Molecular phylogeny of large miliolid foraminifera - University of ...

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64M. Holzmann et al. / Marine Micropaleontology 43 2001) 57±74Fig. 1. Phylogenetic tree based on the complete SSU rRNA gene of two alveolinid genera and eleven soritacean genera using maximumlikelihood analysis. The tree is rooted in Alveolinidae. Bootstrap values are based on 100 resampling. 1664 out of a total of 3006 sites were usedfor analysis.of 50 ml. The thermal cycle parameters consisted of40 cycles of 30 s at 948C, 30 s at 508C and 120 s at728C, followed by 5 min at 728C for ®nal extension.The ampli®ed PCR products were puri®ed using HighPure PCR Puri®cation Kit Roche Diagnostics), thenligated into pGEM-T Vector system Promega) andcloned in XL-2 Ultracompetent Cells Stratagene).Sequencing reactions were prepared by using ABI-PRISM Big Dye Terminator Cycle Sequencing Kitand analyzed with an ABI-377 DNA sequencerPerkin±Elmer), all according to the manufacturer'sinstructions.The complete SSU rDNA was ampli®ed in threeoverlapping fragments by using the followingprimer pairs: sA10±s13, s6r±s17, s14F3±sBfTable 3). Partial SSU rDNA sequences wereobtained by ampli®cation with the primer pairs14F3±sBf Table 3). The new sequences reportedin this paper have been deposited in the EMBL/GenBank database under accession numbersAJ278048-AJ278062 and AJ404294-AJ278062Table 1). The sequences of P. pertususZ69604) and Sorites orbiculus AJ132369) werepublished by Pawlowski et al. 1999).
M. Holzmann et al. / Marine Micropaleontology 43 2001) 57±74 65Fig. 2. Phylogenetic tree based on the complete SSU rRNA gene of two alveolinid genera and eleven soritacean genera using neighbor joininganalysis. The tree is rooted in Alveolinidae. Bootstrap values are based on 1000 resampling.1664 out of a total of 3006 sites were used foranalysis.2.3. Sequence analysisSequences were aligned manually by using thegde 2.2 software Larsen et al., 1993). Selectedsites in homologous regions without gap wereretained for phylogenetic analyses. Analyses arebased on the following methods: the neighbor±joiningNJ) method Saitou and Nei, 1987), applied todistances corrected for multiple hits and for unequaltransition and transversion rates, using Kimura'stwo-parameter model Kimura 1980); the maximumlikelihood ML) method as implemented in the fastDNAm1 program Olsen et al., 1994); and the maximumparsimony MP) method, using paup p 4.0bversion Swofford, 2000). NJanalysis of thecomplete SSU rDNA gene was additionally testedby using the LogDet model Lockhart et al., 1994),because of the biased base composition in MiliolidaG 1 C content of about 30%), which yielded thesame results as with Kimura's two-parametermodel. Parsimony analysis consisted of heuristicsearches with 100 random-addition replicates usingtree bisection±reconnection TBR) branch swappingand stepwise addition of taxa. The reliability ofinternal branches was assessed by bootstrappingFelsenstein, 1988) with 1000 resampling for theNJand 100 resampling for the ML and MP trees,respectively. The phylo_win program Galtierand Gouy, 1996) was used for distance computations,NJand ML tree-building and bootstrapping.
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Molecular phylogeny of large miliolid foraminifera - University of ...

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