Comparative phylogeography of five sympatric Hypseleotris species

ORIGINALARTICLE

Comparative phylogeography of fivesympatric Hypseleotris species (Teleostei:Eleotridae) in south-eastern Australiareveals a complex pattern of drainagebasin exchanges with little congruenceacross species

Christine E. Thacker1*, Peter J. Unmack2�, Lauren Matsui3

and Neil Rifenbark4

1Research 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 and4Department of Biology, University of Southern

California, 3616 Trousdale Parkway, AHF

107A, Los Angeles, CA 90089, USA

*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.

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.

Main conclusions This study is broadly congruent with a previous analysis of

Hypseleotris phylogeny, but the previously observed overall relationship of south-

eastern 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

Journal of Biogeography (J. Biogeogr.) (2007) 34, 1518–1533

1518 www.blackwellpublishing.com/jbi ª 2007 The Authorsdoi:10.1111/j.1365-2699.2007.01711.x Journal compilation ª 2007 Blackwell Publishing Ltd

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 south-

eastern 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. Alter-

natively, 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 drai-

nages [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), com-

monly known as carp gudgeons, is the most speciose gudgeon

genus known in Australia, with 12 species currently recog-

nized. 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

largely incongruent; when congruence was found the populations involved had

quite different genetic divergences.

Keywords

Australia, comparative phylogeography, drainage divides, freshwater biogeogra-

phy, Gobioidei, Eleotridae, sea-level change, sympatric species.

Phylogeography of Hypseleotris

Journal of Biogeography 34, 1518–1533 1519ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

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 south-

eastern 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, Hypse-

leotris 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

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 mitochon-

drial gene, cytochrome b (cyt b). The five species are

Figure 1 Map of sampled localities for

Hypseleotris species and river names in east-

ern Australia. Locality numbers correspond

to those in Table 1. Dotted line denotes

location of the Eastern Highlands.

C. E. Thacker et al.

1520 Journal of Biogeography 34, 1518–1533ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

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 biogeo-

graphical 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 phylogeo-

graphical interpretations.

(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.



MATERIALS AND METHODS

Frozen or ethanol-preserved samples of Hypseleotris species for

DNA analysis were collected from localities across south-

eastern 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 mater-

ial 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¢-GTGGC-

TTGAAAAACCACCGTT-3¢) to HYPSHD (5¢- GGGTTGTTG-

GAGCCAGTTTCGT-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 Tech-

nologies, Rockville, MD, USA), with a profile of 94�C for

3 min, followed by 40 cycles of 94�C/15 s denaturation, 50–

53�C/45 s annealing and 72�C/30 s extension, with a final hold

at 72�C 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 ‘mamma-

lian 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 (Gunther

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

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 likelihood-

ratio 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 phylo-

genetically 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



Table 1 Locality data for all Hypseleotris populations examined.

Population no. Population and province Hypseleotris species Station code

Northern Province

1 Return Ck, Mount Garnet, QLD MID PU97-97

Eastern Province

2 Sheep Station Ck, Lower Burdekin R, QLD MID via C. Perna

3 Murray Ck, Mount Ossa, QLD MID PU02-45

4 Blacks Ck, Mia Mia, QLD MID PU02-44

5 Amity Ck, Wumalgi, QLD MID PU02-46

6 Vandyke Ck, Vandyke, QLD KLU PU01-52

7 Dawson R, Injune, QLD MID PU99-59

8 Maryvale Ck, Maryvale Station, QLD MID PU02-49

9 Baffle Ck, Miriam Vale, QLD KLU PU02-50

10 Oyster Ck, Agnes Waters, QLD KLU PU02-42

11 Reedy Ck, Gillens Siding, QLD MID PU99-57

12 Three Moon Ck, Mulgildie, QLD GAL, KLU, MID PU99-58

13 Elliott R, Elliott, QLD GAL, KLU PU97-51/PU02-38

14 Gregory R, Goodwood, QLD GAL, KLU PU02-37

15 Lenthall Dam, Stoney Ck Station, QLD GAL, KLU PU02-36

16 Cunningham Ck, Gympie, QLD GAL PU02-34

17 Yabba Ck, Imbil, QLD KLU, MID PU99-54

18 Baroon Dam, Mapleton, QLD KLU PU02-31

19 Kilcoy Ck, Conondale, QLD GAL via M. Kennard

20 Waraba Ck, Wamuran, QLD GAL PU02-30

21 Back Ck, Cooyar, QLD GAL, KLU, MID PU99-51

22 Maroon Dam, Maroon, QLD KLU PU02-28

23 Christmas Ck, Lamington, QLD GAL PU02-29

24 Marom Ck, Wollongbar, NSW GAL PU02-18

25 Richmond R, Casino, NSW KLU PU99-42

26 Clarence R, Tabulam, NSW GAL, KLU PU99-43

27 Clouds Ck, Nymboida, NSW GAL PU02-16

28 Orara R, Karangi, NSW GAL PU02-15

29 Corindi R, upper Corindi, NSW GAL PU02-15

30 Hastings R, Wauchope, NSW GAL PU99-38

31 Booral Ck, Stroud, NSW GAL F-FISHY3

32 Nepean R, Wallaca, NSW GAL AMS-36086

33 Georges R, Liverpool, NSW GAL IW94-50

Murray-Darling Province

34 Severn R, Glen Aplin, QLD KLU, MD PU99-49

35 Maranoa R, Mitchell, QLD KLU PU99-60

36 Warrego R, Cunnamulla, QLD LAK, MID PU99-63

37 Paroo R, Yalamurra, QLD KLU PU99-61

38 Dunns Swamp, Rhylstone, NSW KLU, MD PU99-70

39 Turon R, Hill End, NSW KLU PU02-54

40 Bogan R, Nyngan, NSW MD F-FISH21

41 Murray R, Cohuna, NSW KLU PU94-37-2

42 Black Swamp, Cohuna, VIC KLU, LAK, MD, MID PU99-34

43 Salt Ck, Berri, SA MID F-FISHADD7

44 Bremer R, Lake Alexandrina, SA MD IW94-26

Central Australian Province

45 Bulloo R, Quilpie, QLD KLU, MID PU99-62

46 Barcoo R, Tambo, QLD LAK, KLU, MID 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.



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 relation-

ships 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 popula-

tions 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 isola-

tion, 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 accord-

ance with Table 1 and Fig. 2. Letters indicate clades discussed in

more detail in the text.



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 iden-

tified 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 prob-

ably 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 High-

lands, 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



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

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.

(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.



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 communica-

tion), 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 electrophor-

esis (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 popula-

tions. 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 relat-

ive 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



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 Boondo-

oma 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 bewil-

dering 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

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.

Table 2 Comparison of genetic divergences across adjacent drainage divides.

Across drainage divide comparison

Species in both

drainages

Divergences

(p-distances) Comments

Drainage divide

length (km)

Cooper Creek (CAP) vs. Herbert (NP) MID* 0.012 Connection likely occurred

via upper Burdekin River

846 (Burdekin)

Cooper Creek (CAP) vs. Fitzroy (EP) MID, KLU 0.026, 0.041 49

Cooper Creek (CAP) vs. Murray (MDP) MID, KLU* 0.030, 0.015 na

Cooper Creek (CAP) vs. Bulloo (CAP) MID, KLU* 0.027, 0.003 918

Murray (MDP) vs. Bulloo (CAP) MID, KLU* 0.012, 0.014 2398�Paroo (MDP) vs. Bulloo (CAP) KLU� 0.005

Murray (MDP) vs. Fitzroy

(minus Dawson) (EP)

MID, KLU* 0.017, 0.038 922

Murray (MDP) vs. Dawson

(Fitzroy) (EP)

MID* 0.020 922

Murray (MDP) vs. Burnett (EP) GAL/MD*, MID, KLU 0.014, 0.044, 0.034 294

Murray (MDP) vs. Burnett2 (EP) GAL/MD�, KLU� 0.009, 0.002 Movements were in

opposite directions

294

Murray (MDP) vs. Brisbane (EP) GAL/MD, MID, KLU 0.028, 0.048, 0.035 304

Murray (MDP) vs. Clarence (EP) GAL/MD, KLU 0.028, 0.038 384

Murray (MDP) vs. Clarence2 (EP) GAL/MD� 0.013 384

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.

�Due to aridity, only 1272 km of this divide separates watercourses that contain fishes (Wager & Unmack, 2000).



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 Murray-

Darling/H. galii there is a close relationship (p-dis-

tance 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 popula-

tions in Burnett River (Three Moon Creek, 12) and Baffle

Creek (9), and Oyster Creek (10; p-distance ¼ 0.007). Hypse-

leotris galii populations at Mary River (Cunningham Creek,



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-dis-

tance 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 differ-

entiated (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 contin-

ental 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

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 Late-

Pleistocene 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 corres-

ponds 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 sea-

level 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

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 GAL (26, 27, 28 vs. 21), KLU (26 vs. 21) 0.019, 0.009

Brisbane vs. Mary GAL (21 vs. 16, 19), MID* (21 vs. 17), KLU (21 vs. 17, 18) 0.026, 0.050, 0.012

Mary vs. Burnett GAL (16, 19 vs. 12), MID* (17 vs. 12), KLU (17, 18 vs. 19) 0.027, 0.026, 0.007

Burnett vs. Kolan/Baffle MID (12 vs. 11), KLU (12 vs. 9, 10) 0.042, 0.006

Kolan/Baffle vs. Fitzroy MID (11 vs. 7, 8), KLU (9, 10 vs. 6) 0.016, 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.



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 phylogeo-

graphical 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 relation-

ship 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 connec-

tions between rivers, does not appear to be detectable in the

broader phylogeography of Hypseleotris species. Coastal phy-

logenetic 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 popula-

tions, 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, distri-

butional 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 under-

graduate 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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Comparative phylogeography of five sympatric Hypseleotris species