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SESSION 1 : AUSTRALIAN SWGOFAUNAAND WA PROJECT (PILBARA)

7_tII

II

ii

may have involved a past fragmentation event ofa once widespread ancestor, perhaps linked tothe onset of ariditY in Australia.

2. METHODSSpecimens of P. millsiwere collected from tencreek catchments located within two major riverbasins, the Ashburton, and the Fortescue (Fig.1). With some exceptions, between five and tenindividuals per catchment were sequenced'Whole genomic DNA was extracted fromspecimens in 50pl of a proteinase K extractionbuffer for between 10 and 20 hours. A 710 basepair (bp) fragment of the 3'end of the cytochromeoxidase subunit 1 (COl) gene was amplified,using the primers LCO1490 (forward, 5' GGTCAA CAA ATC ATA AAG ATA TTG G 3') andHCO2198 (reverse, 5' TAA ACT TCA GGG TGACCAAAAAAT CA 3'; Folmer et al ' ,1994). The 25pl PCR reactions used 0'2mM dNTPs, 4'0mMMgCl,, 1x buffer, 12.5 pmoles of each primer, 1unit Taq, and 2.5 pl template. Sequences werecleaned using the UltraOleanrM PCR Clean-upDNA purification kit (MoBio Laboratories, Inc.)prior to sequencing. The sequencing reactionwas carried out using the BigDye V3 ReadyReaction Mix (ABl Prism) and the products weresequenced using both primers on an ABI 373automated sequencer (Applied Biosystems)'Sequences were aligned and edited by eye withGeneDoc, version 2.6'0o2, using defaultsettings. Relationships among the sequenceswere analysed using PHYLIP, version 3'57c.Pair-wise nucleotide sequence divergence was

calculated using Kimura's two-parameterdistance model in the DNADIST module. Amaximum likelihood phylogeny was producedusing the module DNAML on 100 multiple datasets (generated in SEQBOOT).

3. RESULTSThe number of haplotypes detected variedamong catchments, ranging from a singlehaplotype at Caves Creek, Roy Hill andSpearhole Creek, to six at Weeli Wolli Creek.lmportantly, haplotypes were not shared amongcatchments. Sequence divergence betweenhaplotypes within the same catchment did notexceed 2.3%. Within the Ashburton, sequencedivergence ranged from 8.9 to 13'1% betweencatchments and within the Fortescue, divergenceranged from 16.4 to26.5o/o between catchments'Sequence divergence between haplotypes indifferent basins ranged from 20.1 to 26'8%. Thephylogenetic analysis showed that geneticstructure within P millsi retlected hydrologicalstructure. Each mtDNA lineage containedhaplotypes from only a single catchment (Fig. 2).While the use of a molecular clock is fraught withassumptions that are often violated, it can give arough estimate of the time frame associated withphylogenetic events. Using an estimate of theevolutionary rate of the COI gene ofapproximately 2o/" divergence per million years'haplotypes from the Ashburton and FortescueRiver basins last shared a common ancestorbetween 10 and 13 mYa.

''l Lower Fortescue

J and Hardey RiverRov Hill (5)

MillstreamHardey River

ina Ck Arna Ck B (2)Marillana Ck C

Weeli Wolli B

Weeli Wolli E Upper Fortescue

0 .1

Figure 2 : CO1 maximum likelihood tree. The tree is rooted using A. subtenuis, (family Ceinidae) and Nedsra sp' (family

Melitidae). confidence tevels > 507o, assessed by 100 replicate bootstraps are shown on branches. Number of individuals

represented by each haplotype is shown for n>1 '

Weeli Wolli /Marillana Ck

lEthel GorgerEthel Gorge. gpearhole

,) '\c k E Ie c k D I; C K B Ie c k A Ie c k c Ii c IA ( 2 ) l

70 SWSB December 2004

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