of
ordination and tree-based approaches. The application of this modern
molecular approach to taxonomic uncertainty in \emph{H. klunzingeri} has
identified the presence of additional candidate species in this nominal
taxon. Moreover, as in our companion study of Australian smelt
(\emph{Retropinna} spp.; Unmack et al., 2022), additional morphological,
phenotypic or ecological data are not required in most cases to validate
the identified candidate species \emph{per se} (the exceptions being KW+
versus KS in \emph{Hypseleotris} , and COO versus MTV
in\emph{Retropinna} ). However, such data remain the foundation for
formal description and naming of candidate species and are of course
valuable for addressing other questions about the biology of individual
species, whether formally named or not.

\textbf{Conclusions}

This study has revealed that carp gudgeons are even more speciose than
previously thought, adding several additional candidates to the existing
six sexual species plus their various hemiclonal relatives (Unmack et
al., 2019; Thacker et al., 2022a; Thacker et al., 2022b). Even ignoring
the complication of sympatric hemiclones, many river basins contain at
least three or more sexual species, with the geographically-extensive
MDB notably harboring six sexual taxa (plus multiple hemiclones).
Moreover, the observed partial mismatch between geographic and
phylogenetic patterns, plus the presence of a natural hybrid zone in the
lower Murray and in at least two Darling tributaries add yet more layers
of complication. Given such complexity, future taxonomic and field
identification efforts will be particularly challenging, and ideally
require the involvement of a molecular identification technology (with
SNPs providing the gold standard of unequivocal identification all
sexual and unisexual forms) as part of a coordinated accumulation of
companion morphological exemplars of each morphotype at each site
surveyed. Our own research group has already adopted this strategy where
resources permit.

The dynamic boom and bust nature of many Australian freshwater
ecosystems highlights the need for monitoring spatial genetic patterns
for all resident species over time, particularly after major climate
events such as have impacted eastern Australia over the past decade
(Hughes, Schmidt \& Finn, 2009; Lintermans, 2013; Legge et al., 2022).
Our study provides future researchers with a framework to pursue such an
endeavor for the \emph{Hypseleotris} of eastern Australia. As a major
component of the biodiversity and ecology of these ecosystems, carp
gudgeons also offer great potential for environmental monitoring,
provided researchers can identify individuals to their correct taxon. In
the past carp gudgeons have often been lumped into one composite `taxon'
(\emph{Hypseleotris} spp.) when included in ecological surveys
(Lintermans, 2007), a custom that precludes any genuine assessment of
whether \emph{Hypseleotris} alpha diversity has declined or shifted
(e.g. hemiclone ratio/presence) at such sites. Finally, this study
further underlines the point that active conservation and management
practices for freshwater fishes, including both the intended (i.e.,
wrong genetic lineage used) and unintended (i.e., where carp gudgeons or
other non-target species unknowingly contaminate the hatchery release
event) consequences of fish stocking programs (Lintermans, 2004), need
to be mindful of the existence of both undescribed candidate species,
and deep phylogeographic structure within all species, to avoid
undertaking or facilitating translocations or mixing of distinct genetic
lineages.

\textbf{Acknowledgements}

We thank the many researchers who assisted on field trips, in the
laboratory, or supplied tissues. We also gratefully acknowledge the
South Australian Museum for access to their tissue collection.

\textbf{Data availability statement}

The allozyme, genomic, and mtDNA sequence data are available on Zenodo
(https://doi.org/10.5281/zenodo.8223411). All novel cyt\emph{b}sequences
have also been deposited in GenBank (accession numbers
OR401349--OR401588).

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