phylogenetic relationships and classification of didelphid marsupials ...

64 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 322
RAG2 locus; the gene product is a large
DNA-binding protein of 1043 amino acids
(Schatz et al., 1989; Oettinger et al., 1990,
1992). Recombination activating genes are
expressed only in the nucleus of developing B
and T lymphocytes, where the RAG1 and
RAG2 proteins act synergistically to make
double-stranded breaks at specific recognition
sequences, initiating recombination of
variable (V), diversity (D), and joining (J)
gene segments; completion of the V(D)J
rearrangement process yields functional immunoglobulin
and T-cell receptor proteins,
an essential step in the development of a
mature immune system (Gellert, 2002).
Nucleotide sequence data from RAG1
have previously been analyzed in several
vertebrate phylogenetic studies (e.g., Groth
and Barrowclough, 1999; Barker et al., 2002;
Baker et al., 2004; Gruber et al., 2007). The
2790 bp fragment that we amplified and
sequenced for this study is homologous with
a single full-length copy in Monodelphis
domestica, where the gene is apparently
located on chromosome 5. Although RAG1
occupies the same chromosome as DMP1,
the considerable distance that separates these
loci (.2 3 10 8 bp) suggests that they belong
to different linkage groups. We obtained fulllength
RAG1 sequences from 41 didelphids
(excluding Caluromysiops and Lestodelphys),
and we downloaded shorter (543 bp) outgroup
sequences from Genbank. Ingroup and
outgroup sequences all translate to open
reading frame. Only two indels (of 6 bp each)
were required to align didelphid RAG1
sequences; one indel is unique to Metachirus
nudicaudatus, whereas the other is shared by
Marmosops parvidens and M. pinheiroi.
von Willebrand Factor
The human vWF gene consists of 52 exons
spanning 178 kb of genomic DNA on the
short arm of chromosome 12 (Mancuso et
al., 1989). The gene is normally active only in
vascular endothelium and in bone-marrow
megakaryocytes (Ruggieri and Zimmerman,
1987). The mature (post-translationally processed)
gene product is a large (2050 amino
acids) glycoprotein that is assembled into
multimers of various sizes; these multimers
are either secreted into the plasma or into the
subendothelial matrix (by endothelial cells),
or they are packaged into the m granules of
platelets (by megakaryocytes). Multimeric
vWF mediates adhesion of platelets to
exposed subendothelium following vascular
injury, and it serves as the plasma chaperone
of blood factor VIII, an important regulatory
protein in the coagulatory cascade (Ruggieri
and Zimmerman, 1987; Ginsburg and Bowie,
1992; Sadler, 1998).
Nucleotide sequence data from exon 28 of
the von Willebrand factor gene have been
analyzed in many previous mammalian
phylogenetic studies (e.g., Porter et al.,
1996; Huchon et al., 1999; Neumann et al.,
2006). The 963 bp fragment that we amplified
and sequenced for this study is homologous
with a single full-length copy in
Monodelphis domestica, where the gene is
apparently located on chromosome 8. We
obtained vWF sequences from 41 didelphids
(excluding Caluromysiops and Lestodelphys)
and seven outgroup taxa, all of which
translate to open reading frame. Only three
indels (two of 3 bp and one of 6 bp) are
required to align didelphid vWF sequences;
all three indels are unique to single taxa.
Summary, Sequence Characteristics, and
Model Fitting
Available information suggests that the
five nuclear genes sequenced for this study
exist as single full-length copies in the only
didelphid genome currently available for
inspection, that they are unlinked (on separate
chromosomes or very widely separated),
that they are active in different tissues, and
that their translated protein products have
widely differing functions (table 10). Therefore,
it is reasonable to expect that taxonomic
patterns of nucleotide substitution at these
loci provide substantially independent evidence
of phylogenetic relationships. Additionally,
the coding sequences we obtained
appear to be fully functional (all translate to
open reading frame), and none exhibits
alignment ambiguities among our ingroup
taxa. Other sequence characteristics that
might affect the utility of these genes for
phylogenetic analysis are considered below.
Base-compositional heterogeneity, as indicated
by significant taxonomic variation in