Amplification and sequencing of mtDNA
In order to augment our microsatellite data, we included mtDNA
previously sequenced from Seal et al. (2015; n = 61) and sequenced new
samples directly for this study (n = 26). A total of 55 samples had both
microsatellite and mtDNA sequenced (Table S1). Again, DNA was extracted
from whole individual ants using a QIAamp DNA Micro Kit (QIAGEN), and a
779-bp sequence was obtained from the COI-tRNA Leucine-COII region of
mtDNA. We used the following primers: C1-J2195 (alias CO1-RLR;
5′-TTGATTTTTTGGTCATCCAGAAGT-3′); and C2-N-3661 (alias Barbara; 5′-
CCACAAATTTCTGAACATTGACCA-3′(Simon et al.,
1994)). PCR mixtures and cycling profiles were identical to Seal et al.
(2015). PCR products were then purified and sequenced at the University
of Texas at Austin’s DNA Sequencing Facility on an Applied Biosystems
3730 DNA Analyzer. Chromatograms were visually checked and resolved in
Geneious v10.2.3 (Kearse et al., 2012), and sequences were aligned using
MEGA v6.06 (Tamura, Stecher, Peterson,
Filipski, & Kumar, 2013) using the ClustalW algorithm
(Thompson, Higgins, & Gibson, 1994).
New sequences are deposited into GenBank under accession numbers
MN088095-MN088120 (Table S1). mtDNA is known to have several properties
that make evolutionary conclusions problematic, such as pseudogenes or
nuclear insertions (numts) (Beckenbach,
2009; Cristiano, Cardoso, &
Fernandes-Salomão, 2014; Martins et al.,
2007; Toews & Brelsford, 2012).
Therefore, we examined for stop codons in our sequences (other than at
the COI-tRNA Leucine transitions) (Seal et
al., 2015). Sequences were also long and frequently readable at
>800bp. Two primary advantages of mtDNA sequences are that
1) sequences can be readily obtained and 2) the problems associated with
mtDNA are understood and can be easily examined, whereas the problems
associated with nuclear markers are more uncertain
(Bowen et al., 2014;
Moreau, 2009). Moreover, a preliminary
examination of the microsatellite markers of T. septentrionalisused in this study supported one of the main findings of mtDNA-based
studies in this species: pronounced genetic differentiation across the
Mississippi River Valley (Matthews et al.,
2020), thus making it unlikely that the COI sequences analyzed were
numts, which usually lack variation because of purifying selection in
nuclear genomes (Martins et al., 2007).
Therefore, mtDNA sequences in this species are likely robust genetic
tools (Matthews et al., 2020;
Mikheyev et al., 2008;
Seal et al., 2015).