Marine Biology (2002) 141: 541–547 DOI 10.1007/s00227-002-0836-3 S.F. Chenoweth Æ J.M. Hughes Æ R.C. Connolly Phylogeography of the pipefish, Urocampus carinirostris, suggests secondary intergradation of ancient lineages Received: 19 April 2001 / Accepted: 1 February 2002 / Published online: 16 August 2002 Ó Springer-Verlag 2002 Abstract We assayed the pattern of mitochondrial DNA speciation. Particularly noteworthy is the undeniable evolution in the live bearing, seagrass specialist pipefish, influence of historical environmental changes on pat- Urocampus carinirostris, in eastern Australia. These life terns of gene flow among conspecific populations (Avise history attributes were predicted to result in strong 1994). Extrinsic forces such as glaciation and sea level phylogeographic structure in U. carinirostris. Phyloge- changes appear to have affected the pattern of neutral netic analysis of cytochrome b sequences detected two molecular evolution in many codistributed species in monophyletic mtDNA clades that differed by 8.69% similar ways regardless of subtle differences in intrinsic sequence divergence – a large level of intraspecific di- life history characteristics (e.g. Australia’s wet tropics: vergence for a marine fish. The geographical distribution Schneider and Moritz 1999; and coastal marine taxa in of clades was non-random and resembled clinal sec- the southeastern United States: Avise 1994). However, ondary intergradation over a 130-km stretch of coast- there are exceptions where life history attributes appear line. Contrary to phylogeographic predictions, this large to be the predominant factor leading to genetic differ- phylogeographic break does not occur across a tradi- ences among populations. For example, very strong tionally recognised biogeographic boundary. Analyses genetic differences are maintained among populations of of historical demography suggested that individuals be- the coral reef fish, Acanthachromis polyacanthus, which longing to the most widespread clade underwent a is exceptional among coral reef fishes due to its lack of a population expansion from a small refuge population pelagic larval phase. Compared with other presently during the Pleistocene. codistributed coral reef fishes that exhibit pelagic larval dispersal, A. polyacanthus exhibits significantly stronger genetic differentiation among Great Barrier Reef popu- lations (Doherty et al.1995). Because these codistributed Introduction species are likely to have experienced similar historical biogeographic forces, these patterns suggest that the Molecular systematics has facilitated several extensions unique life history of A. polyacanthus is a major deter- of our understanding of the interplay between the ge- minant of population genetic patterns. netic differentiation of populations and geographic It has been suggested that patterns of genetic differ- entiation among populations of marine fishes may be associated with degree of habitat specialisation (Smith Communicated by G.F. Humphrey, Sydney and Fujio 1982), and life history dispersal characteristics (Waples 1987). Marine fishes display phylogeographic S.F. Chenoweth (&) Æ J.M. Hughes structures that span a continuum, from the apparent Australian School of Environmental Studies, Griffith University, Nathan, 4111, Brisbane, Australia panmixis displayed by Atlantic eels (Avise et al. 1986) to the secondary intergradation of divergent lineages in the E-mail: [email protected]_edu.au menhaden, Brevoortia tyrannus (Bowen and Avise 1990). Tel.: +61-7-3365 2855 Fax: +61-7-3365 1655 However, more often than not, marine fishes display weak phylogeographic structuring, and intraspecific R.C. Connolly School of Environmental and Applied Sciences, mitochondrial DNA clades are shallow compared with Griffith University, Gold Coast, 9726, Australia freshwater fishes (Shulman and Bermingham 1995; Present address: S.F. Chenoweth Avise et al. 1998). Department of Zoology and Entomology, Hairy pipefish (Urocampus carinirostris Castelnau) University of Queensland, Brisbane, Qld 4072, Australia are small, cryptic fish from the family Syngnathidae, 542 which also includes seahorses and seadragons. U. ca- rinirostris is the most common syngnathid along much of the eastern Australian coast (Howard and Koehn 1985; Gray et al. 1996). It shares a specialised repro- ductive strategy with other syngnathids, involving the female passing eggs to the underside of the male, in the case of U. carinirostris into a brood pouch under the abdomen (Dawson 1985). Young juveniles hatch from the pouch fully formed. U. carinirostris is typically found in shallow meadows of the seagrass, Zostera capricorni, in estuaries and sheltered embayments. The lack of a dispersive egg and larval phase and the weak swimming ability of adults suggest that movement among estuaries is limited and that closure of the life cycle commonly occurs over a very localised geographical scale. We have assayed mitochondrial DNA variation in eastern Australian populations of U. carinirostris, a marine species representing an extreme in habitat spe- cialisation and limited dispersal potential, to determine if such an extreme suite of intrinsic biological attributes has resulted in strong phylogeographic patterns. The absence of such a pattern would further emphasise the potency of extrinsic environmental forces in determining the genetic structure of marine populations. Fig. 1. Map of seagrass beds sampled for Urocampus carinirostris. Pie diagrams are frequency distributions of two major mtDNA Materials and methods clades A (grey) and B (black) based on samples of 25 individuals per site assayed by direct sequencing of the cytochrome b gene and Collections subsequent diagnostic PCR- RFLP assay. Site codes are GD Gladstone Harbour; EH Elliott Heads; BH Burrum Heads; TI Tin We surveyed previously mapped and identified seagrass meadows Can Bay; BC Bells Creek; NI Ningi; CL Cleveland; BW for U. carinirostris using seine nets. The sampling covered the east Broadwater; LM Lake Macquarie coast distribution from Lake Macquarie in the south to the known northern extent of the range (Fig. 1) Samples were analysed from nine different seagrass beds spanning a total of 1,200 km of coastline (Fig. 1). clade B individuals and did not cleave any positions in clade A individuals. Two restriction digests were carried out on all indi- viduals (one per enzyme) using 10ll of PCR product following the Molecular methods manufacturer’s directions. Digests were later run on agarose gels to determine the presence or absence of a diagnostic restriction site. Our molecular analysis involved two phases. First, to identify To allow unbiased comparisons among sites, during both phases of major lineages in the intraspecifc mtDNA phylogeny of U. ca- the analysis, an equal number of individuals were analysed from rinirostris, for each of ten individuals per population, we se- each site. quenced a portion of the mitochondrial cytochrome b gene. Mitochondrial DNA was isolated using the alkaline lysis technique outlined by Tamura and Aotsuka (1988). A fragment of the mi- Statistical methods tochondrial cytochrome b gene was amplified via the polymerase chain reaction using primers GLU-L and CB2H (Palumbi et al. Sequences were aligned by eye. Identical sequences were assigned 1991) which correspond to positions 14725 and 15175 in the hu- the same haplotype identity. Phylogenetic analyses were performed man mtDNA genome. The thermal profile for PCR amplifications using distance-based, (neighbor-joining; Saitou and Nei 1987) involved 40 cycles of 30 s at 940 C, 30 s at 550 C and 1 min at maximum parsimony, and maximum likelihood (quartet puzzling; 720 C. Sequencing of both heavy and light strands was carried out Strimmer and von Haeseler 1996) methods. The mtDNA diversity on an Applied Biosystems 377 automated sequencing machine distributed within and among populations was calculated using the using the Big DyeÒ Terminator chemistry. For each individual, a package REAP (McElroy et al. 1992). A Mantel’s test was used to minimum of 300 bp of high-quality sequence data were obtained test for a relationship between geographic distance and nucleotide from the approximately 450 bp amplified fragment. This sequence divergence among sites (Mantel 1967). data provided numbers sufficient for estimating nucleotide diver- The historical demography of each major mtDNA clade was sity and allowed statistical tests of historical demography to be investigated using mismatch distributions, which are the distribu- performed. tion of pairwise genetic differences among individuals in a popu- Second, to better characterise the geographical distribution of lation (Rogers and Harpending 1992). We used simulations to test two major mtDNA lineages that were detected, a diagnostic re- the significance of the raggedness statistic r, for each mismatch striction enzyme assay was developed and performed on an addi- distribution (program provided courtesy of H. Harpending). Pop- tional 135 individuals (15 per site) (sensu Slade et al. 1993). The ulations that have undergone a large expansion are expected to restriction enzyme Rsa1 cleaved the analysed fragment at position exhibit smooth, unimodal mismatch distributions and low rag- 292 in all clade A individuals and did not cleave any positions in gedness values. More ragged mismatch distributions tend to result clade B individuals. The enzyme Alu1 cleaved position 139 in all from large stable populations (Harpending 1994). 543 cytochrome b sequences, an extremely large value for a Results marine fish. Typing