phylogenetic relationships and classification of didelphid marsupials ...

10 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 322
TABLE 3
Primers Used to Amplify and Sequence BRCA1 and vWF
Primer name Sequence
BRCA1- F1 59 TCATTACTGCCTGAGATCACCAG
BRCA1- F47 59 TATTGCCTAACACAGACAGCAT
BRCA1- F593 59 CAACAATATTGAAGACAAAATATTAGGAAA
BRCA1- F1163 59 ATGARACWGAACTACWGATCGATAG
BRCA1- F1163a 59 AATGAGACTGAACTACAGATCGAT
BRCA1- F1697 59 TTWGATGRTTGTTCATCYRAAAACAC
BRCA1- R743 59 TTGATGAAATCCTCAGGCTGYAGGT
BRCA1- R1218 59 GAAGYCTTCTGCTGCGTCTGA
BRCA1- R1343 59 CTAACATTTGATCACTATCAGTAG
BRCA1- R1780 59 TAAATAYTGGGTRTCRAGTTCACT
BRCA1- R2078 59 GAAATTTCCTGGTTGTTTCCAGCAA
BRCA1- R2151 59 TCCTTTTGATYAGGAACTTGTGAAATT
VWF- F104 59 GGTGTGATGGAGCGTTTACACATCTC
VWF- F120 59 GACTTGGCYTTYCTSYTGGATGGCTC
VWF- F557 59 CCTGGGCTACCTCTGTGACCTGGT
VWF- R655 59 CTTCTAGCACAAACACCACATCCAGAACCA
VWF- R743 59 CTCACATCCATYCGTTGCATCA
VWF- R1141 59 ATCTCATCSGTRGCRGGATTGC
down protocol as described in Jansa and
Voss (2000). Reamplification reactions were
performed using Taq DNA polymerase (Promega
Corp.) in 25 ml reactions for 30 PCR
cycles. The resulting PCR products were
sequenced in both directions using amplification
primers and dye-terminator chemistry on
an AB 3700 automated sequencer.
We searched the draft Monodelphis domestica
genome using the BLAT (modified
BLAST; Kent, 2002) algorithm to determine
the copy number and chromosomal location
of query sequences from M. brevicaudata. As
reported on the Ensemble database (www.
ensemble.org/index.html), the available reference
genome (assembly MonDom5) was
released on October 2006 and has a base
coverage of approximately 7.33.
ALIGNMENT AND PHYLOGENETIC ANALY-
SIS: We aligned DNA sequences with reference
to translated amino-acid sequences
using MacClade 4.08 (http://macclade.org).
Aligned sequences were then analyzed phylogenetically
using maximum parsimony
(MP) as implemented by PAUP* ver.
4.0b10 (Swofford, 1998), maximum likelihood
(ML) as implemented by GARLI ver.
0.95 (Zwickl, 2006), and Bayesian inference
as implemented by MrBayes ver. 3.1.1
(Ronquist and Huelsenbeck, 2003). For MP
analyses, all molecular characters were treated
as unordered and equally weighted, and
all tree searches were heuristic with at least 10
replicates of random stepwise taxon addition
followed by tree bisection-reconnection
(TBR) branch swapping. To choose the best
models of nucleotide substitution for ML and
Bayesian analyses, we examined the fit of
various models separately for each of our five
gene partition (IRBP, vWF, BRCA1, DMP1,
and first and second codon positions of
RAG1) based on neighbor-joining trees of
Jukes-Cantor-corrected distances using both
hierarchical likelihood-ratio tests (hLRTs)
and the Akaike Information Criterion (AIC)
as implemented in ModelTest 3.7 (Posada
and Crandall, 1998). Where the two approaches
disagreed on model choice, we used
the model selected by the AIC for reasons
outlined by Posada and Buckley (2004). For
both ML and Bayesian searches, the best-fit
model was specified, but model parameter
values were not fixed. For ML analyses, we
conducted three independent runs of geneticalgorithm
searches in GARLI, with random
starting topologies and automatic termination
after 10,000 generations with no improvement
in log-likelihood scores. For
Bayesian analysis of each gene partition, we
conducted two independent runs of Metrop-