Genetic and morphological evidence for reproductive isolation between sympatric populations of Galaxias (Teleostei: Galaxiidae) in South Island, New Zealand

Biological Journal of the Linnean Society (2001), 73: 287–298. With 5 figures doi:10.1006/bijl.2001.0541, available online at http://www.idealibrary.com on

Genetic and morphological evidence for reproductive isolation between sympatric populations of Galaxias (Teleostei: Galaxiidae) in South Island, New Zealand JONATHAN M. WATERS∗, YUZINE B. ESA† and GRAHAM P. WALLIS 1

Department of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand

Received 30 November 2000; accepted for publication 12 March 2001

New Zealand’s South Island houses a flock of closely related stream-resident fish taxa (Galaxias vulgaris sensu lato), including a number of species recently described on the basis of subtle morphological differences. The taxonomic status of some members of the species complex remains uncertain. This study examines the degree of reproductive isolation between recently recognized morphotypes from Southland (G. ‘southern’, flatheads; G. gollumoides, roundheads) which co-occur in Bushy Creek, a tributary of the Mataura R. Although these morphotypes are broadly sympatric in Southland and Stewart Island, Bushy Creek is their only documented zone of contact. Molecular (microsatellite, isozyme and mtDNA markers) and morphological analyses of 139 fish samples across a 500-m transect (seven stations) reveal a cline from predominantly G. ‘southern’ (N=85) to predominantly G. gollumoides (N=54), corresponding with a gradual increase in stream gradient. Multivariate analyses of genotypic and morphological data independently reveal distinct clusters that are completely congruent with mtDNA type, suggesting an absence of mtDNA introgression. Our data support the separate species status of G. ‘southern’ and G. gollumoides under both biological and phylogenetic species concepts. We suggest that the speciation of these taxa occurred in allopatry through independent losses of diadromy, with sympatry resulting from secondary contact.  2001 The Linnean Society of London

ADDITIONAL KEY WORDS: hybridization – microsatellites – FCA – morphology – population genetics – control region – phylogeny – speciation.

valley lakes (Sturmbauer & Meyer, 1992) and smelt in North America (Taylor & Bentzen, 1993). Galaxias is the most speciose genus of freshwater fish in New Zealand, comprising at least 17 species. Although most New Zealand Galaxias are streamresident, some species have a migratory marine larval stage (diadromy; McDowall, 1990). The loss of diadromy is thought to be a major initiator of speciation in galaxiid fishes (Ovenden & White, 1990; Waters, Lo´pez & Wallis, 2000). Members of the G. vulgaris (sensu lato) complex are morphologically and genetically similar to diadromous G. brevipinnis (McDowall, 1990; Allibone & Wallis, 1993). Phylogenetic analysis suggests that the radiation of the G. vulgaris complex was initiated by three independent losses of diadromy from a brevipinnis-like ancestor (Waters & Wallis, 2001b). Recent taxonomic revisions of the Galaxias vulgaris

INTRODUCTION Sympatric zone analysis is one of the few means by which biologists can objectively test the species status of closely related taxa (Arnold, 1997). Although hybridization is relatively common among freshwater fish species (Hubbs, 1955), there are many cases in which closely related fish taxa coexist with no evidence of hybridization. For example, analyses of sympatric taxa suggest complete reproductive isolation of sympatric Comephorus dybowski and C. baicalensis in Lake Baikal (Brooks, 1950), cichlids in African rift

∗ Corresponding author. E-mail: [email protected] † Current address: Faculty of Resource Science & Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia. 0024–4066/01/070287+12 $35.00/0

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Study area

Bushy

7 Creek

5 3 1 6 4 2

Al

len

Cr e

ek

Transect location (stations 1–7)

N

Mataura

0

River

1 km

Figure 1. Map of the upper Mataura system in South Island, New Zealand. Bushy Creek (study site) flows into Allen Creek, part of the south-flowing Mataura R. drainage. The Bushy Creek transect includes seven sampling stations, spanning approximately 500 m.

complex have led to the identification of six streamresident species from southern South Island. Taxonomic changes include the description of G. depressiceps (flatheads), the redescription and reinstatement of G. anomalus (roundheads) and the description of G. gollumoides from Stewart Island (McDowall & Wallis, 1996; McDowall & Chadderton, 1999). However, recent phylogenetic analyses indicate that Southland populations of flathead and roundhead Galaxias are genetically distinct from their morphological counterparts in Otago (Waters & Wallis, 2001a,b). Specifically, mitochondrial DNA (mtDNA) sequences indicate that Southland flatheads represent an undescribed southern lineage (G. ‘southern’ ) whereas Southland roundheads are essentially indistinguishable from G. gollumoides. In the Mataura drainage (Southland), flathead and roundhead Galaxias morphotypes coexist in Bushy Creek, a small tributary of Allen Creek (Fig. 1). These sympatric populations may persist because Bushy

Creek’s connection with Allen Creek is often severed during dry periods, preventing upstream movement of introduced brown trout. Elsewhere in the upper Mataura system, trout apparently exclude roundhead Galaxias morphotypes (unpublished data). Preliminary mtDNA data (Waters & Wallis, 2001a) and apparent morphological differences suggest that these forms represent distinct species. However, detailed analyses of morphological and genotypic data are required to verify this suggestion. A lack of hybridization and introgression would support the null hypothesis of complete reproductive isolation and justify separate species status under the biological species concept (BSC; Mayr, 1942). Contact zone studies rely heavily on the availability of diagnostic characters to discriminate between sympatric taxa (Szymura & Barton, 1986; Dowling, Smith & Brown, 1989; Verspoor & Hammar, 1991). Both morphological markers (Hewitt, 1988; Crespin, Berrebi & Lebreton, 1999) and molecular genetic markers (e.g.

REPRODUCTIVE ISOLATION IN SYMPATRIC GALAXIAS

289

Freshwater R Nokomai R Catlins R

G. gollumoides

Bushy Ck st 2 Eyre Ck Waiau R Bushy Ck st 2, 5, 7 Nokomai R Robertson R 98 Mokoreta R MacLennan R Bushy Ck st 4, 7

Bullock Head Ck Allen Ck Rakeahua R 100

G. 'southern'

Aparima R

Bullock Head Ck Nokomai R G. brevipinnis

0.005 changes

Figure 2. Phylogram showing relationships between Galaxias control region haplotypes based on distance analysis. Bootstrap values exceeding 80% are indicated. Underlined haplotypes are sourced from outside the Mataura R. drainage; Bushy Creek haplotypes are indicated in bold.

Roy et al., 1994; Harrison & Bogdanowicz, 1997; Esa, Waters & Wallis, 2000) are commonly used for species discrimination. Indeed, there are several cases in which both molecular and morphological techniques have been applied (e.g. Eisenhour, 1995; Dowling, Broughton & DeMarais, 1997). Here we employ molecular and morphological analyses in an effort to detect and quantify any hybridization between Galaxias morphotypes in the Bushy Creek contact zone.

MATERIAL AND METHODS FISH SAMPLING

A transect analysis, sampling 139 fish from seven stations (c. 20 fish per station) over a distance of approximately 500 m, was carried out in Bushy Creek (Fig. 1). Each sample station spanned 10–20 m in

stream-length, with gaps of 50–60 m between stations. The transect area encompassed a transition from low gradient (riffle; lower stations) to high gradient (pool/ cascade; upper stations) habitats, with riparian vegetation increasing upstream. Fish were caught by electrofishing and placed on ice prior to storage at −80°C. GENETIC ANALYSIS

Crude homogenates were prepared for isozyme electrophoresis by grinding a piece of muscle tissue in a 1.5-ml microfuge tube containing an equal volume of homogenizing buffer (0.05 mM NADP, 1 mM EDTA, 70 mM Tris-HCL pH 8.0). Electrophoresis was carried out using cellulose acetate plates (Titan III) as described by Hebert & Beaton (1989). A total of seven loci from six enzyme systems were screened; AAT-1∗, AAT-2∗, G3PDH-B∗, MPI∗, PEP-A∗, PGI-B∗ and PGM∗

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Table 1. Forty-three variable nucleotide sites in Mataura R. (including Bushy Creek) partial control region sequences (758 bp). Nucleotide numbers give distance from the 5′ end. Dots indicate nucleotide identity with the reference sequence (G. gollumoides from Nokomai R.). Symbols R and A represent variants in diagnostic restriction sites for Rsa I and Alu I respectively R A 111111111222222344445566666667 146678999023445589011366444550055899992 1235924673028324571307512802339245656545796 G. gollumoides Nokomai Nokomai Eyre Mokoreta Bushy Bushy G. ‘southern’ Nokomai Bullock Bullock Allen Bushy

CGTAGATTGTTATTCGAGAATCATCTACATCCTCTATAAAGTT ????......A.....G......A............C...A.. ????......A.........G.CA............CG...A? ???..G....AT..T.....G..A.C....T.A.C.CG..... ..........A.........G.CA...........???????? ..........A.....G......A..........C???????? ????A.CCACA.CCTAGAC..G.CTC.TCCTTA..GC?????? ???GA.CCACA..CTAGAC..G.CTC..CCTTA..GCCGG..C ????A.CCACA.CCTAGACG.G.CTC..CCTTAT.GCCGG..? GCAGA.CCACA.CCTAGACG...CTCGTCCTTAT.???????? GCAGA.CCACA.CCTAGAC..G.CTC.TCCTTA..????????

1.0 mtDNA Gvu4 Gvu5 Gvu7

Frequency

0.8

0.6

0.4

0.2

0

1

2

3

4 Station

5

6

7

Figure 3. Graphical representation of the cline in Galaxias mtDNA type and microsatellite allele frequencies across the seven Bushy Creek stations. The combined frequency of G. gollumoides private alleles (associated with G. gollumoides mtDNA) is presented for each of the three most variable microsatellite loci.

(Allibone et al., 1996). All enzyme systems were developed using Tris-glycine buffer system at pH 8.5. Enzyme staining recipes were employed as described elsewhere (Shaw & Prasad, 1968; Harris & Hopkinson, 1976; Hebert & Beaton, 1989). Total DNA was extracted from muscle tissue using CTAB (see Waters, Esa & Wallis, 1999) and used

for microsatellite and mtDNA analyses. Microsatellite analyses were carried out using four polymorphic loci: Gvu4, Gvu5, Gvu7 and Gvu9. Primers and details of amplification conditions are described in Waters et al. (1999). Microsatellite amplification products were resolved on 9% non-denaturing polyacrylamide gels at 100 V for at least 16 h, stained with SYBRGREEN 1 (Roche) and photographed under UV light. The mitochondrial control region was amplified and sequenced as described in Waters & Wallis (2001a). After sequence alignment (758 bp), we identified restriction sites that discriminate between G. ‘southern’ and G. gollumoides mtDNA lineages. Two restriction enzymes (Alu I and Rsa I), each targeting a diagnostic site, were used to digest control region amplicons. PCR products were purified in 100
l 100% ethanol with centrifugation (13 000 rpm, 20 min), followed by a 70% ethanol wash and resuspension in 20
l dH2O. Five
l of purified PCR product were combined with an equal volume of digestion mix containing 1.5 U of restriction enzyme and incubated at 37°C overnight. Digested fragments were separated on 2% agarose gels (containing 7
g.ml−1 ethidium bromide) for 2 h at 75 V. MORPHOLOGICAL ANALYSIS

Morphological analysis was generally carried out following procedures described in McDowall (1970) and McDowall & Wallis (1996). The following morphometric measurements were performed using calipers: head length (HL), head width (HW), head depth (HD), snout length (SNL), diameter of eye

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Table 2. Allele frequencies (across all seven Bushy Creek stations) for three polymorphic isozyme loci and four microsatellite loci. Frequencies are presented with samples partitioned according to mtDNA type (G. gollumoides, N=54; G. ‘southern’ N=85) Locus

Allele

G. gollumoides

G. ‘southern’

Aat-2∗

90 100

0.02 0.98

— 1.00

Pgi-b∗

65 89

0.06 0.94

— 1.00

Pgm∗

100 120 140

0.87 0.11 0.02

0.99 0.01 —

Gvu4

136 140 152 154 156 160

0.45 0.08 0.31 0.02 0.14 —

— — — — 0.62 0.38

Gvu5

127 130 133 136 139 142 145 148 154 160 163 166

0.01 0.53 0.15 0.09 0.04 0.18 0.01 — — — — —

0.14 — — 0.04 — 0.34 0.04 0.15 0.05 0.07 0.05 0.14

Gvu7

84 86 88 90 92 94 96 98 100 102 108 112 132

0.02 0.32 0.03 0.14 0.04 0.09 0.02 0.07 0.02 0.13 0.03 0.02 0.08

0.04 — — — 0.26 0.43 0.26 — 0.01 0.01 — — —

Gvu9

133 136

0.23 0.77

— 1.00

(DOE), length of upper and lower jaw (LUJ, LM), postorbital head length (POHL), and width and depth of gape (WG, DG). In addition, we measured the width of the upper lip (ULW; see McDowall, 1998) and the length of the gap between the lateral ethmoid and hyomandibular (LEH; see Esa, 2001). Fish

specimens were stained with alizarin (Potthoff, 1984) to facilitate meristic analysis. Meristic counts were made for the following characters: rays in caudal, dorsal, pectoral, pelvic and anal fins; and gill rakers, following procedures described in McDowall (1970) and McDowall & Wallis (1996).

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J. M. WATERS ET AL. Table 3. Estimates of Hardy–Weinberg exact P-values for isozyme and microsatellite loci. Tests were performed across all seven Bushy Creek stations, both with all samples pooled and with samples partitioned according to mtDNA type (G. gollumoides or G. ‘southern’) Locus

G. gollumoides

G. ‘southern’

Pooled samples

Aat-2∗ Pgi-b∗ Pgm∗ Gvu4 Gvu5 Gvu7 Gvu9 Multi-locus

1.0000 0.1362 0.0012∗∗∗ 0.6851 0.6232 0.0216∗∗ 0.0482∗∗ 0.0029∗∗∗

— — — 0.0680 0.5751 0.4913 — 0.2451

1.0000 0.0536 0.0001∗∗∗∗ 0.0000∗∗∗∗ 0.0000∗∗∗∗ 0.0000∗∗∗∗ 0.6013 0.0000∗∗∗∗

∗∗P<0.05; ∗∗∗P<0.01; ∗∗∗∗P<0.001.

Table 4. Estimates of genotypic disequilibrium showing exact P-values for 21 locus pairs. Tests were performed across all seven Bushy Creek stations, both with all samples pooled and with samples partitioned according to mtDNA type (G. gollumoides or G. ‘southern’ ) Locus 1

Locus 2

G. gollumoides

G. ‘southern’

Pooled samples

Gvu4 Gvu4 Gvu5 Gvu4 Gvu5 Gvu7 Gvu4 Gvu5 Gvu7 Gvu9 Gvu4 Gvu5 Gvu7 Gvu9 Aat-2∗ Gvu4 Gvu5 Gvu7 Gvu9 Aat-2∗ Pgi-b∗

Gvu5 Gvu7 Gvu7 Gvu9 Gvu9 Gvu9 Aat-2∗ Aat-2∗ Aat-2∗ Aat-2∗ Pgi-b∗ Pgi-b∗ Pgi-b∗ Pgi-b∗ Pgi-b∗ Pgm∗ Pgm∗ Pgm∗ Pgm∗ Pgm∗ Pgm∗

0.2185 0.5192 0.0093∗∗∗ 0.3222 0.7095 0.2780 0.5553 0.7155 1.0000 0.2098 0.7655 0.2699 0.8210 1.0000 1.0000 0.1966 0.0218∗∗ 0.0529 0.1532 1.0000 1.0000

0.1012 0.5678 0.1010 — — — — — — — — — — — — 1.0000 0.8635 1.0000 — — —

0.0000∗∗∗∗ 0.0000∗∗∗∗ 0.0003∗∗∗∗ 0.0000∗∗∗∗ 0.0144∗∗ 0.0000∗∗∗∗ 0.1000 0.7973 0.4598 0.0308∗∗ 0.0278∗∗ 0.3331 0.4002 0.3031 1.0000 0.0122∗∗ 0.5260 0.0000∗∗∗∗ 0.0006∗∗∗∗ 1.0000 1.0000

∗∗P<0.05; ∗∗∗P<0.01; ∗∗∗∗P<0.001.

STATISTICAL ANALYSIS

Allele frequencies for isozyme and microsatellite loci were calculated using GENEPOP 3.1b (Raymond & Rousset, 1997). Deviations from Hardy–Weinberg proportions were tested with the 2 test (Hartl & Clark, 1989) implemented in GENEPOP. Genotypic disequilibrium tests for each pair of nuclear loci were performed using GENEPOP. In cases where multiple

independent tests were performed, a sequential Bonferroni adjustment (Rice, 1989) was used to modify significance levels to account for experiment-wide error. Phylogenetic trees based on control region sequences were constructed using the distance (minimum evolution) method implemented in PAUP 4.0b4 (Swofford, 1999). Distances were calculated using the Kimura (1980) two-parameter model, and phylogenetic

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1

Axis 2

0

*

–1

–2

–3 0

–0.5

0.5 Axis 1

1.0

1.5

Figure 4. Multivariate (FCA) analysis of Bushy Creek samples based on allelic data from four microsatellite loci and three isozyme loci. Fish with G. gollumoides mtDNA are represented by dark squares, while specimens with G. ‘southern’ mtDNA are represented by light squares. A genotypically ‘intermediate’ G. gollumoides individual is indicated with an asterisk. Table 5. Summary of morphometric data (12 characters) across all seven Bushy Creek stations. Samples are partitioned according to mtDNA type (G. gollumoides or G. ‘southern’) HW/HL

HD/HL

HW/HD

SNL/HL

POHL/HL

DOE/HL

69.8 5.7 80

57.7 5.4 80

121.5 9.0 80

21.9 2.9 80

56.3 5.6 80

23.1 2.4 80

G. gollumoides Mean 63.4 SE 5.7 N 48

56.5 6.4 48

113.4 14.1 48

22.3 3.4 48

53.5 5.3 48

30.7 4.1 48

LUJ/HL

LM/HL

WG/HL

ULW/HL

DG/WG

LEH/HL

33.5 3.5 80

31.1 3.4 80

54.8 5.7 80

12.8 1.4 79

78.1 7.2 79

36.4 6.4 78

G. gollumoides Mean 33.2 SE 3.7 N 48

30.9 3.9 48

45.2 5.9 48

10.2 1.5 40

92.6 13.3 40

28.1 5.4 37

G. ‘southern’ Mean SE N

G. ‘southern’ Mean SE N

confidence was assessed with 1000 bootstrap replicates (Felsenstein, 1985). A control region sequence from Tasmanian G. brevipinnis (GenBank accession AF267338; Waters & Wallis 2001b) was included as an outgroup for phylogenetic analysis. Reference sequences from Southland and Stewart Island were also included in the analysis: G. ‘southern’ (Mataura R., GenBank accession AF234673, AF313943–44, AF313946; Rakeahua R., AF159849; Aparima R.,

AF234675); G. gollumoides (Mataura R., AF313937, AF313940–42; Freshwater R., AF313920; Robertson R., AF159845; Catlins R., AF313918; MacLennan R., AF313917; Waiau R., AF313921). Factorial correspondence analysis (FCA) of genotypic data was performed with Genetix v4.01 (Belkhir et al., 1999). This canonical technique can be used to project individuals into multidimensional space on the basis of allelic information, with each allele analysed as an

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PCII

4.0 2.0 0.0 –2.0 –7.5

–5.0

–2.5

0.0 PCI

2.5

5.0

7.5

Figure 5. Multivariate (PCA) clustering of Bushy Creek samples based on morphological characters (12 morphometric measurements and one meristic character). Fish with G. gollumoides mtDNA are represented by dark squares, while specimens with G ‘southern’ mtDNA are represented by light squares.

independent variable (She et al., 1987; see Roques, Se´vigny & Bernatchez, 2001). Multivariate analyses were also performed on morphological data. Raw data for morphological characters were log transformed prior to Principal Component Analysis (PCA) implemented in SAS statistical software (SAS Institute, 1989).

RESULTS MITOCHONDRIAL DNA

Phylogenetic analysis of partial control region sequences (758 bp) indicated that G. ‘southern’ and G. gollumoides are represented by reciprocally monophyletic mtDNA lineages, each supported by high bootstrap values (Fig. 2). Sequence divergences between these clades ranged from 3.4% to 5.3% (K2P). Haplotype divergences within the G. ‘southern’ clade were generally less than 1% (maximum 1.4%) whereas divergences within G. gollumoides frequently exceeded 1% (maximum 1.7%). Two haplotypes were detected within Bushy Creek G. gollumoides (N=4 sequences; GenBank AF313937, AF313939), whereas a single haplotype was detected in Bushy Creek G. ‘southern’ (N=2 sequences; AF313947; Fig. 2). Bushy Creek samples (139 fish) were classified as either G. ‘southern’ or G. gollumoides based on their mtDNA type as determined by digestion with Alu I and Rsa I. Across all seven stations, 85 fish yielded restriction fragments consistent with G. ‘southern’ mtDNA, while 54 fish yielded fragments consistent with G. gollumoides mtDNA (diagnostic restriction sites indicated in Table 1). Across the 500 m transect, we observed a marked cline in the relative frequencies of the two mtDNA types (Fig. 3): G. ‘southern’ mtDNA dominated the lower stations 1 and 2 (frequencies of 0.89 and 0.90), while the upstream stations 6 and 7 were dominated by G. gollumoides mtDNA (0.71, 0.93). Each mtDNA lineage was well-represented in the middle of the transect (Fig. 3).

NUCLEAR DNA

Isozyme analysis of Bushy Creek samples revealed minor variation at AAT-2∗, PGI-B∗ and PGM∗ (Table 2), but no polymorphism at AAT-1∗, G3PDH-B∗, MPI∗ and PEP-A∗. In contrast, three of the four microsatellite loci (Gvu4, Gvu5 and Gvu7 ) were highly polymorphic, segregating six, 12 and 13 alleles respectively (Table 2). The seven polymorphic nuclear loci (isozymes and microsatellites) yielded a total of 40 alleles. When samples were partitioned according to mtDNA type (Table 2), 24 alleles were ‘private’ to one or other mtDNA group, some with high frequencies (e.g. gollumoides: Gvu4-136, Gvu4-152, Gvu5-130, Gvu7-86; ‘southern’: Gvu4-160; Table 2). High allelic diversity was evident in the G. gollumoides group, with 34 alleles, compared to just 22 alleles detected in G. ‘southern’, despite the larger sample size of the latter mtDNA group (Table 2). Tests for conformity to Hardy–Weinberg expectations revealed highly significant heterozygote deficits (after sequential Bonferroni adjustment) at four of the seven loci when all samples (across all stations) were pooled (P<0.001; Table 3). However, when samples were partitioned according to mtDNA type, the deviation from Hardy–Weinberg expectations was considerably reduced, with no significant values for G. ‘southern’, and three for G. gollumoides (Table 2), only one of which remained significant after Bonferroni adjustment. Similarly, tests for genotypic disequilibrium (with all samples pooled) revealed significant values for 11 of 21 locus pairs (all involving microsatellite loci), including seven highly significant values (P<0.001; Table 3). By contrast, with samples partitioned according to mtDNA type, no significant values were observed for G. ‘southern’, while two were observed for G. gollumoides (Table 4), only one of which was significant after Bonferroni correction. Multivariate analysis (FCA) of combined microsatellite and isozyme data yielded two distinct genotypic clusters, each completely congruent with mtDNA-

REPRODUCTIVE ISOLATION IN SYMPATRIC GALAXIAS

based allocation (Fig. 4). The G. ‘southern’ group was represented by a well-defined cluster, whereas the G. gollumoides group was more diffuse, reflecting substantially more allelic diversity (Fig. 4). Alleles contributing substantially to the resolution of the two groups (by Factorial 1) included Gvu4-136, Gvu4-152, Gvu4-160, Gvu5-130, Gvu7-86 and Gvu9-133. Similar results were produced from microsatellite data alone, whereas separate analysis of isozyme loci yielded little resolution (data not shown). MORPHOLOGY

Morphometric data produced several ratios with discriminatory power for the two mtDNA types. The mean HW/HL, WG/HL and LEH/HL were considerably higher in G. ‘southern’ than in G. gollumoides while DOE/HL and DG/WG were higher in the latter (Table 5). Meristic data yielded relatively little variation, with both species exhibiting very similar fin rays counts (data not shown). Gill raker numbers were the only informative meristic character. All G. ‘southern’ exhibited gill raker counts >10 (overall=11–14) whereas 77.8% of G. gollumoides exhibited gill raker counts <10 (overall=9–13). Accordingly, gill raker counts were the only meristic character included with morphological characters in PCA. For PCA, individuals were classified as either G. ‘southern’ or G. gollumoides, according to their mtDNA type. The first principal component (PCI), which produced equal loading in all variables, accounted for 82.9% of the total variance. The second principal component (PCII) accounted for 6.8% of the remaining variance. Variables that loaded heavily on PCII include gill raker counts (−0.58) and eye diameter (DOE; 0.52). A plot of PCI versus PCII yielded distinct morphological clusters, completely consistent with mtDNA-based allocation (Fig. 5). Despite the congruence of morphological, genotypic and mtDNA data, a minor degree of hybridization could not be ruled out. For instance, the G. gollumoides specimen that was genotypically closest to G. ‘southern’ (Fig. 4; asterisk) exhibited two ‘intermediate’ morphological character states (DOE/HL 26.7; WG/HL 56.3; see Table 5). Such intermediate morphometric values could conceivably indicate a hybrid/backcross origin for this individual, but a suite of fully diagnostic nuclear genetic markers is required to confirm this suggestion.

DISCUSSION REPRODUCTIVE ISOLATION

Multivariate analyses of genotypic data (Fig. 4) and morphological data (Fig. 5) independently revealed two clusters of Bushy Creek samples, each completely

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consistent with mtDNA type. The congruence of morphotype, genotype and mtDNA groupings indicates the absence of mtDNA introgression, and the probable absence of F1 hybrids among Bushy Creek G. ‘southern’ and G. gollumoides. Thus the null hypothesis of reproductive isolation between these forms is supported. The highly significant deviation from Hardy– Weinberg equilibrium (Table 3) and strong genotypic disequilibrium (Table 4) observed for pooled Bushy Creek samples confirms the presence of highly distinct gene pools (see above). The more subtle genotypic disequilibrium (Gvu5 and Gvu7, P=0.0093; Fig. 2) and deviation from Hardy–Weinberg expectations (P= 0.0029 across loci; P=0.0012 at PGM∗; Table 3) observed for G. gollumoides samples might reflect null alleles, localized inbreeding or even selection for certain genotypes. Alternatively, it might be suggested that the departures from equilibrium reflect a low incidence of hybridization. Although this seems an unlikely explanation given the apparent absence of mtDNA introgression (see above), a small degree of hybridization cannot be rejected without the availability of completely diagnostic nuclear genetic markers. This study demonstrates the utility of both molecular and morphological techniques in resolving subtle biological differences. Morphological analysis proved highly informative, with several characters such as gill raker counts, diameter of eye (DOE) and width of gape (WG) useful for species discrimination. In addition, the inclusion of data from four microsatellite loci facilitated genetic resolution that was unavailable from isozyme markers. Microsatellite loci may become increasingly popular for future studies of sympatric zones. Microsatellites have already been applied successfully in several cases where isozymes provided little discriminatory power for closely related sympatric taxa (e.g. Roy et al., 1994; Moulin et al., 1996). The lack of genetic resolution provided by isozymes might reflect the relatively slow rates of evolution that characterize these loci. A similar lack of isozyme resolution is apparent among some other closely related New Zealand Galaxias that are easily distinguished by mtDNA analysis (Waters & Wallis, 2001a). For example, no fixed isozyme differences were detected for G. eldoni, G. vulgaris and the diadromous G. brevipinnis (Allibone & Wallis, 1993). Little is known about the reproductive biology and ecology of either G. ‘southern’ or G. gollumoides, so we can only speculate about any biological mechanisms that might isolate these taxa. Reproductive barriers among closely related sympatric taxa might be prezygotic (e.g. spawning behaviour: Allendorf & Leary, 1988; Allibone & Townsend, 1997) or postzygotic (e.g. hybrid inviability: Barton & Hewitt, 1985; outbreeding depression: Rand & Harrison, 1989). The observed

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cline in species frequency (Fig. 3) across the Bushy Creek transect may reflect increasing stream gradient, suggesting ecological divergence between the taxa. Furthermore, the observed differences in morphological characters such as width of gape (WG/HL) and eye diameter (DOE/HL; Table 5) may reflect divergence in feeding behaviour (e.g. Schluter & McPhail, 1993; McDowall, 1998).

In conclusion, the reproductive isolation evident between G. ‘southern’ and G. gollumoides supports their separate status under the biological species concept (BSC; Dobzhansky, 1937; Mayr, 1942). Reciprocal monophyly of mtDNA, and consistent differences in genotypic and morphological characters also support their separate status under a phylogenetic species concept (PSC; Cracraft, 1983).

SYMPATRIC SPECIATION?

ACKNOWLEDGEMENTS

In the upper Taieri R. system, artificial (humanmediated) secondary contact of allopatric G. vulgaris complex lineages has resulted in bi-directional introgression of mtDNA and nuclear genes (Esa et al., 2000). However, natural and ongoing sympatry has been documented for only two pairs of stream resident lineages of the G. vulgaris complex: ‘southern’/gollumoides (Mataura R. system; current study) and depressiceps/anomalus (Taieri R. system; Allibone et al., 1996; unpublished data). In both cases, analyses using a variety of molecular markers indicate that consistent morphological differences correspond with highly distinct gene pools, with no evidence of mtDNA introgression. There is evidence for convergent morphological evolution, in that both natural contact zones house ‘flathead’ (depressiceps, ‘southern’ ) and ‘roundhead’ (anomalus, gollumoides) morphotypes. It might be inferred that ecological processes (e.g. trophic specialization; McDowall, 1998) have promoted morphological divergence in these zones of sympatry. The co-occurrence of morphologically and genetically divergent Galaxias could be interpreted as evidence of sympatric (ecological) speciation, as recently suggested for other freshwater fishes (e.g. Echelle & Kornfield, 1984; Schliewen, Tautz & Pa¨a¨bo, 1994; Schluter, 1996). This scenario would conflict with the view that geographical isolation is a prerequisite for speciation (Mayr, 1942). A phylogenetic analysis based on 5039 bp of mtDNA indicates that the radiation of the G. vulgaris complex has been facilitated by three independent losses of migratory ability from a diadromous ancestor (G. brevipinnis; Waters & Wallis, 2001b). Furthermore, G. ‘southern’ and G. gollumoides represent independently-derived stream-resident lineages rather than sister taxa. This finding implies that diversification of these lineages was initiated by the loss of diadromy rather than by sympatric speciation. Therefore we suggest that the speciation of G. ‘southern’ and G. gollumoides (through independent losses of diadromy) occurred in allopatry, with subsequent secondary contact (e.g. Bernatchez & Dodson, 1990). Nevertheless, this hypothesis does not rule out the possibility of some subsequent ecomorphological divergence in sympatry (e.g. character displacement; Schluter, 1996; Taylor, 1999).

This study was initiated following discussions with staff of New Zealand’s Department of Conservation (Otago and Southland conservancies). Lyndsay Chadderton, Winston Cooper, Eric Edwards, John Hollows and Murray Nielson assisted with the collection of specimens. The staff of Greenvale station kindly allowed access to Bushy Creek. Bob McDowall provided constructive criticism of the manuscript. The work was funded by contract No. UOO705 from the Marsden Fund, and fish were collected under University of Otago Animal Ethics Approval 43/99.

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