Past and present geographic distributions
of plant taxa have been widely studied and have provided numerous insights
regarding evolutionary history. In concert, phylogenetic studies examining unique
and disjunct distribution patterns have further clarified relationships within
groups of plants (Drew and Sytsma, 2012). East Asian/North American
disjunctions are present in approximately 65 plant genera (e.g., Deng et al.,
2015; Tiffney and Manchester, 2001; Wen, 1999). Current evidence suggests that
these disjunctions have occurred since the mid-Miocene, approximately 12
million years ago (Tiffney and Manchester, 2001). This distribution pattern represents
an ongoing opportunity to examine divergence and dispersal patterns of various
taxa. Robust phylogenetic analyses, as opposed to classical morphological
studies, are needed determine the degree of genetic differentiation that has
occurred since a given dispersal event. The subtribe Nepetinae (Lamiaceae; mint
family) is an ideal model to address questions related to East Asian/North
American disjunctions (Harley et al., 2004; Vogelmann, 1984).

The subtribe Nepetinae (sensu Drew &
Sytsma, 2012) contains 13 genera that are almost exclusively found in Eurasia
and the Mediterranean region (Zielinska and Matkowski, 2014). Within the
subfamily, three genera, Agastache, Dracocephalum, and Meehania, share
a disjunct Eurasian/North American distribution. Two of the Nepetinae genera
found within North America, Dracocephalum
and Meehania, are represented by a
single species in North America, with the remaining species occurring in
Eurasia. Agastache is the only member
of the subtribe with a primarily New World distribution, with only one species
found in eastern Asia (Fuentes-Granados et al., 1998; Lint and Epling, 1945).
Hence, Agastache provides an ideal study group to examine relationships and dispersal
events because of its unique distribution (within Nepetinae) and gene regions can
be easily amplified (B. Drew, personnel observation). Agastache is comprised of 22 species of herbaceous
perennials (Zielinska and Matkowski, 2014) and divided into two morphologically
distinct sections, Brittonastrum and Agastache (Sanders, 1987; Lint and Epling,
1945). These sections are primarily defined by staminal orientation and
geographical distribution (Sanders, 1987; Lint and Epling, 1945). The 14 species of section Brittonastrum
are native to the southwestern United States and central and western Mexico.
Section Agastache consists of 8 species, which are mostly native to Eastern, North-Central,
and Western, North America. The exception is A. rugosa, which is native to eastern Asia (Fuentes-Granados et al,
1998; Lint and Epling, 1945).

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In this study, evolutionary processes and
relationships within the subtribe Nepetinae were examined by creating a
phylogenetic tree using the nuclear ribosomal internal and external transcribed
spacer regions (ITS and ETS) and two pentatricopeptide repeat (PPR) gene regions. This study
represents the most comprehensive phylogenetic study of Nepetinae, both in
terms of sequence data and taxonomic coverage, to date, and will provide
insights into relationships within the subtribe and distribution patterns of taxa
within. Specifically, this study (1) creates a more detailed phylogeny of the
subtribe using sequences from herbarium and field collected specimens, (2) determines
the time of dispersal to North America for the three genera with East
Asian/North American disjunctions, (3) proposes a method of dispersion (e.g.,
Bering strait or similar landbridge) for Agastache, and (4) determines
if the dispersal events of Dracocephalum, Meehania, and Agastache
from Eurasia to North America have the same pattern and timing.





Materials and Methods


Sampling and Outgroups


total of 120 accessions, recovered from herbaria and field collections, were
included in this study. Of these accessions, all were sampled from the tribe
Mentheae, and 116 were sampled from within subtribe Nepetinae. All 13 genera in
Nepetinae were sampled: Agastache,
Cedronella, Dracocephalum, Drepanocaryum, Glechoma, Hymenocrater, Hyssopus,
Lallemantia, Lophanthus, Marmoritis, Meehania, Nepeta, Schizonepeta (Drew
and Sytsma 2012). When possible, multiple representations of a genus were used.
Effort was made to collect and include
multiple accessions from genera that are large or have broad geographic
distributions (e.g., Dracocephalum,
Nepeta). The outgroup used was composed of Lycopus uniflorus, Lycopus maackianus, Horminum pyrenaicum, and Prunella vulgaris (Drew & Sytsma


DNA extraction,
amplification, and sequencing


was extracted from herbarium specimens and collected silica-dried plant
material that was collected using the DNeasy Plant Mini Kit (Qiagen, Valencia,
California, USA) according to the manufacturer’s specifications. One
modification to the protocol was made and involved heating the extracts at 65?C
for 30 minutes (instead of 10) to break down secondary compounds that could
interfere with subsequent PCR. Cycle sequencing reactions were performed
following the procedure of Drew and Sytsma (2012). The data were analyzed using
PE-Biosystems of Sequencing Analysis at University of Arizona. Two pentatricopeptide
repeat (PPR) gene regions, AT1G09680 and AT3G09060 were amplified using the
primers as described in Yuan et al. (2010; AT1G09680) and Drew and Sytsma
(2013; AT3G09060). Internal transcribed spacers (ITS) were amplified using the
primers Leu1 (Baldwin, 1992) and ITS4 (White et al., 1990). For amplification
of herbarium specimens, internal primers ITS2 and ITS3 (White et al., 1990)
were used. External transcribed spacers (ETS) were amplified and sequenced as
described in Drew and Sytsma (2012).


Sequence analyses, Phylogenetic
analysis, and divergence time estimation


sequences were edited in Sequencher 5.4.6 (Gene Codes, Ann Arbor, Michigan,
USA) prior to being manually aligned using Mesquite 3.3 (Maddison and Maddison,
2016). Gaps were treated as missing data and indels were not coded.
Phylogenetic analyses for both data sets were performed using two approaches –
Bayesian Inference (BI) and Maximum likelihood (ML). BI utilizing Markov Chain
Monte Carlo (MCMC) was conducted using MrBayes v.3.2.6 (Ronquist and
Huelsenbeck, 2003), with defaults used for the related parameters. All
characters were unordered and equally weighted, with gaps being treated as
missing data. Garli was used for the ML analyses of the data set. Divergence
times were estimated using BEAST v1.8.3 (Drummond et al., 2012), which
estimates phylogenies and divergence times simultaneously.


Calibration points


points were based off of the 95% CI of both the nrDNA analyses from Drew and
Sytsma (2012). We constrained the crown of subtribe Nepetinae with an age range
of 27.4-37.4 myr (Drew and Sytsma, 2012).





Ancestral area estimation was conducted using
Statistical Dispersal-Vicariance Analysis (S-DIVA). S-DIVA compliments DIVA and
statistically evaluates the alternative ancestral ranges at each node in a tree
accounting for phylogenetic uncertainty and uncertainty in DIVA calculations
(Yu et al., 2010). The nrDNA
Bayesian Inference tree was used to reconstruct ancestral area for all members
of the subtribe.



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