Journal of Biogeography (J. Biogeogr.) (2007) 34, 1518–1533
ORIGINAL
ARTICLE
Comparative phylogeography of five
sympatric Hypseleotris species (Teleostei:
Eleotridae) in south-eastern Australia
reveals a complex pattern of drainage
basin exchanges with little congruence
across species
Christine E. Thacker1*, Peter J. Unmack2 , Lauren Matsui3
and Neil Rifenbark4
1
Research and Collections – Ichthyology,
Natural History Museum of Los Angeles
County, 900 Exposition Boulevard, Los
Angeles, CA 90007, USA, 2Arizona State
University, School of Life Sciences, PO Box
874601, Tempe, AZ 85287, USA, 3Department
of Biology, Santa Monica College, 1900 Pico
Boulevard, Santa Monica, CA 90405, USA and
4
Department of Biology, University of Southern
California, 3616 Trousdale Parkway, AHF
107A, Los Angeles, CA 90089, USA
ABSTRACT
Aim To determine biogeographical patterns in five closely related species in the
fish genus Hypseleotris, and to investigate the relative roles of drainage divide
crossings and movement during lowered sea levels between drainage basins and
biogeographical provinces based on the phylogeographical patterns within the
group. The high degree of overlap in the distributions and ecology of these species
makes them ideal candidates for comparative phylogeographical study.
Location Eastern, central and south-eastern Australia.
Methods A total of 179 Hypseleotris individuals were sequenced from 45
localities for the complete mitochondrial cytochrome b gene and the first 30 base
pairs of the threonine transfer RNA for a total of 1170 bp. Phylogenetic
relationships were hypothesized using parsimony and Bayesian analyses.
Results Phylogenetic analysis resolves the five species into three clades. The first
corresponds to the species Hypseleotris klunzingeri (Ogilby, 1898); within it two
clades are resolved, one consisting of individuals from the Eastern Province (EP),
plus two eastern Murray-Darling Province (MDP) localities, and the other
including the remainder of the MDP localities, along with the Lake Eyre Basin
(Central Australian Province, CAP) individuals. The other two clades include a
mixed Hypseleotris galii (Ogilby, 1898)/Hypseleotris sp. 3 Murray-Darling clade,
with EP and MDP lineages mostly segregated and differentiations in populations
spread along the EP, and a mixed Hypseleotris sp. 4 Lake’s and Hypseleotris sp. 5
Midgley’s clade, with two groups of MDP localities and two CAP lineages
indicated, interspersed with EP lineages as well as those from the Northern
Province.
*Correspondence: Christine Thacker, Research
and Collections – Ichthyology, Natural History
Museum of Los Angeles County, 900 Exposition
Boulevard, Los Angeles, CA 90007, USA.
E-mail: [email protected].
Present address: Brigham Young University,
Integrative Biology, 401WIDB, Provo, UT
84602, USA.
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Main conclusions This study is broadly congruent with a previous analysis of
Hypseleotris phylogeny, but the previously observed overall relationship of southeastern Australian provinces [EP(MDP+CAP)] was not confirmed and is more
complicated than hitherto thought. This highlights the necessity of obtaining a
sufficient number of sampling localities to identify potential connectivity between
populations in order to demonstrate congruent biogeographical patterns. We
identified many instances of drainage divide crossings, which were the major
means of movement between provinces. Despite the commonness of movement
across drainage divides, very few of these were found to be exactly congruent
among the species. Most occurred in different places, or if in the same location,
apparently at different times, or in at least one case, in opposite directions.
Patterns of movement between adjacent coastal drainages were also found to be
www.blackwellpublishing.com/jbi
doi:10.1111/j.1365-2699.2007.01711.x
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
Phylogeography of Hypseleotris
largely incongruent; when congruence was found the populations involved had
quite different genetic divergences.
Keywords
Australia, comparative phylogeography, drainage divides, freshwater biogeography, Gobioidei, Eleotridae, sea-level change, sympatric species.
INTRODUCTION
Comparative phylogeography is the investigation of the
geographical distributions of genealogical lineages within and
among species using genetic data across multiple groups of
taxa with similar distributions (Avise, 2000, 2004). These
studies can provide powerful tests of vicariant patterns and
area relationships. Avise (2004) reviewed 26 comparative
phylogeographical studies representing a broad range of taxa
with various temporal and geographical ranges. Of the papers
reviewed, the degree to which congruence among phylogenetic
lineages and geography was identified varied widely. Probably
the best documented example of general congruence is from
both marine and freshwater species occurring in the southeastern USA (Avise, 1992). However, there is some variation in
the degree of separation and in the geographical location that
separates the clades. In contrast, other studies in the North
American central highlands found no phylogeographical
congruence among species of darters (Turner et al., 1996).
Similar studies on southern Central American freshwater fish
obtained evidence for congruence when only larger-scale
patterns were considered (Bermingham & Martin, 1998).
While studies frequently find a lack of congruence, it should be
noted that many factors can influence the ability to detect
similar patterns, especially different rates of molecular
evolution between taxa, population sizes, demographic factors,
gene coalescence and hybridization (Hickerson et al., 2006).
In addition, dispersal is increasingly being re-recognized as
playing a broader role in biogeographical patterns (McGlone,
2005), which will tend to decrease the likelihood of finding
congruent patterns resulting from vicariance.
In this study, we examine the phylogeography of five species
of freshwater gudgeons or sleepers (genus Hypseleotris) known
from south-eastern Australia. This monophyletic group of
species are closely related and often co-occur, making this
group ideal for examining independent but overlapping
diversification in the same drainages. We also sought to
re-examine a pattern of drainage relationships identified in a
previous study (Thacker & Unmack, 2005), using a different
DNA marker and more extensive sampling among drainages.
We sampled localities in four Australian biogeographical
provinces and sought to identify the route via which lineages
had moved both within and between provinces. Most of these
provinces are separated by long drainage divides, several of
which have low relief headwater areas, which may facilitate
transfer among drainages during periods of flooding. Alternatively, movement between coastal drainages can occur
during low sea-level drainage connections, and considerable
variation exists in the continental shelf width, which may affect
the ease with which fishes can move between different
drainages. Our analysis was undertaken to investigate the
influence of movement either across drainage divides, or
between adjacent coastal drainages around the coast of
Australia relative to the colonization of these provinces.
Australia is a stable continent, which has experienced little
major geological activity during the Tertiary and Quaternary.
All the major drainage basins in Australia were established
prior to this time (Unmack, 2001). The major drainage system
of south-eastern Australia is the Murray-Darling Basin,
encompassing 1,073,000 km2. To the north, east and south it
is bounded by the Eastern Highlands, which separate it from
coastal drainages. These coastal drainages occur in a narrow
band around the Murray-Darling Basin and form a number of
shorter major rivers draining to the eastern and southern
coasts. To the west of the Murray-Darling is the large
endorheic Lake Eyre Basin (1,140,000 km2), an arid region
lacking perennial stream flow (Fig. 1). Despite the existence
of long-term drainage basin boundaries, a number of fish
species in south-eastern Australia show a common distribution
pattern, with populations being shared between east coast drainages [Eastern Province (EP) and Northern Province (NP)],
the Murray-Darling Basin [Murray-Darling Province (MDP)]
and Lake Eyre Basin [Central Australian Province (CAP)],
despite major differences in habitat, stream flow and climate.
Over half the 29 strictly freshwater fishes in the MDP are
shared with EP; nine of those same shared species also occur in
CAP (Unmack, 2001). This high degree of faunal similarity
suggests either that the taxonomy of Australian freshwater
fishes is poorly characterized and that these widespread forms
represent multiple, as-yet unrecognized species, or that there
has been relatively recent movement of a large proportion of
the fauna between these provinces. The limited evidence
obtained so far suggests both may be true (Crowley &
Ivantsoff, 1990; Musyl & Keenan, 1992; Rowland, 1993;
McGlashan & Hughes, 2001; Thacker & Unmack, 2005).
The freshwater fish genus Hypseleotris (Eleotridae), commonly known as carp gudgeons, is the most speciose gudgeon
genus known in Australia, with 12 species currently recognized. Ten of these species are endemic, and one (H. compressa
(Krefft 1864)) is extremely widespread across Australia and has
also been recorded from southern New Guinea (Allen et al.,
2002; Thacker & Unmack, 2005). The twelfth species,
H. cyprinoides (Valenciennes 1837), is broadly distributed,
occurring in South Africa, Madagascar, Japan and throughout
Journal of Biogeography 34, 1518–1533
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C. E. Thacker et al.
Figure 1 Map of sampled localities for
Hypseleotris species and river names in eastern Australia. Locality numbers correspond
to those in Table 1. Dotted line denotes
location of the Eastern Highlands.
the Indo-Pacific region. Individuals of Hypseleotris species are
generally small (< 6 cm long), with a laterally compressed
head and body, small mouth and two dorsal fins. The
phylogeny of the genus indicates that there have been two
radiations within Australian Hypseleotris, with five species each
being endemic to either north-western or south-eastern
Australia (Thacker & Unmack, 2005). All but two of the
north-western species are found in separate drainage basins
and most have limited distributions. In contrast, all the southeastern species are widespread and abundant (Fig. 2). They are
usually found in sympatry, with up to four, three and one to
three species being captured in the same seine net haul in the
MDP, CAP and EP, respectively (Unmack, 2000).
Despite their abundance, surprisingly little is known of the
biology of south-eastern Hypseleotris spp., in part due to their
confusing taxonomic status. Prior to Hoese et al. (1980) it
was generally thought that there was one species within MDP,
H. klunzingeri (Ogilby, 1898), and three in EP, H. galii (Ogilby,
1898), H. klunzingeri and the widespread H. compressa. Hoese
et al. (1980) then recognized Midgley’s carp gudgeon, Hypseleotris sp. 4 and Lake’s carp gudgeon, Hypseleotris sp. 5. Later,
Unmack (2000) recognized a fifth species, Murray-Darling
carp gudgeon, Hypseleotris sp. 3, which is restricted to MDP
(Fig. 2b) and is closely related to its allopatric sister species
H. galii. The latter three species are not formally described, but
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are well known by their common names (Allen et al., 2002).
Many authors continue to treat all Hypseleotris spp. together as
they can be difficult to distinguish (e.g. Harris & Gehrke,
1997). This practice hinders understanding of the species,
because any biological data gathered cannot be attributed to a
specific taxon. Further confusing matters, Bertozzi et al. (2000)
demonstrated that hybrids were found, to the extent of a
quarter of all Hypseleotris individuals they examined from the
lower Murray River, involving three of the four species, and
that Lake’s carp gudgeon was always found as a hybrid
genotype. They proposed that several of these hybrids may in
fact be hemi-clonal hybridogenic lineages (Bertozzi et al.,
2000).
A recent phylogenetic study of Hypseleotris species (Thacker
& Unmack, 2005) included sampling of multiple individuals
within several Australian species, and demonstrated a repeated
phylogeographical pattern among south-eastern Australian
drainage groups: [(EP(MDP,CAP)]. The phylogeny in that
study was based on a combined analysis of morphological
characters and mitochondrial DNA sequence data (the
complete ND2 gene). The aim of this study is to increase
greatly the sampling within the five south-eastern Australian
Hypseleotris species, and to compare the results of Thacker &
Unmack (2005) with those obtained using another mitochondrial gene, cytochrome b (cyt b). The five species are
Journal of Biogeography 34, 1518–1533
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
Phylogeography of Hypseleotris
(a)
(b)
(c)
(d)
Figure 2 Map showing the distribution of each Hypseleotris species in south-eastern Australia. Dark lines represent province boundaries.
Province names are given in (d). White points indicate samples sequenced within each species (some points are difficult to see when close to
province boundaries). (a) H. klunzingeri (Ogilby, 1898); (b) Hypseleotris sp. 3 Murray-Darling (lighter shading) and H. galii (Ogilby, 1898)
(darker shading); (c) Hypseleotris sp. 5 Midgley’s; (d) Hypseleotris sp. 4 Lake’s. Arrow in (b) indicates the outlying population from
Waterpark Creek.
H. klunzingeri (EP, MDP and CAP), H. galii (EP only),
Hypseleotris sp. 3 Murray-Darling (MDP only), Hypseleotris sp.
4 Lake’s (MDP and CAP only), and Hypseleotris sp. 5 Midgley’s
(EP, MDP, CAP and a small portion of NP) (Fig. 2).
Collectively, these five species represent multiple instances
of diversification throughout south-eastern Australia. The
increased sampling density of this study with respect to
Thacker & Unmack (2005) allows us to examine the biogeographical patterns of Hypseleotris species at a much finer scale.
Given that all five species are typically widespread, abundant and
commonly co-occur, they provide an excellent test of congruent
phylogeographical patterns in south-eastern Australia, and
demonstrate how increased sampling scale influences phylogeographical interpretations.
Journal of Biogeography 34, 1518–1533
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1521
C. E. Thacker et al.
MATERIALS AND METHODS
Frozen or ethanol-preserved samples of Hypseleotris species for
DNA analysis were collected from localities across southeastern Australia (Fig. 2), primarily using seine nets (Table 1).
Most specimens were identified to species in the field while
alive, based on Unmack (2000). Representative genetic material was deposited in the Evolutionary Biology Unit of the South
Australian Museum, while formalin-fixed and fixed and
preserved representatives were deposited in the Australian,
Victorian and South Australian museums. These samples can
be identified based on their station code (Table 1).
Muscle tissue from each specimen was used for total
genomic DNA extraction, performed with the DNeasy Tissue
Kit (Qiagen, Chatsworth, CA, USA). Amplification of the cyt b
gene was achieved in two portions, using Hypseleotris-specific
primer pairs designed for this study: HYPSLA (5¢-GTGGCTTGAAAAACCACCGTT-3¢) to HYPSHD (5¢- GGGTTGTTGGAGCCAGTTTCGT-3¢) for the 5¢ end, and HYPSL510
(5¢-AGATAATGCAACCCTMACCCG-3¢) or HYPSL500 (5¢CTTYTCMMTAGATAATGCAACCC-3¢) to PH15938 (5¢CGGCGTCCGGTTTACAAGAC-3¢) for the 3¢ end. PCR was
performed using Platinum Taq DNA polymerase (Invitrogen,
Rockville, MD, USA) or Gibco Taq polymerase (Life Technologies, Rockville, MD, USA), with a profile of 94C for
3 min, followed by 40 cycles of 94C/15 s denaturation, 50–
53C/45 s annealing and 72C/30 s extension, with a final hold
at 72C for 7 min. PCR products were electrophoresed on a
low-melting-point agarose gel, visualized and photographed,
then excised and purified with the QIAquick gel extraction kit
(Qiagen). Using the same primers (1 lm rather than 10 lm),
the PCR fragments were cycle-sequenced using the Big Dye
terminator/Taq FS Ready Reaction Kit version 3.1, purified by
passing the reactions through 750 lL Sephadex columns (2.0 g
in 32.0 mL ddH2O), and visualized on an ABI 377 automated
sequencer (Applied Biosystems, Foster City, CA, USA). The
heavy and light strands were sequenced separately. The
resultant chromatograms were reconciled using sequencher
4.1.2 (GeneCodes Corp., Ann Arbor, MI, USA) to check base
calling, translated to amino acid sequence using the ‘mammalian mtDNA’ code, concatenated for each taxon, and aligned
by eye. There were no ambiguities or gaps in the alignment; all
the gaps present in the final matrix were due to missing data
and treated as such (coded as ? rather than a new character
state) in the analysis. Aligned nucleotide sequences were
exported as nexus files from sequencher.
In addition to newly sequenced taxa, six additional
sequences were obtained from GenBank. In accordance with
the basal gobioid phylogeny of Thacker & Hardman (2005),
cyt b sequences from the taxa Calumia godeffroyi (Günther
1877) (AY722194) and Gobiomorphus australis (Krefft 1864)
(AY722216, AY722218) were included and used to root
the phylogeny. Three ingroup sequences from Thacker &
Hardman (2005) were also included: Hypseleotris klunzingeri
(AY722189), H. compressa (AY722188) and H. aurea (Shipway
1950) (AY722187). The latter two sequences were included to
1522
determine whether or not the phylogenetic conclusions of this
study, based on cyt b, would confirm results based on ND2
presented by Thacker & Unmack (2005). Phylogenetic analyses
using both Bayesian and parsimony methods were performed.
Bayesian analyses were run using MrBayes ver. 3.1.1
(Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck,
2003). Analyses were conducted by first determining the
appropriate model for nucleotide change with the likelihoodratio test (LRT) and Akaike’s information criterion (AIC), as
implemented in MrModeltest 2.0 (Nylander, 2004), then
specifying that model in a MrBayes 3.1.1 search run for
1,000,000 generations with four simultaneous chains. This
length of search ensured that the runs converged. Trees were
sampled every 100 generations, and the first 500 trees (50,000
generations) were discarded as burn-in. The Bayesian estimates
of posterior probabilities were included to indicate support for
clades. The same data matrix was also analysed under the
parsimony criterion with paup* ver. 4.0b8 (Swofford, 2003).
One thousand replications of a heuristic search were run, using
tree bisection–reconnection branch swapping, and with the
data designated as equally weighted. Due to the many
intraspecific comparisons, few informative characters were
available at the tips of the tree, and many most parsimonious
trees resulted. To prevent exhausting the memory space
available, a maximum of 100 trees was saved for each
replication. A strict consensus was constructed using paup*.
Pairwise distances among lineages were used as estimates of
the degree of relatedness; these were calculated using
mean between-group p-distances in mega 3.1 (Kumar et al.,
2004).
RESULTS
A total of 179 Hypseleotris individuals were sequenced and 182
were analysed, including three sequences derived from a
previous study (Thacker & Hardman, 2005). Taxa analysed
included 17 Hypseleotris sp. 4 Lake’s, 42 Hypseleotris sp. 5
Midgley’s, 13 Hypseleotris sp. 3 Murray-Darling, 50 H. galii,
58 H. klunzingeri, and one each of H. compressa and H. aurea.
The matrix consisted of 1170 aligned positions, comprising the
complete cyt b gene and the first 30 bp of the threonine
transfer RNA (sequences available in GenBank; accession
numbers DQ468143–DQ468321). Of these, 362 were phylogenetically informative. MrModeltest indicated that the
GTR + I + G model was most appropriate for these data,
based on both the LRT and AIC. Results from the parsimony
analysis (not shown) were generally congruent with those of
the Bayesian analysis, with a difference in the basal nodes of
the hypothesis. In the parsimony analysis, H. compressa and
H. aurea were placed outside the remainder of Hypseleotris.
These results are not concordant with Thacker & Unmack
(2005), in which H. compressa and H. aurea are placed as sister
to south-eastern Hypseleotris exclusive of H. klunzingeri, but
in both cases the alternative placements of H. compressa and
H. aurea are weakly supported. The Bayesian results do not
resolve the placement of H. aurea and H. compressa, placing
Journal of Biogeography 34, 1518–1533
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Phylogeography of Hypseleotris
Table 1 Locality data for all Hypseleotris populations examined.
Population no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Population and province
Northern Province
Return Ck, Mount Garnet, QLD
Eastern Province
Sheep Station Ck, Lower Burdekin R, QLD
Murray Ck, Mount Ossa, QLD
Blacks Ck, Mia Mia, QLD
Amity Ck, Wumalgi, QLD
Vandyke Ck, Vandyke, QLD
Dawson R, Injune, QLD
Maryvale Ck, Maryvale Station, QLD
Baffle Ck, Miriam Vale, QLD
Oyster Ck, Agnes Waters, QLD
Reedy Ck, Gillens Siding, QLD
Three Moon Ck, Mulgildie, QLD
Elliott R, Elliott, QLD
Gregory R, Goodwood, QLD
Lenthall Dam, Stoney Ck Station, QLD
Cunningham Ck, Gympie, QLD
Yabba Ck, Imbil, QLD
Baroon Dam, Mapleton, QLD
Kilcoy Ck, Conondale, QLD
Waraba Ck, Wamuran, QLD
Back Ck, Cooyar, QLD
Maroon Dam, Maroon, QLD
Christmas Ck, Lamington, QLD
Marom Ck, Wollongbar, NSW
Richmond R, Casino, NSW
Clarence R, Tabulam, NSW
Clouds Ck, Nymboida, NSW
Orara R, Karangi, NSW
Corindi R, upper Corindi, NSW
Hastings R, Wauchope, NSW
Booral Ck, Stroud, NSW
Nepean R, Wallaca, NSW
Georges R, Liverpool, NSW
Murray-Darling Province
Severn R, Glen Aplin, QLD
Maranoa R, Mitchell, QLD
Warrego R, Cunnamulla, QLD
Paroo R, Yalamurra, QLD
Dunns Swamp, Rhylstone, NSW
Turon R, Hill End, NSW
Bogan R, Nyngan, NSW
Murray R, Cohuna, NSW
Black Swamp, Cohuna, VIC
Salt Ck, Berri, SA
Bremer R, Lake Alexandrina, SA
Central Australian Province
Bulloo R, Quilpie, QLD
Barcoo R, Tambo, QLD
Hypseleotris species
Station code
MID
PU97-97
MID
MID
MID
MID
KLU
MID
MID
KLU
KLU
MID
GAL, KLU, MID
GAL, KLU
GAL, KLU
GAL, KLU
GAL
KLU, MID
KLU
GAL
GAL
GAL, KLU, MID
KLU
GAL
GAL
KLU
GAL, KLU
GAL
GAL
GAL
GAL
GAL
GAL
GAL
via C. Perna
PU02-45
PU02-44
PU02-46
PU01-52
PU99-59
PU02-49
PU02-50
PU02-42
PU99-57
PU99-58
PU97-51/PU02-38
PU02-37
PU02-36
PU02-34
PU99-54
PU02-31
via M. Kennard
PU02-30
PU99-51
PU02-28
PU02-29
PU02-18
PU99-42
PU99-43
PU02-16
PU02-15
PU02-15
PU99-38
F-FISHY3
AMS-36086
IW94-50
KLU, MD
KLU
LAK, MID
KLU
KLU, MD
KLU
MD
KLU
KLU, LAK, MD, MID
MID
MD
PU99-49
PU99-60
PU99-63
PU99-61
PU99-70
PU02-54
F-FISH21
PU94-37-2
PU99-34
F-FISHADD7
IW94-26
KLU, MID
LAK, KLU, MID
PU99-62
PU97-103
MID ¼ Hypseleotris sp. 5 Midgley’s; KLU ¼ H. klunzingeri (Ogilby, 1898); GAL ¼ H. galii (Ogilby, 1898); MD ¼ Hypseleotris sp. 3 Murray-Darling;
LAK ¼ Hypseleotris sp. 4 Lake’s.
Population number refers to localities shown in Fig. 1. The population column provides the name of the creek or river, then the nearest local place
name, followed by the state abbreviation (NSW ¼ New South Wales; QLD ¼ Queensland; SA ¼ South Australia; VIC ¼ Victoria).
Station codes can be used to track references to genetic material deposited in the South Australian Museum and morphological samples deposited in
the Australian, Queensland, South Australian and Victorian Museum collections.
Journal of Biogeography 34, 1518–1533
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C. E. Thacker et al.
them in a basal polytomy. The Bayesian hypothesis is
presented in Fig. 3. Posterior probability values are indicated
for major nodes; most values are 100%.
DISCUSSION
Relationships among Hypseleotris clades
The phylogeny shown in Fig. 3 indicates strong support for the
monophyly of the genus Hypseleotris, and relationships among
Hypseleotris species that do not conflict with the previous
phylogenetic study of Thacker & Unmack (2005), although
there is some uncertainty in the Bayesian hypothesis, as
represented by the polytomy among H. compressa, H. aurea,
H. klunzingeri and the remainder of the species. All
H. klunzingeri individuals were resolved as a monophyletic
group with two distinct clades. All individuals of H. galii and
Hypseleotris sp. 3 Murray-Darling are grouped together, most
forming a clade in which the species are not completely
separated; two of the sampled H. galii fall within a sister clade
containing most of the Hypseleotris sp. 3 Murray-Darling. All
of the Hypseleotris sp. 4 Lake’s and Hypseleotris sp. 5 Midgley’s
form a clade, with some mixing of species; eight of the sampled
Hypseleotris sp. 4 Lake’s, plus one individual of Hypseleotris sp.
5 Midgley’s, are resolved as a clade sister to the remainder of
Hypseleotris sp. 5 Midgley’s and the remaining nine of the
Hypseleotris sp. 4 Lake’s. The concordance of these relationships with the Australian drainage systems is discussed below
for each clade in turn.
Hypseleotris klunzingeri
The Bayesian hypothesis of Fig. 3 resolves the oldest separation
within H. klunzingeri into two geographical groups: most MDP
populations plus those in CAP (clade A, Fig. 3) vs. all other EP
drainages plus two localities from MDP (clade B, p-distance of
A vs. B ¼ 0.037). Within the MDP/CAP clade (clade A of
Fig. 3), the largest separation is between most MDP populations and those from CAP (p-distance ¼ 0.014). One MDP
individual from Paroo River (37) is grouped with the CAP
clade; Paroo River is the westernmost river in MDP, and thus
is geographically closest to CAP. The existence of closely
related haplotypes in Paroo River and CAP suggests that there
has been a recent connection between these drainages
(p-distance ¼ 0.006). Despite their current hydrological isolation, populations in CAP drainages Bulloo River (45) and
Cooper Creek (Barcoo River, 46) were almost identical
(p-distance ¼ 0.003), suggesting that connectivity was more
recent than hypothesized by Unmack (2001).
The second H. klunzingeri clade (clade B, Fig. 3) consists of
all EP populations plus MDP samples from Maranoa (35) and
Figure 3 Bayesian estimate of phylogeny for Hypseleotris species,
based on 1170 bp of sequence data, including the complete
cytochrome b gene and partial threonine transfer RNA. Numbers
on nodes are posterior probability values of clades. Sampled
individuals are identified by species, with abbreviations as for
Table 2. The number indicates the collection locality, in accordance with Table 1 and Fig. 2. Letters indicate clades discussed in
more detail in the text.
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Journal of Biogeography 34, 1518–1533
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Phylogeography of Hypseleotris
Turon (39) rivers. Phylogeographical breaks are evident among
EP localities, the largest of which separates the population
from Fitzroy River (Vandyke Creek, 6) from the rest of EP
(p-distance ¼ 0.021). Fitzroy River is the northernmost EP
drainage from which samples of H. klunzingeri were obtained.
Lack of more detailed sampling within and around Fitzroy
River precludes detailed interpretation of this pattern. Clearly,
though, it has been isolated from other EP populations for
some considerable time. The northernmost population identified by us in the field is from Herbert Creek, about 100 km
north of the mouth of Fitzroy River. Pusey et al. (2004) listed
other records from further north, including the Plane, Pioneer
and Burdekin basins; however, we feel these are most probably
based on misidentifications and/or introductions. We have
undertaken some sampling in each of these basins and have
found only Hypseleotris sp. 5 Midgley’s.
The next largest split within EP (p-distance ¼ 0.012) is
between (i) populations at the Brisbane River and south (21,
22, 25, 26), and (ii) those found at Mary River and north, plus
one individual from Maroon Dam (22, Brisbane River), and
two MDP populations (Maranoa River, 35 and Turon River,
39) from tributaries of the Darling River (Fig. 1). Within EP,
virtually all H. klunzingeri individuals/populations segregate
and cluster based on the geographical proximity of the river
drainages. The MDP individuals found in clade B are most
closely related to EP samples from Burnett River drainage
(Three Moon Creek, 12), and one Gregory River (14)
individual. Gregory River is a coastal stream slightly south of
Burnett River (Fig. 1), and connections between them probably occur during lowered sea levels. This result suggests recent
movement of individuals from Burnett River across the
Eastern Highlands into MDP (p-distance ¼ 0.002, including
one identical shared haplotype). The concordance between the
phylogenetic hypothesis and a map of sampling sites is shown
in Fig. 4a.
Hypseleotris galii and Hypseleotris sp. 3
Murray-Darling
Individuals of Hypseleotris sp. 3 Murray-Darling occur in three
lineages within clade C, the clade containing Hypseleotris
exclusive of H. klunzingeri, H. compressa and H. aurea (Fig. 3).
Three individuals of Hypseleotris sp. 3 Murray-Darling are
resolved outside the majority of H. galii and Hypseleotris sp. 3
Murray-Darling, and form a polytomy with these species and
the lineage containing Hypseleotris sp. 4 Lake’s and Hypseleotris
sp. 5 Midgley’s (Fig. 3). These unresolved Hypseleotris sp. 3
Murray-Darling individuals include one individual from
Dunns Swamp (38) and two individuals from Black Swamp
(42). These unusual haplotypes could be a result of ancestral
polymorphism or hybridization, and/or their persistence may
be facilitated within hemi-clonal hybrid lineages speculated to
occur within this species (Bertozzi et al., 2000). The remainder
of Hypseleotris sp. 3 Murray-Darling is found within clade D
(Fig. 3). Two lineages within Hypseleotris sp. 3 Murray-Darling
and H. galii (clades D and E) are not well resolved and consist
of related (p-distance ¼ 0.015) but geographically distinct
populations from MDP and northern EP populations centred
on the Burnett River (Three Moon Creek, 12) and two minor
coastal drainages to the south (Elliott River, 13; Gregory River,
14 and Lenthall Dam, 15, a tributary to Gregory River)
(Fig. 2). In addition, haplotypes from two H. galii individuals
were found within Clade D: one each from Burnett River
(Three Moon Creek, 12) and Clarence River (26). This suggests
movement, after the initial separation of H. galii and
Hypseleotris sp. 3 Murray-Darling across the Eastern Highlands, in two places from MDP into EP. The phylogeographical
structure revealed within Hypseleotris sp. 3 Murray-Darling
(clade D) and H. galii (from clade E only) is consistent with
the absence of a morphological character unique to H. galii
(Unmack, 2000). Hoese et al. (1980) used the presence of a
prominent black spot on the anus of H. galii females to identify
this species. However, it is absent from EP H. galii populations
north of Mary River (it remains unclear whether Mary River
populations have the anal spot or not; see the discussion of
Mary River H. galii below). This trait is also absent from
Hypseleotris sp. 3 Murray-Darling, suggesting that northern H.
galii populations may need to be re-identified as Hypseleotris
sp. 3 Murray-Darling, a result also consistent with our
phylogenetic analyses (Fig. 3).
Within the remainder of H. galii, an unusual mix of
populations was resolved, often with little correspondence to
geographical distance between drainages, and in some cases
with significant differences within drainages. Clade G includes
the southernmost populations (Nepean River, 32 and Georges
River, 33), some populations centred around the New South
Wales–Queensland (NSW–QLD) border (Waraba Creek, 20;
Christmas Creek, 23 and Clarence River, 26), plus one
population from Mary River (19). Sister clade F is weakly
supported (posterior probability of 60%). Within clade F,
population 16 (Cunningham Creek), from Mary River, is sister
to the other clade F lineages. The remainder of clade F contains
another group of populations mostly also centred on the
NSW–QLD border (Back Creek, 21; Marom Creek, 24; Clouds
Creek, 27; Orara River, 28 and Corindi Creek, 29), plus
Hastings River (30) and Karaugh River (31) in central NSW.
A second individual from Hastings River (30) was resolved
outside clades F and G. Three populations within Clarence
River were sampled (Clarence River, 26; Clouds Creek, 27 and
Orara River, 28), and each was not closely related. The same
was true for Mary River (Cunningham Creek, 16 and Kilcoy
Creek, 19). Multiple individuals from Hastings River (30) were
also unrelated, and to a lesser extent so were individuals in
Christmas Creek (23), while Clarence River (26) also had a
haplotype in clade D. These results may indicate that there has
been irregular mixing and isolation of various populations,
with sufficient retention of ancestral polymorphism that
multiple distinct lineages/haplotypes become fixed in different
parts of the drainages, and thus persist. Lineage relationships
are superimposed on a sampling map in Fig. 4b.
Two groups of populations within clades F and G require
additional discussion. The type locality of H. galii is the Sydney
Journal of Biogeography 34, 1518–1533
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
1525
C. E. Thacker et al.
(a)
(b)
(c)
Figure 4 Correlation between phylogenetic hypotheses and sampling locations for (a) Hypseleotris klunzingeri (Ogilby, 1898); (b) H. galii
(Ogilby, 1898) and Hypseleotris sp. 3 Murray-Darling; (c) Hypseleotris sp. 4 Lake’s and Hypseleotris sp. 5 Midgley’s. For clarity, some nodes
have been rotated relative to Fig. 3; numbers on nodes are posterior probability values.
Botanical Gardens, which is located in the vicinity of the
Georges and Nepean rivers (32, 33). Phylogenetically, these
populations are most closely related to those from the NSW–
QLD border. The population from which H. galii was
originally described was thought to have been introduced,
although the original source remains unclear (Ogilby, 1898). It
remains possible that current populations in the vicinity of
Sydney have been introduced. Individuals sampled from Mary
1526
River (Cunningham Creek, 16) were not originally identified as
H. galii, but instead appeared to be Hypseleotris sp. 5 Midgley’s.
The second population obtained from Mary River (Kilcoy
Creek, 19) was provided by M. Kennard, and we were
unable to identify them alive. Despite sampling many localities
within Mary River, we have never clearly field-identified any
H. galii, despite being able to do so in all surrounding
drainages.
Journal of Biogeography 34, 1518–1533
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
Phylogeography of Hypseleotris
Hypseleotris sp. 4 Lake’s
A primary conclusion of these data, as well as the previous
study of Thacker & Unmack (2005), is that the taxa known as
Hypseleotris sp. 4 Lake’s and Hypseleotris sp. 5 Midgley’s are
not distinguishable based on mitochondrial DNA haplotypes.
The identifications of taxa based on morphology are not in
doubt: Hypseleotris sp. 4 Lake’s may be separated easily from
Hypseleotris sp. 5 Midgley’s, based on the absence of scales on
the head, nape and anterior body (Hoese et al., 1980).
However, this character does not correspond with groupings
based on haplotype data, and may therefore represent a
polymorphism, found in individuals in interior MDP and CAP
drainages, the only ones with Hypseleotris sp. 4 Lake’s
populations (Fig. 2).
Bertozzi et al. (2000) examined collections of Hypseleotris
individuals from the lower Murray River, where H. klunzingeri,
Hypseleotris sp. 3 Murray-Darling, Hypseleotris sp. 4 Lake’s,
and Hypseleotris sp. 5 Midgley’s co-occur. Using allozyme
electrophoresis of 20 variable loci, they identified four groups
they called HA, HB, HC and HX, as well as the hybrid classes
HAxHB, HAxHX and HBxHX. HC was shown to correspond
to H. klunzingeri, and did not participate in any detectable
hybridization. All individuals of Hypseleotris sp. 4 Lake’s they
examined were F1 hybrids between either Hypseleotris sp. 5
Midgley’s (later determined to equal HB; M. Adams, personal
communication) or Hypseleotris sp. 3 Murray-Darling (later
determined to equal HA; M. Adams, personal communication), and what they termed HX, a taxon not observed in its
pure form. They speculated that the most likely explanation for
this pattern was the existence of multiple hemi-clonal lineages,
although they lacked sufficient evidence to demonstrate this
clearly.
The data presented in Fig. 3 are derived from analysis of the
mitochondrial genome, and so reveal only the pattern of the
maternal lineages. However, the allozyme data of Bertozzi et al.
(2000) are consistent with the results presented in this study.
The allozyme patterns predict that morphological samples of
Hypseleotris sp. 4 Lake’s could have one of several mtDNA
types, either a pure Hypseleotris sp. 4 form, or a contribution
from the female hybrid parent. Clade H, the most distinct
group (p-distance to Hypseleotris sp. 5 Midgley’s ¼ 0.094),
sister to the remainder and containing primarily Hypseleotris
sp. Lake’s morphotypes plus a single Hypseleotris sp. Midgley’s
from Salt Creek (43), may represent the original mtDNA type
for Hypseleotris sp. 4 Lake’s (HX, Fig. 3). Several Hypseleotris
sp. 4 Lake’s haplotypes are also found within Hypseleotris sp. 5
Midgley’s populations, including fish from MDP (Warrego
River, 36 and Black Swamp, 42) and CAP (Barcoo River, 46).
This is what would be expected as a result of a male
Hypseleotris sp. 4 Lake’s mating with a female Hypseleotris sp.
5 Midgley’s. Other hybrids between both Hypseleotris sp. 4
Lake’s and Hypseleotris sp. 5 Midgley’s with Hypseleotris sp. 3
Murray-Darling have been detected via allozyme electrophoresis (Bertozzi et al., 2000), although no haplotypes were found
to be shared between these three lineages in our analysis. This
is probably due to our limited sampling of individuals and
populations, especially within MDP, which is where the
Bertozzi et al. (2000) study was based.
Hypseleotris sp. 5 Midgley’s
The final Hypseleotris species examined, Hypseleotris sp. 5
Midgley’s, was placed primarily in a series of four clades
(clades I, J, K and L; one individual was resolved in clade H, as
described above, and another singleton from Back Creek, 21
fell outside the four clades). Hypseleotris sp. 5 Midgley’s is
more widespread than other Australian Hypseleotris examined,
occurring in all three zones, EP, MDP and CAP, as well as in
the southernmost portion of NP (Fig. 2). The largest break
within Hypseleotris sp. 5 Midgley’s (p-distance ¼ 0.047, or
0.042 including individuals only within clade I) separates
Brisbane River (Back Creek, 21) from remaining EP populations. A second large break (p-distance ¼ 0.044) separates
populations from Burnett and Mary rivers (clade J: Three
Moon Creek, 12, Yabba Creek, 17, and a solitary haplotype
from CAP: Barcoo River, 46) from remaining Hypseleotris sp. 5
Midgley’s populations. The rest of Hypseleotris sp. 5 Midgley’s
formed an unresolved trichotomy with clades K, L and the
population from Burdekin River (2). These three lineages were
all separated by p-distances of between 0.022 and 0.026. Clade
L consisted of a group of closely related populations from
Kolan River (Reedy Creek, 11, immediately north of Burnett
River) north to Murray Creek (3), plus one CAP individual
(Bulloo River, 45), a distinct haplotype from Fitzroy River
(Dawson River, 7), and populations from MDP (Warrego
River, 36; Black Swamp, 42 and Salt Creek, 43). Burdekin River
(2) was the next most northerly population sampled after
Murray Creek (3), but the two are separated by about 250 km,
and Herbert River, the northernmost population, was another
c. 180 km from Burdekin River. The lack of geographically
intermediate populations makes interpretations difficult relative to the significance of the phylogenetic separations we found
between these northern populations. Overall, most individuals
from the same localities and larger drainages were usually
grouped together. Two exceptions were noted: first, the single
Barcoo River haplotype (46) found within clade I that did not
group with other Barcoo River haplotypes (clade K); and
second, the Dawson River (7) and Maryvale Creek (8)
populations both within the Fitzroy River, but they did not
group together although they were within the same clade (L)
(Figs 3 & 4c).
Movement across drainage divides and congruence
among taxa
The genus Hypseleotris occurs along both sides of several long
drainage divides between EP and CAP, EP and MDP, and
MDP and CAP (Fig. 2). The length of each drainage divide is
provided in Table 2. Based on existing geological data, there
have been few if any river captures across the Eastern
Highlands for a considerable time span, well beyond the time
Journal of Biogeography 34, 1518–1533
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1527
C. E. Thacker et al.
Table 2 Comparison of genetic divergences across adjacent drainage divides.
Across drainage divide comparison
Species in both
drainages
Divergences
(p-distances)
Cooper Creek (CAP) vs. Herbert (NP)
MID*
0.012
Cooper Creek (CAP) vs. Fitzroy (EP)
Cooper Creek (CAP) vs. Murray (MDP)
Cooper Creek (CAP) vs. Bulloo (CAP)
Murray (MDP) vs. Bulloo (CAP)
Paroo (MDP) vs. Bulloo (CAP)
Murray (MDP) vs. Fitzroy
(minus Dawson) (EP)
Murray (MDP) vs. Dawson
(Fitzroy) (EP)
Murray (MDP) vs. Burnett (EP)
Murray (MDP) vs. Burnett2 (EP)
MID, KLU
MID, KLU*
MID, KLU*
MID, KLU*
KLU MID, KLU*
0.026,
0.030,
0.027,
0.012,
0.005
0.017,
MID*
0.020
GAL/MD*, MID, KLU
GAL/MD , KLU 0.014, 0.044, 0.034
0.009, 0.002
Murray (MDP) vs. Brisbane (EP)
Murray (MDP) vs. Clarence (EP)
Murray (MDP) vs. Clarence2 (EP)
GAL/MD, MID, KLU
GAL/MD, KLU
GAL/MD 0.028, 0.048, 0.035
0.028, 0.038
0.013
Comments
Connection likely occurred
via upper Burdekin River
Drainage divide
length (km)
846 (Burdekin)
0.041
0.015
0.003
0.014
49
na
918
2398à
0.038
922
922
Movements were in
opposite directions
294
294
304
384
384
The abbreviation for each region being compared is provided after the drainage name. Species codes: GAL ¼ Hypseleotris galii (Ogilby, 1898);
KLU ¼ H. klunzingeri (Ogilby, 1898); MD ¼ Hypseleotris sp. 3 Murray-Darling; MID ¼ Hypseleotris sp. 5 Midgley’s.
All MDP calculations excluded Paroo River (37) and MDP haplotypes from populations 35 and 39 that occurred in the EP clade B of H. klunzingeri.
Similarly, both unusual H. galii haplotypes from EP populations 12 and 26 that occurred in the MDP clade D were not included in distance
calculations, nor were the three basal haplotypes from Hypseleotris sp. 3 Murray-Darling in clade C (populations 38, 42). The unusual haplotype from
Hypseleotris sp. 5 Midgley’s Barcoo River (46) that occurred in clade J was also excluded. All drainage divide distances were derived from Hutchinson
et al. (2000).
*Populations being compared are sister to each other.
Populations that have had more recent secondary mixing.
àDue to aridity, only 1272 km of this divide separates watercourses that contain fishes (Wager & Unmack, 2000).
frame involved within Hypseleotris (Unmack, 2001). However,
there are numerous areas between many drainages that have
areas of particularly low relief, which may allow fishes to cross
drainage divides without geomorphic modification during wet
climatic periods (Unmack, 2001). For instance, connection
between MDP and Burnett River is most likely to have
occurred across an essentially flat plain that exists between the
upper Boyne River (a Burnett River tributary) near Boondooma and Durong and the upper reaches of Burra Creek, an
eventual tributary to Condamine River (MDP) (Fig. 1). This
area has no discernible drainage divide and could easily allow
movement during wetter climatic periods. A large proportion
of fishes (Unmack, 2001) and other aquatic biota such as
turtles (Georges & Adams, 1996; Georges et al., 2002) and
shrimps (Murphy & Austin, 2004; Cook et al., 2006) are
shared, or have sister species between MDP and EP as well as
between MDP and CAP, suggesting that many species have
been able to cross the Eastern Highlands and the drainage
divide separating MDP and CAP. However, few studies have
clearly identified which drainages these exchanges occurred
between, except within Paratya shrimps, which had a bewildering pattern that suggested many drainage divide crossings
(Cook et al., 2006). Given the high degree of sympatry and
extensive overlap in distributions, it might be expected that
each Hypseleotris species would show a similar phylogenetic
1528
pattern of drainage divide crossings. However, in most
instances this does not appear to be the case, as outlined
below. Genetic distances for all comparisons across drainage
divides are provided in Table 2.
Central Australian Province comparisons
In H. klunzingeri there is a close relationship between CAP
populations from Bulloo River (45) and Cooper Creek (Barcoo
River, 46) populations, which are sister to MDP populations
(Fig. 3). However, in Hypseleotris sp. 5 Midgley’s this is not the
case. Cooper Creek (CAP; Barcoo River, 46) is most closely
related to Herbert River (Return Creek, 1), a northern coastal
drainage and Bulloo River (45; CAP) is related to MDP
populations (Warrego River, 36; Black Swamp, 42 and Salt
Creek, 43) as well as one EP population (Dawson River, 7;
Fig. 3). Presumably the connection between Herbert River
(Return Creek, 1) and Cooper Creek (Barcoo River, 46) in
Hypseleotris sp. 5 Midgley’s must have been via the upper
Burdekin River (EP) (which was not sampled), as Herbert
River borders Burdekin River, which in turn shares a long
drainage divide with Cooper Creek (Fig. 2). There appear to be
several possible areas with low divides separating Burdekin
River and Cooper Creek that could have facilitated past
movement across this drainage divide.
Journal of Biogeography 34, 1518–1533
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Phylogeography of Hypseleotris
Eastern Province comparisons with Murray-Darling
Province
In H. klunzingeri there is a larger separation between MDP and
EP populations (p-distance ¼ 0.035), but with some recent
exchange (p-distance ¼ 0.002) of haplotypes from the Burnett
River (EP; Three Moon Creek, 12) to MDP (Maranoa River,
35 and Turon River, 39; Fig. 3). In Hypseleotris sp. 3 MurrayDarling/H. galii there is a close relationship (p-distance ¼ 0.014) between Burnett River (EP; Three Moon
Creek, 12) and MDP (Severn River, 34; Dunns Swamp, 38,
Black Swamp, 42 and Bremer River, 44), with additional
more recent exchange of MDP haplotypes into Burnett River
(p-distance ¼ 0.002). This is the opposite direction to
exchanges within H. klunzingeri (Fig. 3), and the genetic
divergence for H. klunzingeri is slightly larger (p-distance of
0.002 vs. 0.009). In contrast, Hypseleotris sp. 5 Midgley’s
populations in Burnett River (EP; Three Moon Creek, 12)
show no relationship to MDP populations at all (Fig. 3)
(p-distance ¼ 0.044). The most likely source of movement of
Hypseleotris sp. 5 Midgley’s from EP into MDP was via Fitzroy
River (EP; Dawson River, 7), which is the next major drainage
north of Burnett River that has a shared drainage divide with
MDP (Fig. 2). Note that H. klunzingeri populations from
Fitzroy River (EP; Vandyke, 6) show a closer phylogenetic
relationship than any other EP population to those from MDP
(Fig. 3), but with a larger genetic distance (p-distance of 0.017
vs. 0.038; Table 2). Lastly, we also found evidence for
movement of an Hypseleotris sp. 3 Murray-Darling haplotype
from MDP into Clarence River (26; clade D), but none was
found for the sympatric H. klunzingeri (Fig. 3).
Summary of congruence in movement across drainage divides
We obtained evidence that Hypseleotris have crossed all coastal
drainage divides along the Eastern Highlands between the
Burdekin and Clarence rivers, except for the Brisbane River
(spanning 2800 km of drainage divide). In addition, the
drainage divides between CAP and MDP have also been
crossed. However, virtually all crossing events appear to have
taken place in different places, or if at the same place, at a
different time and/or in opposite directions. Across the Eastern
Highlands we have H. galii/Hypseleotris sp. 3 Murray-Darling
moving between Clarence River and MDP as well as between
Burnett River and MDP (the latter at two different times).
Hypseleotris klunzingeri has probably moved between Fitzroy
River and MDP as well as having recent connections between
Burnett River and MDP. Hypseleotris sp. 5 Midgley’s has
moved between Herbert River (presumably via the Burdekin
River) and Cooper Creek (CAP) and also possibly between
Fitzroy River and MDP. The one divide crossing that may be
congruent in time and space was between MDP and Bulloo
River (CAP) for H. klunzingeri and Hypseleotris sp. 5 Midgley’s
(Table 2). Thus, except for Brisbane River, every shared
drainage divide has seen movement across it, but usually only
involving one or two of the species that occur within those
drainages and, usually, the divergence estimates between those
species that have common regions of movement vary. If these
fishes are indeed crossing at these low points between
drainages, then it is not surprising that there would be little
congruence. It is easy to imagine that species might be
differentially able to take advantage of rare short-term weather
events that result in sheet flow, by means of which fishes could
move between drainages. It seems clear that all the species have
been able to take advantage of this type of drainage divide
crossing at one time or another. These results suggest that
some drainage divides may not be strong barriers to the
movement of aquatic organisms in the long term. This has
important implications for the study of Australian freshwater
biogeography, as many species share similar ranges and
distribution patterns to Hypseleotris (Unmack, 2001). In
addition, similar topographical settings exist in many parts
of the world that are older and lack recent orogenic
movements (including much of the USA east of the Rocky
Mountains). Over time, due to erosion, the topography of the
landscape becomes more subdued, which reduces elevational
differences between drainage basins and increases chances for
movement via flooding.
Coastal phylogenetic breaks and congruence among
taxa
The continental shelf of Australia adjacent to the distribution
of Hypseleotris varies considerably in width, which in turn
influences the degree to which present-day rivers may coalesce
during periods of lower sea level (Unmack, 2001). Continental
shelf width is especially narrow (20–40 km measured to 200 m
below sea level) along most of coastal NSW, and gradually
broadens (50–80 km) from Brisbane River north to between
the Burnett and Fitzroy rivers. From the Fitzroy River north,
the continental shelf broadens greatly, to up to 250 km wide,
and then slowly narrows to 120 km at the Burdekin River and
100 km at the Herbert River. Most geographical overlap
between Hypseleotris species is between the Clarence River
(northern NSW) north to Fitzroy River, an area over which
continental shelf width gradually widens. The differences in
potentially connectivity between drainages should be reflected
in phylogenetic patterns. However, there appeared to be little
evidence of congruence, as discussed below. Genetic distances
between major EP drainages are presented in Table 3.
The largest genetic difference between coastal populations
was found within Hypseleotris sp. 5 Midgley’s between Brisbane
River (Back Creek, 21) and Mary River (Yabba Creek, 17)
(p-distance ¼ 0.050) and also between Burnett River (Three
Moon Creek, 12) and Kolan River (Reedy Creek, 11)
(p-distance ¼ 0.042) (Figs 1 & 3). Hypseleotris klunzingeri also
displays greater genetic divergence between Brisbane River
(Back Creek, 21) and Mary River (Yabba Creek, 17 and Baroon
Dam, 18) (p-distance ¼ 0.012), than it does between populations in Burnett River (Three Moon Creek, 12) and Baffle
Creek (9), and Oyster Creek (10; p-distance ¼ 0.007). Hypseleotris galii populations at Mary River (Cunningham Creek,
Journal of Biogeography 34, 1518–1533
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1529
C. E. Thacker et al.
Table 3 Comparison of genetic divergences between the six larger adjacent coastal drainages in Eastern Province.
Drainage comparisons
Species comparisons
Divergences (p-distances)
Clarence vs. Brisbane
Brisbane vs. Mary
Mary vs. Burnett
Burnett vs. Kolan/Baffle
Kolan/Baffle vs. Fitzroy
GAL (26, 27, 28 vs. 21), KLU (26 vs. 21)
GAL (21 vs. 16, 19), MID* (21 vs. 17), KLU (21 vs. 17, 18)
GAL (16, 19 vs. 12), MID* (17 vs. 12), KLU (17, 18 vs. 19)
MID (12 vs. 11), KLU (12 vs. 9, 10)
MID (11 vs. 7, 8), KLU (9, 10 vs. 6)
0.019,
0.026,
0.027,
0.042,
0.016,
0.009
0.050, 0.012
0.026, 0.007
0.006
0.021
Species codes: GAL ¼ Hypseleotris galii (Ogilby, 1898); KLU ¼ H. klunzingeri (Ogilby, 1898); MD ¼ Hypseleotris sp. 3 Murray-Darling;
MID ¼ Hypseleotris sp. 5 Midgley’s.
Numbers after species represent populations compared in distance calculations.
Both unusual H. galii haplotypes from EP populations 12 and 26 that occurred in the MDP clade D were not included in distance calculations.
Hypseleotris galii and H. klunzingeri were also examined from smaller intermediate drainages; these populations were not considered in these
comparisons.
*Populations being compared are sister to each other.
16 and Kilcoy Creek, 19) and Burnett River (Three Moon
Creek, 12) are also quite divergent (p-distance ¼ 0.027). This
divergence parallels that seen in Hypseleotris sp. 5 Midgley’s,
and with a similar genetic distance, 0.027 vs. 0.026. The
remaining populations of H. galii had an unusual mix of
haplotypes from two clades that made comparisons between
adjacent drainages difficult. Patterns different from the other
species were found in the sympatric H. klunzingeri. Samples
from Fitzroy River (Vandyke Creek, 6) H. klunzingeri were
clearly segregated from more southern coastal populations
(p-distance 0.021), although Hypseleotris sp. 5 Midgley’s
showed similar differentiation across that region (p-distance ¼ 0.016) (Figs 1 & 3), but with different phylogenetic
patterns of relatedness to surrounding drainages.
Clearly, there is little congruence in the genetic divergences
of Hypseleotris species between adjacent rivers, with the
exception of between Mary and Burnett rivers in H. galii and
Hypseleotris sp. 5 Midgley’s. The only other spatially congruent
break appeared to be between Brisbane and Mary rivers in
Hypseleotris sp. 5 Midgley’s and H. klunzingeri, although
divergences were quite different (Table 3). This lack of
congruence could be due to differences in the species ecology,
or perhaps there is greater randomness as to how and when
fishes move between drainages during lower sea levels.
Qualitatively, the phylogenetic patterns of Hypseleotris sp. 5
Midgley’s most closely approximate patterns of continental
shelf width. Populations south of Burnett River, where the
continental shelf is narrow (50–80 km), are the most differentiated (Table 3). North of the Burnett River, as far as the
Pioneer River, most populations group together closely in the
phylogeny; this is the area where the shelf is widest (250 km).
North of the Pioneer River, where the continental shelf
narrows again (100–120 km), divergences of Burdekin and
Herbert river populations are larger, but not as large as shown
within the southernmost populations. Most populations of
H. galii and H. klunzingeri occur along the narrower continental shelf, but clearly have been able to move between coastal
drainages more recently than Hypseleotris sp. Midgley’s, based
on their considerably smaller genetic divergences (Table 3).
This suggests that some species are able to move between
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drainages despite quite narrow continental shelf widths. None
of these species is ever found in estuarine conditions, but
H. galii and H. klunzingeri may be abundant in some aquatic
habitats in close proximity to the ocean, whereas, based on our
field experience, Hypseleotris sp. Midgley’s may be more
commonly found a little further upstream.
Congruence with other taxa
Given the number of significant phylogeographical breaks
found within this study, it is not surprising to find that some
of them are consistent with results found in other species,
although many exceptions exist that show no consistency.
Numerous aquatic invertebrate groups (crayfishes, shrimps,
mussels) have been examined between CAP and western MDP,
and most of these groups show some evidence of Mid- to LatePleistocene connections between these provinces (Hughes &
Hillyer, 2003; Carini & Hughes, 2004; Hughes et al., 2004).
However, only the crayfish, Cherax destructor Clark 1936,
appears to display evidence for particularly recent movement
of haplotypes between MDP and CAP (Hughes & Hillyer,
2003). The close relationship of H. klunzingeri and Hypseleotris
sp. 5 Midgley’s between Bulloo River and MDP may be
congruent with these results for invertebrates. The region
between Brisbane and Mary rivers was identified as having a
spatially congruent genetic divergence in H. klunzingeri and
Hypseleotris sp. 5 Midgley’s. This divergence broadly corresponds spatially to additional minor divergences found in this
general area within the freshwater fish species Nannoperca
oxleyana Whitley 1940, Pseudomugil signifer Kner 1866 and
Rhadinocentrus ornatus Regan 1914 (Hughes et al., 1999; Page
et al., 2004; Wong et al., 2004), presumably due to a low sealevel drainage barrier that prevents coalescence of these rivers.
A divergence also occurs between Burnett and Kolan rivers in
Hypseleotris sp. 5 Midgley’s that may be congruent with the
separation of P. signifer populations between these same rivers
(Wong et al., 2004). Undoubtedly, as more taxa are examined,
further evidence for congruent breaks will be obtained, but at
this stage the majority of the evidence suggests that a few
Journal of Biogeography 34, 1518–1533
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Phylogeography of Hypseleotris
phylogenetic breaks are spatially congruent and that very few
are temporally congruent across taxa.
The influence of increased sampling scale on
biogeographical interpretations in Hypseleotris
The general hypothesis of Thacker & Unmack (2005), based on
large-scale sampling, is largely consistent with the results
obtained here. The relationships among species shown in the
Bayesian phylogeographical hypothesis correspond to those
shown by Thacker & Unmack (2005), with slightly less
resolution. Within species, they proposed that biogeographical
relationships could be summarized as EP[(MDP)(CAP)] for
H. klunzingeri, [EP(MDP)] for H. galii/Hypseleotris sp. 3
Murray-Darling, and [EP(MDP)(northern EP)(CAP)] for
Hypseleotris sp. 5 Midgley’s. In the present paper, denser
sampling has revealed two MDP groups within H. klunzingeri,
one within a larger, predominantly EP clade, and the other
with a group of CAP lineages nested within it. Similarly,
although most of the Hypseleotris sp. 3 Murray-Darling and H.
galii samples are arrayed in the same pattern as by Thacker &
Unmack (2005), three individuals were identified that fell
outside the major clade, and details of structuring within
provinces were thus revealed. Hypseleotris sp. 4 Lake’s and
Hypseleotris sp. 5 Midgley’s exhibited an array of phylogeographical relationships that echoed some of the characteristics
found in Thacker & Unmack (2005), but with much greater
complexity.
For each species there was an apparently congruent relationship between EP and MDP. However, with the added details
from smaller-scale (denser) among- and within-population
sampling, the interpretations of these earlier results have
changed. When broadly interpreted, these distributions are still
congruent at the larger scale; however, it is clear that when the
smaller-scale details are examined, none of the relationships
between MDP and EP for each species are congruent relative to
when and where individuals crossed drainage divides. In one
case where movement occurred between the same areas
(Burnett River and MDP) in H. klunzingeri and H. galii/
Hypseleotris sp. 3 Murray-Darling, haplotypes were exchanged
in opposite directions.
CONCLUSIONS
Analysis of the phylogeography of five eastern Australian
Hypseleotris species has demonstrated that drainage divides
between EP, CAP and MDP have mostly been crossed at
different places and times. The alternative explanation of
haplotype transfer among drainages, via low sea-level connections between rivers, does not appear to be detectable in the
broader phylogeography of Hypseleotris species. Coastal phylogenetic breaks among populations of the three species
inhabiting EP correlate with one another only slightly,
indicating that each species has had a different history in
those coastal areas. Overall, phylogeographical congruence in
this group is minimal.
In addition, this study underscores the importance of
utilizing as fine a sampling scale as possible when evaluating
phylogeographical relationships. The findings based on sparse
sampling of Thacker & Unmack (2005) were found to be part
of a much more complex pattern when additional sampling
was undertaken. Several additional transfers among provinces
and drainage basins were detected with our increased
sampling, although there appears to be little evidence for
congruent patterns among Hypseleotris taxa. In order to test
properly for potential routes of movement between populations, one must undertake a sufficiently dense sampling to
determine if patterns are geographically congruent. Within
obligate aquatic organisms that are relatively mobile (like
many fishes), the standard scale of sampling to demonstrate
congruence should be based on obtaining not less than one
sample per discrete river basin. In larger river basins and those
with more complex geological history, more samples should be
obtained, as demonstrated by the multiple clades found within
certain river basins in our study, as well as by others elsewhere
in Australia (Hurwood & Hughes, 1998; McGlashan & Hughes,
2000), North America (Gerber et al., 2001) and Europe (Sanjur
et al., 2003).
ACKNOWLEDGEMENTS
The authors thank the many people who provided tissue
samples and assistance with field work in Australia, especially
M. Adams, M. Baltzly, M. Hammer, M. Kennard, C. Perna,
R. Remington, and the various state fisheries agencies who
provided collecting permits. C.E.T. thanks the Australian
Museum, Sydney (AMS), for a Collection Fellowship enabling
study of collections of Hypseleotris species, and AMS staff
J. Leis, M. McGrouther, K. Parkinson and T. Trinski for their
assistance. This study was supported by a grant from the
National Science Foundation (NSF DEB 0108416) and by
grants from the W. M. Keck and R. M. Parsons Foundations to
the Program in Molecular Systematics and Evolution at the
Natural History Museum of Los Angeles County.
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BIOSKETCHES
Christine Thacker is Associate Curator of Ichthyology at the
Natural History Museum of Los Angeles County. Her research
focuses on the evolution and relationships of species and
populations throughout the Indo-Pacific, including both
marine and freshwater groups.
Peter Unmack is currently a postdoctoral fellow at Brigham
Young University. He specializes in the biogeography, distributional ecology, systematics and conservation of freshwater
fishes.
Lauren Matsui is currently an undergraduate at Humboldt
State University; she specializes in bird ecology and behaviour,
and participated in this research through an undergraduate
research fellowship from the Natural History Museum of Los
Angeles County.
Neil Rifenbark is currently a medical student at the
University of Southern California. He completed his undergraduate degree in Biology at USC, during which time he also
received an undergraduate research fellowship from the
Natural History Museum of Los Angeles County to participate
in this project.
Editor: Bob McDowall
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