phylogenetic relationships and classification of didelphid marsupials ...

2009 VOSS AND JANSA: DIDELPHID MARSUPIALS 9
presence and absence were both commonly
observed for a given taxon). However,
whereas we formerly (Voss and Jansa, 2003)
treated such polymorphism as a separate state
intermediate to the two fixed conditions, with
transformations to and from the polymorphic
state weighted as half-steps (the ‘‘scaled’’
option discussed by Wiens, 2000), we here
conform with the prevailing custom of coding
polymorphisms as taxonomic ambiguities
(e.g., as ‘‘0/1’’ for a binary character). This
coding change has minimal impact on our
results (only weakly supported relationships
are affected, even in separate analyses of
morphology) but it facilitates likelihood
modeling of morphological character evolution
(Lewis, 2001) in Bayesian analyses of our
combined datasets (see below).
MOLECULAR SEQUENCING AND HOMOLO-
GY COMPARISONS: The laboratory procedures
we used for DNA amplification and sequencing
(including the names and locations of
primers used in PCR reactions) have already
been described for three of the protein-coding
nuclear loci analyzed in this report: IRBP
(Jansa and Voss, 2000), Dentin Matrix
Protein 1 (DMP1; Jansa et al., 2006), and
Recombination Activating 1 Gene (RAG1;
Gruber et al., 2007). In addition, we
sequenced two other protein-coding nuclear
genes as described below.
TABLE 2
(Continued)
Percent complete character data b
Nonmolecular Molecular Combined
Dasyuridae
Murexia longicaudata 95.3 57.9 58.5
Sminthopsis crassicaudata
Microbiotheriidae
96.1 42.1 43.0
Dromiciops gliroides
Peramelidae
99.2 63.3 63.9
Echymipera kalubu 95.3 63.2 63.8
Perameles gunnii 93.0 63.3 63.8
a Nomenclature follows Wilson and Reeder (2005) and Gardner (2008) except as noted.
b Number of filled (total minus empty) cells in the corresponding row of each data matrix, divided by the total number
of cells (N5 129, 7320, and 7449 for nonmolecular, molecular, and combined data, respectively) 3 100. For the
nonmolecular data, empty cells include those scored as missing (‘‘?’’) and those scored as inapplicable (‘‘-’’). For the
molecular data, only unsequenced base pairs were counted as missing (‘‘?’’); gaps (‘‘-’’) were counted as filled data cells.
c Micoureus was formerly treated as a full genus.
d Formerly identified as Monodelphis adusta (e.g., by Jansa and Voss, 2000; Voss and Jansa, 2003), our material is
referable to M. peruviana, a distinct species recently resurrected from synonymy by Solari (2007).
We amplified 2.1 kb of BRCA1 exon 11
from genomic DNA in two fragments using
the primers listed in table 3. The first
fragment, comprising the upstream 1.2 kb
of the exon, was amplified using primers F1
paired with R1218 or F47 paired with R1343.
The second, downstream fragment (ca. 1 kb)
was amplified with primers F1163 or F1163a
paired with R2078 or R2151. These amplification
products were then used in a second
round of PCR to generate smaller pieces of
suitable size for sequencing. For the upstream
fragment, either F1 or F47 was paired
with R743, and F593 was paired with either
R1218 or R1343. For the downstream
fragment, F1163 or F1163a was paired with
R1780, and F1697 was paired with either
R2078 or R2151. A single fragment of ca.
1 kb was amplified from vWF exon 28 using
either F104 or F120 paired with R1141. This
product was then used in a second round of
PCR in which F1 or F47 was paired with
either R665 or R742, and F557 was paired
with R1141.
Initial amplifications using genomic DNA
as template were performed as 20 ml reactions
using Ampli-Taq Gold polymerase
(Perkin-Elmer Corp.) and recommended
concentrations of primers, nucleotides, buffer,
and MgCl 2. These genomic amplifications
were performed using a four-stage touch-