Phylogenetic Relationships of Australian Members of the Family Percichthyidae Inferred from Mitochondrial 12S rRNA Sequence Data

Molecular Phylogenetics and Evolution Vol. 18, No. 3, March, pp. 335–347, 2001 doi:10.1006/mpev.2000.0871, available online at http://www.idealibrary.com on

Phylogenetic Relationships of Australian Members of the Family Percichthyidae Inferred from Mitochondrial 12S rRNA Sequence Data Dean R. Jerry,* Martin S. Elphinstone,† and Peter R. Baverstock† *CSIRO Livestock Industries, Armidale, New South Wales 2350, Australia; and †Animal Conservation Genetics, Southern Cross University, Lismore, New South Wales 2380, Australia Received May 18, 2000; revised August 14, 2000; published online February 7, 2001

Phylogenetic relationships among 20 Australian species of the family Percichthyidae were investigated from sequence data of two portions of the mitochondrial 12S rRNA gene. The molecular data indicate that Australian genera within this family cluster into three distinct clades. The first clade is composed of some species currently ascribed to the genus Macquaria, along with Nannatherina, Nannoperca, and Bostockia, the second of Maccullochella and two catadromous Macquaria species, and the third of Gadopsis. However, the positioning of Gadopsis within this family was unresolved. Monophyly within each genus was well supported, except for Macquaria, which is clearly polyphyletic. The molecular data were used to examine two hypotheses of Australian percichthyid evolution and favor a freshwater origin for the family. © 2001 Academic Press

Key Words: Percichthyidae; mtDNA; phylogenetics; inland sea model; punctuated marine invasion model.

INTRODUCTION Classification of fishes to the family level within the suborder Percoidei has long been enigmatic for fish taxonomists. Of particular concern has been that members of this suborder generally lack specializations which would allow them to be placed into familial groups that reflect meaningful ancestral interrelationships (Gosline, 1966). Included within this taxonomic conundrum that is the Percoidei is a small assemblage of freshwater and brackish perciform genera from Australia. Consensus on where to place this group of fishes within the suborder has been varied, with genera shuffled among the basal percoid families of the Kuhlidae (Regan, 1940; Norman, 1957), the Maccullochellidae (Katayama, 1960), and the Serranidae (Greenwood et al., 1966). However, in many cases these families merely represent repositories for genera that could not adequately be classified elsewhere.

Gosline (1966) attempted to unravel the disorder within the Percoidei by provisionally placing the Australian perciforms into a new family—Percichthyidae. This family was defined on the basis of a number of generalized basal percoid character states and comprised around 25 genera of freshwater, estuarine, and oceanic species from temperate and subtropical regions of the world. However, when defining the Percichthyidae, Gosline (1966) made no mention of the ancestral affinities of its constituent genera and acknowledged that it was likely to be polyphyletic. With this in mind, Johnson (1984) used osteological and anatomical characteristics to restrict the Percichthyidae to include only species inhabiting brackish and freshwaters of Australia and South America. Furthermore, he stated that the genera within his revised Percichthyidae were monophyletic, due to the possession of a series of nested synapomorphies, namely, scales with simple, slightly amputated, needle-like ctenii, enlarged sensory pores on the dentary, and a separate inner division of adductor mandibulae section A 1. Some authors argue, however, that these synapomorphies are not firm enough evidence for monophyly (Nelson, 1994). Accordingly, Johnson’s (1984) revision of the family Percichthyidae is still not universally accepted among fish taxonomists. The family Percichthyidae comprises approximately 22 species and two subspecies from eight genera (Johnson, 1984). All but two are endemic to the Australian continent. It is considered that most percichthyid genera presumably represent a secondary radiation from marine protopercoid ancestors (Darlington, 1965; Whitley, 1959; McDowall, 1981; Allen, 1989); fossil records for the existence of percichthyids in Australia are known from Eocene, mid-Oligocene, and Miocene deposits (Hills, 1934, 1943, 1946; Estes, 1984; Allen, 1989). The Australian component of the Percichthyidae includes 3 species of freshwater cod (genus Maccullochella), 5 species of brackish and freshwater perches (genus Macquaria), 2 species of river blackfishes (genus Gadopsis), 6 species of pygmy perches (genera Nanno-

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perca and Nannatherina), and the monotypic nightfish (Bostockia porosa). In addition to the above taxa, there are several subspecies and 3 further as yet undescribed species: a cryptic species of Macquaria ambigua (Musyl and Keenan, 1992), a cryptic species of Macquaria australasica (Duffy, 1986), and a newly discovered percichthyid from the Bloomfield River, Queensland (S. Brooks and B. Pusey, unpublished). Despite the endemism of Australian percichthyids, and the likelihood that they may comprise a monophyletic assemblage, little is actually known of the genealogical connections among genera. The only attempt to explore generic relationships among taxa was that of MacDonald (1978), where morphological and protein information were used to determine relationships among Maccullochella, Macquaria, Percalates, and Plectroplites. Based on this data, MacDonald (1978) synonymized Percalates and Plectroplites with Macquaria and postulated that Maccullochella and Macquaria probably represented two separate invasions into freshwater by marine ancestors that were already distinct. In addition to this work, however, there has been no subsequent attempt to resolve phylogenetic relationships among Australian percichthyids, particularly among the additional genera included by Johnson (1984). In this study, two contiguous portions of the mitochondrial 12S rRNA gene were sequenced to assess the phylogenetic relationships among Australian members of the family Percichthyidae. The molecular data were used to evaluate two models of evolution of the Australian fish fauna. This study is the first to present a phylogeny of all the Australian percichthyids based on molecular data. MATERIALS AND METHODS Specimens Examined Phylogenetic relationships were examined among all extant Australian percichthyids, except for the cryptic species of Macquaria australasica. The Australian percichthyids examined were Gadopsis bispinosus (Stony Creek, VIC), Gadopsis marmoratus (Stony Creek, VIC), Gadopsis marmoratus (Little Forester River, TAS), Maccullochella peelii peelii (Murrumbidgee River, NSW), Maccullochella peelii mariensis (Mary River, QLD), Maccullochella ikei (Nymboida River, NSW), Maccullochella macquariensis (Murrumbidgee River, NSW), Bostockia porosa (WA), Nannatherina balstoni (WA), Nannoperca vittata (Donnelly River, WA), Nannoperca oxleyana (Fraser Island, QLD), Nannoperca obscura (Glenelg River, VIC), Nannoperca australis (Tookayerta Creek, SA), Nannoperca variegata (Glenelg River, VIC), Macquaria ambigua sp. (undescribed cryptic species of M. ambigua; Coopers Creek, SA), Macquaria ambigua (Murrumbidgee River, NSW),

Macquaria australasica (Dartmouth Dam, VIC), Macquaria novemaculeata (Clarence River, NSW), Macquaria colonorum (Hopkins River, VIC), and Bloomfield River cod (Bloomfield River, QLD), a newly discovered but as yet undescribed species. Where possible, two or more specimens of each species were sequenced. When Gosline (1966) originally split the Percichthyidae from the Serranidae, he included Morone saxatilis (along with others) in his “estuarine and freshwater percichthyids” based on the absence of what he termed characteristic evolutionary advances present in the latter group. Later, Johnson (1984) placed the genus Morone into a new percoid family, Moronidae. Because of their supposed similarities, Morone saxatilis (Colorado River, Arizona, USA) and a Serranidae species, Epinephelus tauvina (Clarence River, NSW), were considered appropriate outgroup taxa with which to resolve phylogenetic relationships among the Australian Percichthyidae. DNA Extraction Depending on the sample, either caudal fin-clips or muscle tissue were digested in 500 ␮l extraction buffer (100 mM NaCl, 50 mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 0.5% SDS, 0.2 mg Proteinase K) at 55°C for 3 h. DNA purification was by standard phenol:chloroform extraction (Sambrook et al., 1989), followed by ethanol precipitation in 0.1 vol of 3 M sodium acetate and 2 vol of absolute ethanol at ⫺70°C for 2 h. Samples were spun at 10,500 rpm for 5 min; the DNA pellet was washed in 70% ethanol and then resuspended in 500 ␮l TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). DNA Amplification and Sequencing Two contiguous segments of the 12S rRNA mitochondrial gene (approximately 400 bp each) were amplified by PCR using the 12S a/b and 12S c/d primers described in Fuller et al. (1998). Initial amplification was from gDNA in a total reaction volume of 25 ␮l containing 100 ␮M dNTPs, 100 nM each primer, 0.5 units of Taq polymerase (Boehringer Mannheim), Taq 10⫻ enzyme buffer (Boehringer Mannheim), and 100 ng of template DNA. Amplification was performed in a MJ Research Inc. Minicycler and consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min. Band lengths of PCR products were visualized in 1% agarose stained with ethidium bromide. The PCR products were purified using the QIAquick PCR purification kit (Qiagen Gmbh) before elution into 30 ␮l of distilled water. Purified DNA was used for direct cycle sequencing with dye-labeled terminators (Applied Biosystems) on an ABI 373A automated sequencer (Applied Biosystems). Sequences were obtained from both strands of the two gene segments and in most cases were sequenced at least twice for verifi-

PHYLOGENETIC RELATIONSHIPS OF AUSTRALIAN PERCICHTHYIDAE

cation and to resolve any ambiguous positions. DNA sequences have been deposited into Genbank (Accession Nos. AF294443–AF294459 and AF295045– AF295062. Secondary Structure-Based Alignment Preliminary multiple alignment of sequences was achieved using Clustal-W (Thompson et al., 1994) with default gap penalties. The resultant alignment was then adjusted by eye, with special attention paid to aligning loops (nonpairing regions) and stems (basepairing regions) according to the proposed 12S rRNA secondary structure models for carp (Cyprinus carpio) (van de Peer et al., 1994). Here, Watson–Crick bonds and comparatively stable purine–pyrimidine bonds were considered indicative of base-pairing. Phylogenetic Analysis Pairwise comparisons, statistical information, and phylogenetic trees were generated using Paup* 4.0d65 (provided by D. L. Swofford) and MEGA (ver 1.01; Kumar et al., 1993). Absolute numbers of transitions and transversions were calculated and plotted against genetic distance to determine whether there was any evidence for saturation of these two character states in the data. The phylogenetic information content of the data set was evaluated by the skewness of 10,000 randomly generated tree distributions and the g 1 statistic (Hillis and Huelsenbeck, 1992). Phylogenetic analyses consisted of both maximumlikelihood and neighbor-joining approaches. Maximum-likelihood was performed using the PUZZLE option in Paup* 4.0d65, constrained by the transition/ transversion ratio determined in MEGA, and using approximate likelihood calculations. The number of quartet puzzling steps was set at 10,000. Neighborjoining was likewise performed in Paup* 4.0d65, with support for branches evaluated by 10,000 bootstrap replications. In both analyses insertion/deletion events were ignored and considered missing data instead of a fifth character state. Treating the data in this way circumvented the problem of whether or not insertions/ deletions longer than 1 bp represented single or multiple substitution events. Jukes and Cantor’s (1969) model was used as the pattern of sequence evolution. RESULTS Sequence Variation Little adjustment was needed to compensate for differences in secondary structure alignment, with all corresponding stems and loops in carp mitochondrial 12S rRNA confidently found. This demonstrates that secondary structure of the rRNA of this gene is not only conservative among percichthyids, but also among percichthyids and carp.

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Aligned unambiguous sequence data from the two contiguous fragments of the 12S rRNA gene were combined to give a total of 566 bp sites for phylogenetic analyses (Fig. 1). Of these, 188 were variable. The total nucleotide composition was A ⫽ 31.5%, T ⫽ 21.4%, C ⫽ 24.5%, and G ⫽ 22.7%. This indicated that the Lstrand base composition for the region sequenced in percichthyids is adenine-rich and guanine-poor. Sequence divergences (uncorrected) within the Percichthyidae ranged from 0.0% for the two sister species of M. ambigua, as well as for M. ikei and M. peelii mariensis, to 15.5% between these later two Maccullochella species and G. marmoratus (forest) (average divergence between all Percichthyidae taxa ⫽ 9.5 ⫾ 3.7%) SE. The greatest divergence observed between two species within a genus was that between M. novemaculeata and M. ambigua (10.3%) (average species divergence within genera ⫽ 4.0%). Ingroup and outgroup taxa sequence divergences ranged between 11.6 and 18.8% (average 14.6 ⫾ 1.7%) SE (Table 1). Estimated g 1 values for analyzed sequences were skewed proportionately to the left, indicating that the data were more structured than random data (g 1 ⫽ ⫺0.91, P ⬍ 0.01). Likewise, pairwise comparisons of transitions and transversions versus genetic distance did not appear to plateau (Fig. 2). This demonstrates that the data provided useful information for phylogenetic analyses across the full spectrum of taxonomic diversity represented. The average transition/transversion ratio between taxa was 2.44. Phylogenetic Analyses Neighbor-joining and maximum-likelihood phylogenetic analyses produced trees with similar topologies (Figs. 3 and 4), with five higher-level clades evident. These five major clades form what appears to represent three major evolutionary lineages. The first lineage has Macquaria as the sister genus to Bostockia, Nannatherina, and Nannoperca, and the second lineage has Maccullochella as the sister genus to Macquaria novemaculeata and Macquaria colonorum. However, due to a prolonged branch length between these later two genera (0.045), the M. novemaculeata clade may itself represent a distinct evolutionary radiation within the Percichthyidae. The final lineage is Gadopsis, which formed essentially an unresolved trichotomy with the other clades. Consequently, phylogenetic positioning of this problematic genus within the Percichthyidae remains unresolved. Overall, monophyly of species groups within the five clades is strongly supported by neighbor-joining and maximum-likelihood methods. For example, there is 100% bootstrap support for monophyly of species within Maccullochella and Gadopsis, whereas for the pygmy perches, consisting of the five species of Nannoperca and Nannatherina, there is a minimum of 68%. Interestingly, within Gadopsis, the two specimens

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FIG. 1. DNA sequence data for two segments of mitochondrial 12S rRNA for 20 taxa from the family Percichthyidae and 2 outgroup taxa. Note: Dots denote identity with the first sequence, a dash denotes an alignment gap, and n denotes missing data.

PHYLOGENETIC RELATIONSHIPS OF AUSTRALIAN PERCICHTHYIDAE

FIG. 1—Continued

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FIG. 1—Continued

FIG. 1—Continued

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TABLE 1 Number of Nucleotide Substitutions (above Diagonal) and Uncorrected Percentage Sequence Divergences (below Diagonal) between 20 Taxa from the Family Percichthyidae and 2 Outgroup Taxa for 566 bp of Mitochondrial 12S rRNA G.b G.ms G.mf B.p G.b G.ms G.mf B.p N.b E.v N.a N.ob N.o N.v M.a M.as M.au Bl M.s E.t M.c M.n M.pp M.i M.pm M.m

26 4.8 2.6 13.4 12.6 12.3 11.9 12.3 13.0 13.5 12.6 12.4 11.2 12.7 18.0 15.3 14.2 14.8 14.8 15.0 15.0 13.7

4.6 14.2 13.7 13.4 13.2 13.6 14.5 13.4 14.3 14.1 12.9 14.0 18.8 18.0 13.6 14.3 14.5 14.7 14.7 14.8

14 25 13.4 12.2 12.0 11.7 12.1 12.8 13.3 12.6 12.4 11.0 12.6 17.8 15.9 13.9 14.8 15.3 15.5 15.5 14.6

73 77 73 5.1 6.6 6.8 6.2 7.7 8.0 7.7 7.8 7.0 8.8 15.6 13.7 8.8 9.4 10.2 10.4 10.4 9.9

N.b

E.v N.A N.ob N.o

N.v M.a M.as M.au

69 75 67 28

67 73 66 36 21

74 73 73 44 26 24 19 21 29

3.8 3.1 3.5 4.9 4.7 5.7 5.7 5.7 7.3 13.9 13.4 8.4 9.4 10.8 10.9 10.9 10.6

1.6 1.8 2.7 4.4 6.6 6.6 6.2 7.0 13.0 11.6 8.8 9.4 10.8 10.9 10.9 11.7

65 72 64 37 17 9 0.7 2.7 3.5 6.9 7.0 6.2 7.3 12.6 11.8 9.5 9.9 10.8 10.9 10.9 11.5

67 74 66 34 19 10 4 2.6 3.8 7.1 7.2 6.4 7.7 13.2 12.4 9.5 10.0 11.1 11.3 11.3 11.1

71 79 70 42 27 15 15 14 5.3 7.7 7.8 7.1 7.9 13.9 12.7 10.1 10.1 12.4 12.6 12.6 12.0

7.7 7.8 7.1 7.5 13.7 14.2 9.5 9.6 11.7 11.8 11.8 12.4

69 78 69 42 31 36 38 39 42 42 0.0 4.4 5.5 14.3 13.2 9.1 10.3 11.5 11.7 11.7 10.9

67 76 67 42 31 36 38 39 42 42 0 4.4 5.6 14.4 13.2 9.3 10.4 11.6 11.8 11.8 11.1

61 70 60 38 31 34 34 35 39 39 24 24 3.8 13.6 12.4 9.0 9.4 9.9 10.1 10.1 9.7

Bl 69 76 69 48 40 38 40 42 43 41 30 30 21 14.9 14.0 9.2 10.2 11.0 11.2 11.2 11.2

M.s 98 102 97 85 76 71 69 72 76 75 78 78 74 81 14.6 14.3 15.7 15.4 15.6 15.6 15.8

E.t

M.c M.n M.pp M.i M.pm M.m

83 97 86 74 73 63 64 67 69 77 72 71 67 76 79

77 74 76 48 46 48 52 52 55 52 50 50 49 50 78 77

80 77 80 51 51 51 54 54 55 52 56 56 51 55 85 81 17

14.2 15.1 3.1 15.5 9.9 11.1 15.7 10.1 11.2 15.7 10.1 11.2 15.1 9.7 11.1

81 79 84 56 59 59 59 61 68 64 63 63 54 60 84 84 54 60 0.2 0.2 4.0

82 80 85 57 60 60 60 62 69 65 64 64 55 61 85 85 55 61 1 0.0 4.2

82 80 85 57 60 60 60 62 69 65 64 64 55 61 85 85 55 61 1 0

75 81 80 54 58 64 63 61 66 68 60 60 53 61 86 82 53 60 22 23 23

4.2

Note. G.b, Gadopsis bispinosus; G.ms, Gadopsis marmoratus (stony); G.mf, Gadopsis marmoratus (forest); B.p, Bostockia porosa; N.b, Nannatherina balstoni; E.v, Nannoperca vittata; N.a, Nannoperca australis; N.ob, Nannoperca obscura; N.o, Nannoperca oxleyana; N.v, Nannoperca variegata; M.a, Macquaria ambigua; M.as, Macquaria ambigua sp.; M.au, Macquaria australasica; Bl, Bloomfield River cod; M.s, Morone saxatilis; E.t., Epinephelus tauvina; M.c, Macquaria colonorum; M.n, Macquaria novemaculeata; M.pp, Maccullochella peelii peelii; M.i, Maccullochella ikei; M.pm, Maccullochella peelii mariensis; M.m, Maccullochella macquariensis.

from different populations of G. marmoratus seem not to be monophyletic, with the specimen from Little Forester River (forest) a sister taxon to G. bispinosus. The level of sequence divergence evident between these two specimens is equal to or greater than that exhibited among a few of the “good” biological species examined, lending support to suggestions that G. marmoratus comprises a northern and a southern species (Sanger, 1986). A further discrepancy between our data and current taxonomy is that species within the genus Macquaria are polyphyletic due to exclusion of M. novemaculeata

FIG. 2. Absolute number of transitions (■) and transversions (F) versus percentage divergence (uncorrected) for all pairwise comparisons of the mitochondrial 12S rRNA gene in 20 Australian percichthyid and 2 outgroup taxa.

and M. colonorum from the clade containing M. australasica and M. ambigua. Despite this inconsistency, however, the remaining two sister taxa of M. ambigua and M. australasica, along with the recently discovered Bloomfield cod, are monophyletic. DISCUSSION Phylogeny of the Percichthyidae Phylogenetic analyses indicate that the 20 examined taxa separate into three distinct evolutionary radiations. The first and largest radiation comprises the freshwater species of Macquaria, along with Nannoperca, Nannatherina, and Bostockia. Within this radiation, the larger perches form a sister group to the pygmy perches, which appear the most derived. The second radiation comprises Maccullochella, Macquaria novemaculeata, and Macquaria colonorum. The inclusion of these two species of Macquaria in this radiation was unexpected based on present taxonomy. Finally, the third radiation is that of Gadopsis, which forms an unresolved trichotomy with that of the other two major evolutionary branches. This finding that genera cluster into distinct clades agrees with the only other study that has examined

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FIG. 3. Neighbor-joining tree of 20 percichthyid and 2 outgroup taxa produced using Jukes and Canter’s (1969) model of sequence evolution. Numbers on branches indicate the percentage of bootstrapped trees (10,000 replicates) and scale below tree indicates branch length.

evolutionary relationships among the Australian Percichthyidae. MacDonald (1978), using both isozyme electrophoresis and comparative examination of morphological characters, demonstrated that species within the Maccullochella varied from those he synonymized with Macquaria at two-thirds of body characters and over 60% of protein loci examined. Such large differences led him to surmise that these two genera represented two distinct evolutionary lineages within the family and furthermore that the degree of variation observed may be indicative of separation at the familial level. Although mtDNA information alone cannot effectively establish the boundaries of familial groups, our data indicate that Maccullochella—along with M. novemaculeata, M. colonorum, and Gadopsis—are phylogenetically distinct from the remaining percichthyid members. Therefore, when viewed in conjunction with the significant differences demonstrated by MacDonald’s (1978) morphological and isozyme data set, there is an argument for the family Percichthyidae to be further refined to consist of several subfamilies to fully reflect the distinct evolutionary lineages identified. Generic Relationships Not only is the present investigation the first to scrutinize evolutionary relationships among Austra-

lian Percichthyidae, but it also is the first to examine species interrelationships within each genus. Genealogies among species within each genus are treated separately below. Macquaria. Morphological and isozyme information led MacDonald (1978) to synonymize Percalates (M. novemaculeata and M. colonorum), Plectroplites (M. ambigua), and Macquaria (M. australasica) into a single genus (Macquaria Cuvier and Valenciennes, 1830 being the oldest name available). MacDonald (1978) considered Macquaria to be a closely related monophyletic assemblage. Mitochondrial 12S rDNA sequence data, however, disagrees with MacDonald’s supposition and indicates that M. novemaculeata and M. colonorum are remotely related to M. ambigua and M. australasica. Two explanations may account for this discrepancy between the morphological and isozyme data of MacDonald (1978) and the DNA sequence data generated in the present study. The first possibility is that M. novemaculeata and M. colonorum really are unrelated to other species presently within this genus. This would signify that similarities in morphology and isozymes are the consequence of convergence or retention of ancestral character states. Perhaps, MacDonald (1978) may have erred as others have when classifying basal percoids in that many of the characters used to

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FIG. 4. Maximum-likelihood tree of 20 percichthyid and 2 outgroup taxa produced using Jukes and Canter’s (1969) model of sequence evolution. Quartet support values for each internal branch based on 10,000 puzzling steps are shown as percentages above the branches.

define phylogenetic relationships were “generalised” and therefore susceptible to convergence. This may be particularly true of isozyme data, which becomes increasingly insensitive to true levels of protein variation at or above the generic level (Nei and Chakraborty, 1973; Avise, 1989). The other possible explanation for polyphyly of Macquaria is that the gene trees that we have generated do not mirror true phylogenetic relationships among taxa. This is because gene trees derived from mtDNA sequence data may not always be representative of species trees (Brower et al., 1996). We believe, however, that this is not the case for our data, as the level of sequence divergence observed among M. novemaculeata, M. colonorum, and other Macquaria is around 10%. Consequently, evolution of this gene would be expected to have had enough time to come into equilibrium with the true species phylogeny. To resolve the possible taxonomic confusion within this genus, additional sequence data is desirable, particularly from nuclear genes. Reinstatement of Percalates to characterize M. novemaculeata and M. colonorum may be warranted if the relationships highlighted by 12S rRNA are collaborated by nuclear data. Nannoperca, Nannatherina, and Bostockia. Classification of both pygmy perches and B. porosa to the family level has long posed problems for taxonomists. Over the years, they have been placed in their own family, the Nannopercidae, the Kuhliidae, or, more recently, the Percichthyidae (Regan, 1940; Norman, 1957; Johnson, 1984; Kuiter et al., 1996). Phylogenetic

analyses, however, demonstrate that the pygmy perches are monophyletic with Macquaria, with B. porosa a sister species to Nannatherina. Consequently, Johnson (1984) appears to be correct in placing both pygmy perches and Bostockia into the Percichthyidae. Johnson (1984) also believed that Nannoperca, Nannatherina, and Bostockia shared a number of reductive morphological specializations and that they were the three most derived percichthyid genera. Our data is somewhat consistent with this hypothesis, with taxa from these three genera forming the most derived branches within the M. ambigua clade. Until recently, three genera have been commonly accepted as pygmy perches (i.e., Nannatherina, Nannoperca, and Edelia), with the genus Edelia comprising E. obscura and E. vittata (i.e., N. obscura and N. vitatta in the present study). Kuiter et al. (1996) suggest, however, that the anatomical differences used to differentiate Edelia and Nannoperca are trivial. Accordingly, they submitted that the genus Edelia should be incorporated with Nannoperca. Based on 12S rRNA, we likewise found no justification for maintaining recognition of the genus Edelia, with N. obscura (viz. E. obscura) and N. vittata (viz. E. vittata) clearly not monophyletic and unmistakably sister taxa to N. australis, N. oxleyana, and N. variegata. Consequently, usage of Edelia should be dropped from taxonomic nomenclature. Maccullochella. There is strong bootstrap support for the monophyly of the Maccullochella. However, within this clade, phylogenetic analyses suggest that

PHYLOGENETIC RELATIONSHIPS OF AUSTRALIAN PERCICHTHYIDAE

the two subspecies of M. peelii are not sister taxa, in that M. peelii mariensis is monophyletic to M. ikei, not to M. peelii peelii; this discrepancy, however, is tenuous because it is based on only one unambiguous character change (Table 1). Additional data, however, has been sought from 16S rRNA and cytochrome b with similarly low levels of sequence divergence between the three taxa evident (D. R. Jerry, unpublished). In fact, M. peelii mariensis and M. ikei are indistinguishable at over 1200 bp sampled from three mitochondrial genes (i.e., 12S rRNA, 16S rRNA, and cytochrome b) and are both different from M. peelii peelii by the same 2 bp. The limited number of character differences between M. ikei and the two subspecies of M. peelii indicate that radiation in this group is relatively recent. This view is supported by Rowland (1993), who estimated the time of divergence of these three taxa from isozyme data as being between 1.7 and 0.8 million years ago. Gadopsis. Evolutionary affinities of Gadopsis have particularly been enigmatic. Typically, they have been treated as a monotypic family in the suborder Percoidei (Greenwood et al., 1966; Nelson, 1994), but have been variously placed into the suborders Blennioidei (Richardson, 1848), Gadoidei (Gu¨nther, 1862), Ophidioidei (Gosline, 1968), and Trachinoidei (Rosen and Patterson, 1969). They have even been assigned to their own order, Gadopsiformes (Scott et al., 1974). More recently, Johnson (1984) included them in the family Percichthyidae, based on a number of larval features that they share with other Australian percichthyids, and suggested they were closely related to Bostockia and the pygmy perches. We were unable to adequately resolve the higher-level taxonomic positioning of Gadopsis within the Percichthyidae. However, it is clear from our data that Gadopsis is not a sister genus to Bostockia, Nannatherina, or Nannoperca, as maintained by Johnson (1984). Indeed, Johnson’s (1984) whole argument for the inclusion of Gadopsis in the Percichthyidae was founded on a number of anatomical synapomorphies that this genus shared with Bostockia and the pygmy perches. Many authors have argued, however, that the synapomorphies that Johnson (1984) based this judgment on were by themselves not firm enough evidence for inclusion (Sanger, 1986; Nelson, 1994). If this is true, then it is conceivable that we could not adequately resolve the taxonomic positioning of Gadopsis within the family because the characters used by Johnson (1984) to argue for the inclusion of this genus were the consequence of convergence and not the consequence of a common evolutionary linkage. Evolution and Biogeography of Australian Percichthyids The Australian continental landmass has been isolated since it split fully from Antarctica around 38 million years ago (Veevers, 1984). Furthermore, the

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FIG. 5. Expected phylogenetic relationships of extant Australian freshwater fish genera under (a) the inland sea and (b) punctuated marine invasion models. FW, freshwater genera; M, marine genera.

interior of the continent was inundated for 10 –15 million years during the mid to late Cretaceous by vast marine transgressions (Brunnschweiler, 1984; Veevers, 1984). As a result, the Australian freshwater fish fauna has a high degree of endemism, with a conspicuous absence of primary freshwater fish groups, such as the cyprinids (minnows) and nandids (leaf fishes) (Allen, 1989). The absence of these fish groups suggests that all but a few of Australia’s endemic freshwater fishes are of secondary marine derivation and that they evolved after the fragmentation of Gondwanaland (de Beaufort, 1951; Whitley, 1959; McDowall, 1981; Harris, 1984). Several theories have been offered to explain the evolution of the Australian fish fauna (McDowall, 1981; Crowley and Ivantsoff, 1989; Musyl, 1990; Musyl and Keenan, 1992; Rowland, 1993). Generally, these theories fall into two distinct models—the inland sea model and the punctuated marine invasion model. The inland sea model decrees that as the ocean retreated, marine ancestors became trapped in the resultant landlocked sea. During the ensuing eras, the sea gradually became fresh and its sequestered fish evolved and radiated into the freshwater fauna of today (Crowley and Ivantsoff, 1989). Under this model of evolution, freshwater genera within a family would be more directly related to other freshwater genera than to those of marine genera (Fig. 5a). In the punctuated marine invasion model, however, the evolution and diversity of Australian freshwater fishes are explained as a corollary of multiple, separate invasions by marine ancestors. Fish families, therefore, would be composed of freshwater genera with evolutionary linkages more closely allied to marine fishes than to those of other freshwater genera (Fig. 5b). Although it is impossible with two outgroup taxa to fully resolve this debate, in the absence of any known marine relatives it appears that the Australian Percichthyidae (perhaps with the exception of Gadopsis) are a monophyletic unit, as suggested by Johnson (1984). Therefore, present phy-

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logenies support the inland sea model as the pattern of evolution for this family. However, the future inclusion of several marine taxa in our phylogenetic data set warrants further attention to confirm that there are no marine linkages between the three major clades. Likely candidate species should come from those marine families excluded from the Percichthyidae by Johnson (1984) (i.e., Serranidae, Moronidae, Acropomatidae, etc). Finally, in recent years putative cryptic species of golden perch (Musyl and Keenan, 1992), Macquarie perch (Dufty, 1986), freshwater cod (Rowland, 1993), and river blackfish (Ovenden et al., 1988; Sanger, 1986) have been identified using isozyme electrophoresis and comparative morphometrics. Although mtDNA is not a definitive tool for identifying species complexes, it can provide valuable information on antecedent associations and relative times of divergence between two species. If the morphological and isozyme differences observed between these cryptic species and their taxonomically described sister taxa are truly indicative of species-level splits—and not just geographical variation—then levels of sequence divergence at the mitochondrial 12S rRNA gene indicates that there has been several recent speciation events within the Australian Percichthyidae in the last few million years. These recent speciation events have been explained as the consequence of biogeographic vicariant events, such as stream capture (Rowland, 1993), increasing aridity (Musyl and Keenan, 1992), and/or sea level rises (Ovenden et al., 1988). ACKNOWLEDGMENTS We are extremely grateful to all colleagues who provided sample tissues. In particular, we thank Chris Burridge, Graham Thompson, Mark Adams, Peter Unmack, Steve Brooks, Bob Simpson, Rob Wagner, and Rob Hobson.

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