American Journal of Botany 101(11): 1987–2004, 2014.

CHLOROPLAST DNA SEQUENCE UTILITY FOR THE LOWEST

PHYLOGENETIC AND PHYLOGEOGRAPHIC INFERENCES IN
ANGIOSPERMS: THE TORTOISE AND THE HARE IV1
JOEY SHAW2,3,5, HAYDEN L. SHAFER2, O. RAYNE LEONARD4, MARGARET J. KOVACH2,
MARK SCHORR2, AND ASHLEY B. MORRIS4
2Department of Biological and Environmental Sciences, University of Tennessee at Chattanooga, Chattanooga, Tennessee 37403
USA; 3Botanical Research Institute of Texas, Fort Worth, Texas USA; and 4Department of Biology, Middle Tennessee State
University, Murfreesboro, Tennessee 37132 USA

• Premise of the study: Noncoding chloroplast DNA (NC-cpDNA) sequences are the staple data source of low-level phylogeo-
graphic and phylogenetic studies of angiosperms. We followed up on previous papers (tortoise and hare II and III) that sought
to identify the most consistently variable regions of NC-cpDNA. We used an exhaustive literature review and newly available
whole plastome data to assess applicability of previous conclusions at low taxonomic levels.
• Methods: We aligned complete plastomes of 25 species pairs from across angiosperms, comparing the number of genetic dif-
ferences found in 107 NC-cpDNA regions and matK. We surveyed Web of Science for the plant phylogeographic literature
between 2007 and 2013 to assess how NC-cpDNA has been used at the intraspecific level.
• Key results: Several regions are consistently the most variable across angiosperm lineages: ndhF-rpl32, rpl32-trnL(UAG), ndhC-
trnV(UAC), 5′rps16-trnQ(UUG), psbE-petL, trnT(GGU)-psbD, petA-psbJ, and rpl16 intron. However, there is no universally best
region. The average number of regions applied to low-level studies is ~2.5, which may be too little to access the full discrimi-
nating power of this genome.
• Conclusions: Plastome sequences have been used successfully at lower and lower taxonomic levels. Our findings corroborate
earlier works, suggesting that there are regions that are most likely to be the most variable. However, while NC-cpDNA se-
quences are commonly used in plant phylogeographic studies, few of the most variable regions are applied in that context.
Furthermore, it appears that in most studies too few NC-cpDNAs are used to access the discriminating power of the cpDNA
genome.

Key words: chloroplast; DNA barcode; intergenic spacer; intraspecific; intron; noncoding cpDNA; phylogeography; plastid
region; plastome; tortoise and hare.

The chloroplast genome has long been recognized as the
workhorse for testing relationships between biological and geo-
graphical phenomena in angiosperms (Palmer, 1987; Palmer
et al., 1988; Olmstead and Palmer, 1994; Soltis et al., 1997;
Graham and Olmstead, 2000; Kelchner, 2000). Chloroplast se-
quences have been used successfully to infer relationships at all
taxonomic levels, from the deepest-level relationships of land
plants and angiosperms (Chase et al., 1993; Borsch et al., 2003;
Hilu et al., 2003; Moore et al., 2010), through intermediate tax-
onomic levels of orders and families (Downie et al., 2000;
Potter et al., 2007; Chin et al., 2014), to relationships among
closely related species or populations (Soltis et al., 1997, 2006;
Schaal et al., 1998; Shaw and Small, 2004; Morris et al., 2008).
1 Manuscript received 9 September 2014; revision accepted 17 September
2014.
The authors thank Brad Ruhfel, Stephen Downie, an anonymous
reviewer, and the Associate Editor for their careful consideration of this
article. The authors thank Ed Schilling (University of Tennessee) and
James Beck (Wichita State University) for reviewing early drafts of this
manuscript. They also extend special thanks to R. Small, E. Schilling,
E. Lickey, J. Beck, K. Grubbs, S. Farmer, W. Liu, J. Miller, and C. Winder
for their work and important contributions to the earlier chapters of this
research and the Hesler Foundation at the University of Tennessee for
financially supporting the earlier chapters.
5 Author for correspondence (e-mail: joey-shaw@utc.edu)
doi:10.3732/ajb.1400398
It is at this shallowest taxonomic level, (inter- and intraspecific
studies) that researchers are often challenged with finding suf-
ficient genetic variability to address their questions (Schaal
et al., 1998; Schaal and Olsen, 2000; Holderegger and Abbott,
2003; Petit and Vendramin, 2007). Shaw et al. (2005, 2007)
provided guidance in the search for the most consistently vari-
able noncoding chloroplast regions (NC-cpDNA). In “The Tor-
toise and the Hare II” (TH2) (Shaw et al., 2005), the focus was
to compare the relative number of genetic differences typically
found in NC-cpDNA regions that were prevalent in the litera-
ture at that time. In that study, 21 NC-cpDNA regions were as-
sessed for utility within each of 10 seed plant lineages. Each
lineage comprised three congeneric species. The results were
surprising in that the most commonly used regions at that time
(e.g., trnL intron, trnL-trnF), were among the least informative,
while some uncommonly used regions (trnD-trnT, trnS-trnG,
and rpoB-trnC) appeared to be much more informative. In “The
Tortoise and the Hare III” (TH3) (Shaw et al., 2007), the focus
was to expand genomic sampling to all single-copy NC-cpDNA
regions. Initially, paired plastomes from three angiosperm lin-
eages (Atropa vs. Nicotiana [Solanaceae]: asterid; Lotus vs.
Medicago [Fabaceae]: rosid; and Saccharum vs. Oryza [Poa-
cae]: monocot) were used to screen all single-copy NC-cpDNA
regions to search for any that might have higher variability than
the best regions identified in TH2. The outcome highlighted 13
regions, and these were sequenced across the same three-species
groups as the TH2 study. In the end, nine rarely or never before

American Journal of Botany 101(11): 1987–2004, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America

1987
1988 AMERICAN JOURNAL OF BOTANY
used NC-cpDNA regions were identified as being, on average
across angiosperms, the most informative for low-level studies
(rpl32-trnL(UAG), trnQ(UUG)-5′rps16, 3’trnV(UAC)-ndhC, ndhF-
rpl32, psbD-trnT(GGU), psbJ-petA, 3′rps16–5′trnK(UUU), atpI-
atpH, and petL-psbE).
Before the publication of TH3, very few congeneric species
pairs of whole plastomes were available in GenBank, and we
were fortunate to find species pairs within the same family (as-
terids: Solanaceae; rosids: Fabaceae; and monocots: Poaceae).
There are now ~340 angiosperm chloroplast genomes in Gen-
Bank (January 2014), many of which represent congeneric
accessions. This wealth of recent data allows us the new oppor-
tunity to more thoroughly screen the utility of noncoding re-
gions of the plastome across a wider spectrum of the angiosperm
phylogeny to evaluate inferences made in TH3. As a conse-
quence of the rapidly increasing number of whole plastomes
now available in public databases, we have an opportunity to
learn from the growing availability of such data for the benefit
of the larger community of researchers not yet sequencing chlo-
roplast genomes.
With the rapid advancement of next-generation sequencing
(NGS) approaches, we have entered the “Golden Age of Molecu-
lar Ecology” (Paliy, 2013), and sequencing whole plastomes, or
even hundreds of nuclear loci, for low-level inquiry is certainly
the future (Soltis et al., 2013). It is becoming increasingly feasi-
ble to sequence whole or nearly whole plastomes using high-
throughput methods (Cronn et al., 2012; De Wit et al., 2012; Shi
et al., 2012; Straub et al., 2012; Stull et al., 2013; Uribe-Convers
et al., 2014), and the cost savings over Sanger sequencing can be
significant for large-scale studies involving many taxa (Godden
et al., 2013) or for laboratories that are already established with
the needed laboratory and computational infrastructure. Core se-
quencing facilities now offer opportunities for multiple laborato-
ries to share the cost of an NGS run, further lowering the price
per sample. While there are a few notable cases in which re-
searchers have published phylogenies based on whole plastome
data, most are targeted at resolving deep relationships (Ruhfel
et al., 2014) or tackling questions in model organisms (Eserman
et al., 2014) or economically important groups (Nikiforova et al.,
2013; Njuguna et al., 2013), but this is rapidly changing. At pres-
ent, the majority of whole plastome publications are limited
to one or a few accessions per study (e.g., Martin et al., 2013;
Walker et al., 2014), and these typically come from the larger
research laboratories, reflecting the financial and computational
costs associated with this kind of data generation (Rocha et al.,
2013; Soltis et al., 2013). The start-up costs for hardware and
software alone may prohibit smaller, lesser-funded laboratories
around the world from moving from sequencing a few NC-
cpDNA regions to generating massive quantities of genomic data.
For smaller-scale studies, or studies coming from lesser-funded
or equipped laboratories, the cost of NGS and the complexity of
bioinformatics analyses required for such large data sets may still
be prohibitive (Doyle, 2013; Rocha et al., 2013; Bowen et al.,
2014; but see Straub et al., 2012 for an alternative viewpoint).
Bowen et al. (2014), in summarizing results from a meeting at the
National Evolutionary Synthesis Center (NESCent), suggested
that (1) single mtDNA locus studies (for plants, we would mod-
ify this phrase to “one to several cpDNA loci studies”) provide
powerful first assessments of patterns; (2) genome-wide analyses
are warranted if results from standard markers are discordant
with other aspects of the organisms biology; (3) at this stage of
software development and technology, a judicious rather than
wholesale application of genomics is prudent. Given all of this,
[Vol. 101
we anticipate that Sanger or NGS of a smaller number of indi-
vidual NC-cpDNA regions will continue to be an important ap-
proach for many exploratory inquiries, low-level systematics and
phylogeographic studies, and for barcoding studies, at least for
the near future. Therefore, there is still a need to be able to select
from a few, presumably the most potentially informative, NC-
cpDNA regions.
In the present study, we asked: Which, and how many, of the
“most informative regions” of the TH series have been useful at
the lowest phylogenetic and phylogeographic levels in angio-
sperms? While we are particularly interested in intraspecific data,
there are currently no publicly available whole plastome phylo-
geography data sets, nor are there many publications for such an
assessment (but see Whittall et al., 2010 and Doorduin et al.,
2011). Therefore, we addressed our question by analyzing a large
set of whole plastomes, reflecting as many low-level taxonomic
comparisons as are currently available through the NCBI Organ-
elle Genome Resources (GenBank). We used 25 closely related
species pairs—congeners when possible (20/25)—across 10 ma-
jor lineages of angiosperms (Nymphaeales, magnoliids, mono-
cots, Proteales, eurosid I, eurosid II, Caryophyllales, Ericales,
euasterid I, euasterid II) (Fig. 1) to compare all single-copy NC-
cpDNA regions and matK because it is among the most variable
coding regions and has been a popular region since 1997 (Hilu
and Liang, 1997). We also were able to compare the results
within the major clades to overall results.
In addition to this new data set, we searched the literature cov-
ering the period since the last TH paper (2007–2013). To deter-
mine whether NC-cpDNA sequences are an important tool for
low-level studies, we asked: What percentage of intraspecific
studies of plants used cpDNA sequence data? Beyond this initial
question, we also asked: Which NC-cpDNA regions were chosen
and sequenced? How many NC-cpDNAs were needed to gener-
ate sufficient data? How many papers explicitly reported screen-
ing NC-cpDNAs, and how many were typically screened?
All results are discussed in the context of the trends in the
current literature and findings of our previous TH studies. In the
end, we propose a strategy for investigators of low-level taxo-
nomic or phylogeographic studies using NC-cpDNA sequence
data.
MATERIALS AND METHODS
Taxon selection for genomic comparisons—Initially, we set out to use only
congeneric species pairs in angiosperms; however, doing so, left us with phylo-
genetic gaps in our sampling. To fill in these phylogenetic gaps, we used the
most closely related species pairs available in a few lineages, resulting in an
initial list of 34 angiosperm species pairs for comparison. After we had com-
piled all of the data, four species pairs were removed (Acorus americanus and
A. calamus, Nicotiana sylvestris and N. tabacum, Oryza nivara and O. sativa,
and Phyllostachys edulis and P. nigra var. henonis) because they contained too
few genetic differences between them, as we later explain in detail. We also had
to remove five other species pairs (Wolffia australiana and Wolffiella lingulata,
Colocosia esculenta and Lemna minor, Ranunculus macranthus and Megaler-
anthus saniculifolia, Cuscuta exaltata and C. obtusiflora, and Erodium carvifo-
lium and E. texanum) because they were too variable to be confidently aligned
across the entire genome (the large indels and genomic rearrangements in these
species pairs that made our analyses difficult could certainly provide workers of
these groups with important information in other contexts). The remaining 25
species pairs (including 20 congeneric pairs) used in our analyses are shown in
Fig. 1. We made a strong effort to sample across the phylogenetic breadth of
angiosperms, using nomenclature and topology from the Angiosperm Phylog-
eny Group (http://www.mobot.org/MOBOT/research/APweb/). We made an
effort to sample about the same number of species pairs per major lineage. In
the end, we were able to sample one species pair from Nymphaeales, two from
November 2014] SHAW ET AL.—TORTOISE AND HARE IV 1989

Fig. 1. Phylogenetic breadth of sampling for plastome comparisons. Species pairs (mostly congeners) are listed beside family names and next to major
lineages. Tree topology follows Angiosperm Phylogeny Group III (APG III) (http://www.mobot.org/MOBOT/research/APweb/).

magnoliids, five from monocots, one from Proteales, four from eurosid I, four
from eurosid II, one from Caryophyllales, one from Ericales, three from euaste-
rid I, and three from euasterid II (Fig. 1).
NC-cpDNA selection—Rather than try (1) to determine exactly where the
large single copy (LSC), small single copy (SSC), and inverted repeat (IR) re-
gions occur in each of the 25 lineages and (2) expand the data set to include
uncommon intergenic spacers (i.e., intergenic spacers unique to a given taxon
because of genomic rearrangements unique to that taxon), we used the Gossy-
pium hirsutum genome (Lee et al., 2006) as a model. It was used because it is a
typical chloroplast genome to determine which regions to include or exclude as
well as the starting and stopping points of the LSC, SSC, and IRs.
It is well accepted that the most variable portions of the cpDNA genome are
not in the IR regions (Clegg et al., 1984; Wolfe et al., 1987). The IR regions
have been shown to contain low levels of variability (Maier et al., 1995), and
Presting (2006) confirmed and quantified earlier reports that the IR was signifi-
cantly more resistant to genetic changes compared to the single-copy regions.
We therefore concentrated on the single-copy portions of the genome.
The two single-copy regions consist of the LSC, which is typically about 80
kb long, and the SSC, which is typically 20 kb. These regions contain a combi-
nation of rRNA and tRNA genes, as well as protein-encoding genes. It is com-
mon knowledge that gene-encoding regions will accumulate genetic differences
more slowly than noncoding regions, the exceptions being matK (Steele and
Vilgalys, 1994; Johnson and Soltis, 1995; Fazekas et al., 2008; Lahaye et al.,
2008) and perhaps ycf1 (Dong et al., 2012), which seem to accumulate mutations
at a fast pace for coding regions. Because matK has historically been an impor-
tant region (Hilu and Liang, 1997) and is commonly recognized as being as
variable as many noncoding regions of the plastome, our research effort was
focused on matK and all single-copy, noncoding regions of the plastome. In
total, 107 noncoding regions and matK were compared across 25 species pairs
spanning the breadth of angiosperms.
Sequence alignment—The GenBank nucleotide BLAST function was used for
initial alignment. BLAST aligns local regions of two separate sequences based on
nucleotide similarity. In initial BLAST searches, we found that some of the more
genetically dissimilar species pairs (e.g., Aethionema) often contained too many
gaps to ensure reliable alignments. Thus, we altered the default BLAST algorithm
parameters to increase the standard linear gap cost, as a measure to reduce the pres-
ence of misaligned sequences. BLAST parameters were as follows: Max target
sequences = 100, expected threshold = 10, Max matches in a query range = 0,
Match/Mismatch scores = 1-2, and Gap Costs = Existence: 5; Extension: 2. After
the sequences were aligned with BLAST, the alignments were visualized in Por-
table Document Format, and the gene-encoding regions were unequivocally de-
noted using the annotations in GenBank. Alignments were manually scored for
genetic differences. In a few cases where small sections of the alignments were too
variable to be confidently aligned (e.g., poly A/T regions), gaps were opened up,
and these were conservatively scored as a single genetic difference (an indel).
Data analysis—All noncoding portions of the genome, including intergenic
spacers (IGS) and introns, were analyzed for genetic differences between the
1990 AMERICAN JOURNAL OF BOTANY
species pairs, and these genetic differences were counted as potentially infor-
mative characters (PICs) following Shaw et al. (2005, 2007). (PICs have been
shown to be a good predictor of parsimoniously informative characters [Fior
et al., 2013].) The PICs included indels, nucleotide substitutions, and inversions.
Indels and inversions were scored as binary (presence/absence) characters fol-
lowing Simmons and Ochoterena (2000). The process of alignment, annotation,
and analysis was repeated for each of the 25 species pairs. The length of the
noncoding region was recorded, and the number of PICs tallied for each non-
coding cpDNA region and matK in each of the 25 lineages. All genetic differ-
ences were scored as independent, single characters.
Three types of calculations were performed to evaluate NC-cpDNA variabil-
ity. First, we estimated the proportion of observed genetic differences for each
NC-cpDNA using a modified version of the formula used in O’Donnell (1992),
Gielly and Taberlet (1994), and Shaw et al. (2005, 2007). The proportion of ge-
netic differences (or % variability) = [(NS + ID + IV) / L] × 100, where NS = the
number of nucleotide substitutions, ID = the number of indels, IV = the number
of inversions, and L = the aligned sequence length. Second, we averaged the
number of PICs found within each noncoding chloroplast region across the 25
lineages that contained those NC-cpDNA regions (if the region was missing in a
lineage, the total was divided by 24 rather than 25). Third, to ensure that lineages
containing a greater number of genetic differences between the species pair
(greater evolutionary distance) were not overrepresented (weighted) in global
comparisons, we determined the percentage contribution of each noncoding re-
gion to the overall PIC total within a lineage (what was called “normalized PICs”
in earlier TH papers). In effect, we calculated the percentage contribution of a
NC-cpDNA to the number of genetic differences observed in a species pair.
These values were then used to generate an average value for each noncoding
region so that the regions could be directly compared.
We argue that the percentage contribution values are the most accurate for
comparisons of NC-cpDNA variability across lineages because they reduce the
overrepresentation of average PIC values from the species pairs that evolved at
faster rates or are evolutionarily further apart. For example, Silene species ac-
cumulated 1483 PICs across all regions, while Olea species only had 321 PICs.
Without this “normalization” of the data, the species pair with the greatest over-
all number of genetic differences (e.g., Silene) would more strongly influence
the analysis compared with other lineages that had many fewer genetic differ-
ences between species. A downside to the percentage contribution analysis is
that species pairs with a very low number of genetic differences between them
tend to have overly high scores in this analysis. For example, the Nicotiana
species pair was omitted because there were only two nucleotide substitutions
across the entire alignment, and therefore, each NC-cpDNA containing them
would have the very large percentage contribution PIC score of 0.50. For this
reason, we omitted species pairs with fewer than 100 PICs in the alignment
(discussed earlier in Taxon selection for genomic comparisons).
Adjusted mean PICs—A mixed-model analysis of covariance (ANCOVA)
was used to look for statistical separation between the 10 NC-cpDNAs that
ranked highest in the overall percentage contribution analysis. We chose to ana-
lyze only 10 regions because statistical inference would be weakened with all
108 regions. Furthermore, we argue that if statistical separation among the top
10 is present, then it is sure to exist between these and the rest. The mean PICs/
region was treated as the response variable and the fixed-effects factor was the
NC-cpDNAs; lineage was treated as a block (random-effects factor) and region
length (number of base pairs per region) as a covariate. Data were log-transformed
[log10 (PIC + 1)]; [log10 (length + 1)] to satisfy the parametric test (ANCOVA)
assumptions of normality, variance homogeneity, and linearity. Region-specific
PIC estimates were reported as geometric means ± SE.
A preliminary ANCOVA indicated that there was a direct linear relationship
between the number of PICs/region and length of the region (P < 0.0001) and
that the slopes of PIC-length relationships were similar (P > 0.10), which al-
lowed us to compare slope-adjusted mean PICs/region. Region-specific esti-
mates of adjusted mean PICs (y), which control for the effect of region length
(x, covariate), were determined using linear regression models [log (y+ 1) = a +
b(x + 1)] with a common slope (b = 0.9427) and length value (x = mean length =
~1073 bp). Scheffé’s method was used to make pairwise comparisons (con-
trasts) between region-specific mean PICs and/or groups of mean PICs, based
on patterns exhibited by the data. Scheffé’s procedure, a highly conservative
post hoc test that maintains a familywise type I error rate at or below the alpha
level, is recommended for multiple contrasts between groups of sample means
(Zar, 2010).
Data were analyzed using the UNIVARIATE, REG, GLM, MIXED, and
LSMEANS procedures of the Statistical Analysis System (SAS Institute,
2011). Statistical significance was declared at an alpha level of 0.05.
[Vol. 101
Literature review—To answer the question “What proportion of intraspe-
cific plant studies used cpDNA sequence data?”, we performed a Web of Sci-
ence search in May 2014 by confining the dates to 1987–2013 and searching on
the terms “phylogeography or phylogeographic”, refined to articles, then fur-
ther refined by “*aceae”. Subsequent refinements were performed using either
“nuclear or nDNA” or “mitochondria or mitochondrial or mtDNA” or “chloro-
plast or plastid or cpDNA”.
To address the other questions posited in the Introduction section, we per-
formed a search on Web of Science in January 2014. We first used the topic
search terms “phylogeography or phylogeographic” limited to the years 2007–
2013. We refined this number using the topic search terms “chloroplast or cp-
DNA” to focus specifically on plant phylogeographic studies. While this may
exclude papers that exclusively use nuclear markers, we determined this to be
the most appropriate search strategy for the present survey. The top five source
titles for these plant phylogeographic studies during the selected 7 years were
Molecular Ecology (99), Journal of Biogeography (79), Molecular Phylogenet-
ics and Evolution (45), American Journal of Botany (32), and Plant Systematics
and Evolution (28), representing approximately 41% of total publications under
our search criteria. Because our emphasis here is on low taxonomic level and
population studies, we excluded papers in Molecular Phylogenetics and Evolu-
tion and Plant Systematics and Evolution due to the more traditional systematic
nature of publications therein. Of the total of 210 plant phylogeographic studies
in the remaining three journals (Molecular Ecology, Journal of Biogeography,
and American Journal of Botany), 33 were excluded following our review for
either being a review or focused on an animal, more than three species, or a
nonvascular plant. This exclusion resulted in 178 publications being included in
our review.
We built a database containing the following information for each of these
178 studies: author, year of publication, source title, taxon, family, markers
tested (when available), markers used, and length and PICs for markers used
(when available). We used these data to answer the questions posited in the
introduction.
We were also interested in comparing relative utility of regions within stud-
ies to determine whether patterns predicted by TH2 and TH3 were supported by
the literature. For this comparison, we could only include studies that explicitly
stated the number of PICs/region and used at least two or more regions. Of the
178 studies included in our review, 45 met these criteria. The remaining studies
either reported PICs across all regions combined, or many did not report PICs
at all. For these 45 studies, NC-cpDNAs were ranked by decreasing number of
PICs observed, and trends were qualitatively assessed across studies.
RESULTS
Overview of molecular data set— In all, we manually exam-
ined over 2.5 million base pairs of aligned data from 25 species
pairs, resulting in a data set of 107 single-copy NC-cpDNA re-
gions including 15 introns, 92 IGS, and matK for 25 species
pairs (Appendix S1, see Supplemental Data accompanying the
online version of this article).
Taxon selection, omission, and results on omitted taxa—At
the outset, we identified 34 congeneric or fairly closely related
species pairs; however, five of the pairs were too variable to be
confidently aligned, and four were too invariable to yield reli-
able information. There were only two genetic differences be-
tween Nicotiana sylvestris (NC_007500) and Nicotiana tabacum
(NC_001879); one difference was in the psbM-trnD(GUC) spacer,
and the other was in the petD intron. There were only 22 genetic
differences between Acorus americanus (NC_010093) and
Acorus calamus (NC_007407). These differences were found in
ndhF-rpl32 (1), ccsA-ndhD (1), ndhG-ndhI (1), 5′trnK(UUU)-
3′rps16 (2), rps16 intron (1), trnQ(UUG)-psbK (1), trnC(GCA)-petN
(2), trnD(GUC)-trnY(GUA) (1), trnT(GGU)-psbD (3), trnT(UGU)-
trnL(UAA) (1), trnM(CAU)-atpE (1), atpB-rbcL (1), ycf4-cemA (1),
petA-psbJ (1), psbE-petL (1), psaJ-rpl33 (1), rps18-rpl20 (1),
and rpl16 intron (1). We only observed 26 genetic differences
between Oryza nivara (NC_005973) and Oryza sativa (NC_
008155), and these were found in ndhF-rpl32 (1), ccsA-ndhD
November 2014] SHAW ET AL.—TORTOISE AND HARE IV
(1), ndhA intron (1), psbA-3′trnK(UUU) (1), rps16 intron (2),
trnD(GUC)-trnY(GUA) (2), trnE(UUC)-trnT(GGU) (2), psbZ-trnG(GCC)
(2), ycf3-trnS(GGA) (2), ndhC-trnV(UAC) (3), trnM(CAU)-atpE (1),
accD-psaI (1), petL-petG (1), petD-rpoA (1), rps11-rpl36 (1),
rpl14-rpl16 (2), rpl16 intron (1), and rpl22-rps19 (1). Twenty-
four genetic differences were observed between Phyllostachys
edulis (NC_015817) and Phyllostachys nigra (NC_015826). In
Phyllostachys, differences were found in rpl32-trnL(UAG) (5),
ccsA-ndhD (2), psaC-ndhE (2), matK-5′trnK(UUU) (1), 5′rps16-
trnQ(UUG) (2), rpoB-trnC(GCA) (1), petM-psbM (1), psbM-trnD(GUC)
(1), ycf3 intron1 (1), ycf3-trnS(GGA) (1), trnT(UGU)-trnL(UAA) (2),
ndhC-trnV(UAC) (1), rpl33-rps18 (1), petB intron (1), and rpl14-
rpl16 (2). We also had to remove species pairs because they were
too variable to be confidently aligned; that is, between these spe-
cies pairs, GenBank BLAST opened up large gaps with scattered
unaligned nucleotides, and we could not manually improve on
these alignments. Species pairs in which this was the case in-
clude: Erodium carvifolium (NC_015083) and Erodium texanum
(NC_014569); Cuscuta exaltata (NC_009963) and Cuscuta gro-
novii (NC_009765); Wollfia australiana (NC_015899) and Woll-
fiella lingulata (NC_015894); Colocasia esculenta (NC_016753)
and Lemna minor (NC_010109); and Ranunculus macranthus
(NC_008796) and Megaleranthus saniculifolia (NC_012615).
In a few cases, one or two NC-cpDNAs had to be omitted
from an otherwise neatly aligned genomic comparison because
these NC-cpDNAs were too variable to be confidently aligned.
This was the case in Nuphar/Nymphaea for petN-psbM, Phoe-
nix/Pseudophoenix for trnC-petN and petN-psbM, Gossypium
for psbE-petL, Silene for trnH(GUG)-psbA and clpP-psbB, and
Anthriscus/Daucus for ndhF-rpl32, rpl32-trnL(UAG), 5′trnK-
3′rps16, 5′rps16-trnQ(UUG), and atpH-atpI (Appendix S1).
The most informative noncoding cpDNA regions—Figure 2A
shows the number of genetic differences observed within every
single-copy NC-cpDNA and matK, averaged across 25 species
pairs. In this analysis, the top 10 regions were: 5′rps16-trnQ(UUG),
ndhC-trnV(UAC), ndhF-rpl32, trnT(GGU)-psbD, psbE-petL, petA-
psbJ, rpl32-trnL(UAG), rpl16 intron, ndhA intron, and rpoB-
trnC(GCA) (underlines highlight the regions in common between
this analysis and the analysis of average percentage contribution
to total PICs, below). The matK gene was ranked 12th. Figure 2B
summarizes the PIC data where the value for each NC-cpDNA
represents the average percentage contribution of the total genetic
differences observed in pairwise species comparisons averaged
across the 25 lineages. In this analysis, the top 10 regions were:
ndhF-rpl32, ndhC-trnV(UAC), rpl16 intron, rpl32-trnL(UAG),
5′rps16-trnQ(UUG), 5′trnK(UUU)-3′rps16, psbE-petL, trnT(GGU)-
psbD, petA-psbJ, and psbM-trnD(GUC) (underlines highlight the
regions in common between this analysis and the analysis of over-
all average PICs, above). The matK gene was again ranked 12th.
While the relative positions of the most variable regions may
change between the averaged PIC and percentage contribution
of averaged PIC analyses (or even lineage to lineage, see be-
low), there are eight NC-cpDNAs in the top 10 of both of these
analyses and these are underlined above and throughout the rest
of this paper. These eight regions account for roughly 21% of
the PICs in a given lineage (based on percentage contribution of
each region averaged across the 25 species pairs). The top 12
regions account for >33% of the total PICs.
Percentage variability within each region is shown in Fig. 2C.
Eight of the top 11 most variable regions in this analysis had an
average total length of about ≤250 bp and 9/11 were ≤350 bp,
making the total PICs offered by these regions relatively low even
1991
though their percentage variability was high (compare Fig. 2C
with Fig. 2A and 2B). The rpl32-trnL(UAG) and rps15-ycf1 regions
were the only two of the top regions that were over 500 bp and
only rpl32-trnL(UAG) was over 750 bp. Because choosing the most
informative NC-cpDNAs requires combining the effects of NC-
cpDNA length and percentage variability, Fig. 2D is positioned
next to Fig. 2C to highlight the fact that several NC-cpDNAs that
are highly variable by percentage are also very short.
Because we had more than one species pair within most ma-
jor lineages, we compared the trends in the major clades of an-
giosperms with the overall trends described above (Fig. 3A–F).
While the ranking of the top regions is not perfectly consistent
across the major lineages, there are some NC-cpDNAs that
consistently rank among the top 13 (top 13 shown to incorpo-
rate matK because it has been a popular and informative marker
since the beginning of sequencing cpDNA).
Either ndhF-rpl32 or rpl32-trnL(UAG) was the highest ranked
region in four of six major lineages, in one lineage rpl32-trnL(UAG)
was ranked second behind 5′rps16-trnQ(UUG), and both regions
were too variable to be included in one species comparison (An-
thriscus/Daucus). The 5′rps16-trnQ(UUG) region ranked in the top
two in two of six major lineages and in the top 13 in four of six
lineages, but was excluded from one lineage for being too vari-
able (Daucus/Anthriscus). It should be noted that eurosid I was
the only major lineage in which neither ndhF-rpl32 nor rpl32-
trnL(UAG) ranked in the top 2; however, these NC-cpDNA regions
are missing from Populus and unusually small in Vigna, perhaps
skewing the data because these two lineages account for two of
four species pairs of the eurosid I group. The rpl32-trnL(UAG) re-
gion did rank in the top five in the other eurosid I, Cucumis. An-
other high-ranking region, ndhC-trnV(UAC), ranked in the top five
in four of six of the major lineages.
Analysis of the top eight most variable regions in the 25 indi-
vidual species pair comparisons (Fig. 4) shows that ndhC-
trnV(UAC), rpl32- trnL(UAG), and ndhF-rpl32 all ranked in the top
10 in 16 of 25 lineages, psbE-petL in 15 of 25 lineages (and it
was too variable to be included in Gossypium), trnT(GGU)-psbD
in 14 of 25 lineages, 5′rps16-trnQ(UUG) and petA-psbJ in 11 of
25 lineages, and rpl16 intron in 10 of 25 lineages.
Adjusted mean PICs— Results of the mixed-model AN-
COVA (which accounted for variation across lineages and con-
trolled for the effect of region length) indicate that the mean
log-transformed number of PICs/region differed across the 10
NC-cpDNAs that ranked the highest in the overall percentage
contribution analysis (F9,194 = 3.09, P = 0.0017). Following the
ANCOVA, we performed pairwise contrasts of adjusted means
and/or groups of means using Scheffé’s procedure; selected
comparisons involving combined DNA regions (combined vs.
combined; single vs. combined) revealed significant differ-
ences in the mean PICs/region between certain groups of the 10
regions.
Scheffé’s contrasts revealed significant differences in ad-
justed mean PICs/region among three groups of combined
DNA regions (P < 0.05; Fig. 5). Mean PICs/region (compared
as groups based on visual breaks in the data) was highest for
Group 1 (geometric mean = 19.2 PICs; regions ndhF-rpl32,
rpl32-trnL(UAG)), intermediate for Group 2 (12.7 PICs; regions
ndhC-trnV(UAC), rpl16 intron, petA-psbJ), and lowest for Group
3 (10.2 PICs; regions 5′rps16-trnQ(UUG), psbE-petL, trnT(GGU)-
psbD, ndhA intron, rpoB-trnC(GCA)). Pairwise contrasts within
the three groups yielded no differences in mean PICs/region
(P > 0.05).
1992 AMERICAN JOURNAL OF BOTANY [Vol. 101

Fig. 2. Expected number of genetic differences within the noncoding single-copy portions of the plastome (NC-cpDNA). Gene order is preserved
beginning at the top with the intersection of inverted repeat b (IRb) and the large single-copy (LSC) region; the small single-copy (SSC) region is at the
bottom and begins with rps15-ycf1. The vertical black lines and the four-point stars highlight the 10 regions that on average contain the greatest number of
potentially informative characters (PICs) in those analyses (A, B). (A) Number of PICs averaged across the 25 species pairs. (B) Average percentage of
potentially informative characters (PICs) that each noncoding region contributes to the total PICs observed between species pairs. (C) Percentage variabil-
ity averaged across 25 species pairs. The top 11 most variable regions by percentage are cutoff from the rest by the vertical black line and the four-point
stars (there was a tie for 10th place). Arrows from NC-cpDNAs in (C) point to the same regions in (D) to highlight the fact that 8/11 of the most variable
regions in terms of percentage variability are too short to be of great value. (D) Length of noncoding regions averaged across 25 species pairs. The black
line in (D) marks 250 bp to highlight those regions that may be potentially highly variable in terms of percentage but are less than 250 bp long.
November 2014] SHAW ET AL.—TORTOISE AND HARE IV 1993

Fig. 2. Continued.
1994 AMERICAN JOURNAL OF BOTANY [Vol. 101

Fig. 3. Clade-specific ranks of single-copy noncoding NC-cpDNAs (top 13). Percentage contribution of total potentially informative characters (PICs)
(y-axis) for each NC-cpDNA (x-axis) averaged across the species pairs in the clade. The eight highest-ranking regions in the analysis of all 25 lineages
combined are marked with four-pointed stars (ndhF-rpl32, rpl32-trnL, rps16-trnQ, ndhC-trnV, trnT-psbD, psbE-petL, petA-psbJ, rpl16 intron).

In another contrast (excluding Group 1), the mean PICs/region
for the highest-ranking Group 2 region (geometric mean = 14.7
PICs; region ndhC-trnV(UAC) was higher than that for other
regions in Groups 2 and 3 (10.6 PICs; regions rpl16 intron, petA-
psbJ, 5′rps16-trnQ(UUG), psbE-petL, trnT(GGU)-psbD, ndhA intron,
rpoB-trnC(GCA); P < 0.05). In summary, the ANCOVA and mul-
tiple contrasts of adjusted mean PICs/region within the top 10
regions (averaged across 20–25 lineages) indicated that ndhF-
rpl32, rpl32-trnL(UAG), and ndhC-trnV(UAC) were the most vari-
able frames, respectively.
November 2014] SHAW ET AL.—TORTOISE AND HARE IV
Literature review—The total number of “phylogeography or
phylogeographic” papers published between 1987 and 2013 was
10 196. We refined this number by limiting the selections to arti-
cles only (9286) and refining by the search term “*aceae” to cap-
ture any mention of a plant family in the text, resulting in a total of
1089 papers. Further refining by the search terms “chloroplast or
plastid or cpDNA” resulted in 699 papers; refining by “mitochon-
dria or mitochondrial or mtDNA” resulted in 351 papers; refining
by “nuclear or nDNA” resulted in 253 papers (Fig. 6).
For the literature review, 178 papers published between 2007
and 2013 were reviewed for this study. Of those 178, 123 (69%)
used cpDNA sequence data (Fig. 7). The percentage of papers
using cpDNA sequences varied by year, but ranged between
50% (2007) and 81% (2012). The average number of cpDNA
regions used was two to three, with a minimum of one to a
maximum of six. Only 28 of the 178 papers explicitly reported
screening additional loci, although it was not always intimated
what loci were tested. When this information was provided, we
determined that the mean number of loci screened by year
ranged from four to 15, with a few papers indicating “several”
regions were screened.
Comparing the noncoding regions within studies showed that
in 11 comparisons, trnH(GUG)-psbA provided more PICs than
other regions in the same study. However, in six other studies,
trnH(GUG)-psbA was not the best performer. The region with the
second highest observed PICs was trnS(GCU)-5′trnG(GCC), based
on nine comparisons. There were only two studies (in which it
was included) that it was not the top performer. Of the top re-
gions reported in the present study, two (ndhC-trnV(UAC) and
psbM-trnD(GUC) were not used in any of the studies reviewed for
this comparison. The remaining top regions identified here
were used infrequently in the surveyed papers: rpl32-trnL(UAG)
(best in three of four studies); ndhF-rpl32 (best in the one study
in which it was used); psbE-petL (best in the one study in which
it was used); trnT(UGU)-trnL(UAA) (best in one of two studies);
trnT(GGU)-psbD, rpoB-trnC(GCA), and petA-psbJ were not best in
any of the studies in which they were used (2, 1, and 2 studies,
respectively; Appendix S2, see online Supplemental Data).
DISCUSSION
NC-cpDNAs are important for low-level studies— Today,
data from NC-cpDNA is the most commonly used tool for phy-
logeographic and low-level phylogenetic studies of plants (Fig.
6). Our literature review highlighted the fact that researchers
working at inter- and intraspecific levels still rely heavily on
NC-cpDNA sequence data from one to a few loci (Figs. 6, 7).
Of the more recently published plant phylogeography papers,
those published between 2007 and 2013, nearly 70% used cp-
DNA sequence data (Fig. 7).
There is a body of literature dating back to the 1990s in
which botanists have been searching for the most appropriate
NC-cpDNAs for low-level phylogenetic and phylogeographic
studies (Taberlet et al., 1991; Demesure et al., 1995; Dumolin-
Lapegue et al., 1997b; Small et al., 1998; Hamilton, 1999;
Saltonstall, 2001; Shaw et al., 2005, 2007). In the mid-2000s, a
few studies compared numerous NC-cpDNA regions across a
broad enough taxonomic scope such that general trends about
NC-cpDNA region relative variability could be reported (Small
et al., 1998; Bastia et al., 2001; Grivet et al., 2001; Aoki et al.,
2003; Dhingra and Folta, 2005; Shaw et al., 2005, 2007; Ebert
and Peakall, 2009). Reports of these general trends may have
1995
allowed workers to begin utilizing the most informative NC-cpDNA
regions and push the use of the plastome further toward the
shallowest taxonomic levels.
The most informative noncoding cpDNA regions—A few
NC-cpDNA regions stand out as being the most likely to contain
high levels of variability at the inter- and intraspecific levels, even
though this study (Figs. 2–4) and a body of literature (Mort et al.,
2007; Shaw et al., 2007; Särkinen and George, 2013) reinforces
the observation that there is no universally best NC-cpDNA. That
said, screening what are known to be, on average, the fastest
evolving regions is a good starting point for molecular investiga-
tions of new groups (Cires et al., 2012; Fior et al., 2013). In the
best-case scenario, researchers would have, or could generate, at
least two complete plastomes within their study genus (or study
section of large genera) to screen for the most informative re-
gions, before beginning a phylogeographic or low-level phyloge-
netic study (Doorduin et al., 2011; Fehrmann et al., 2012; Li
et al., 2013; Särkinen and George, 2013). Since there may be as
many as 17 000 genera of angiosperms (http://www.theplantlist.
org) and GenBank presently (August 2014) has only 374 angio-
sperms plastomes representing 205 genera—and a fair number of
these are either species poor, endemic, or genera of parasitic
plants and not likely to be useful predictors of variability across
large numbers of species—it is unlikely that most researchers
will have this advantage for several more years.
While it is true that the cost of NGS approaches are rapidly
declining, and novel and simpler methods for undertaking such
work are being rapidly published, many researchers (particularly
at primarily undergraduate institutions) are still limited by fund-
ing and computational capacity. For these researchers, as well as
others, there still remains a trade-off between collecting “big
data” for a small number of taxa or individuals at the same cost
of collecting “small data” for a larger number of taxa or individu-
als. The cost of software (e.g., Geneious) may be inexpensive to
some, yet to other researchers this cost alone may be prohibitive.
Additionally, for phylogeographic studies, software has not yet
fully developed to be able to handle the computational loads as-
sociated with NGS big data. Therefore, the choice between NGS
and Sanger approaches, while not necessarily mutually exclusive
(Fior et al., 2013), is going to be an independent one based on
funding (both short-term and long-term potential), laboratory in-
frastructure, and computational support in a given laboratory. As
such, there is still a valid need for information such as that pre-
sented here, providing insight into which NC-cpDNA regions
or portions of the plastome are likely to be most informative
(Fig. 8). That is, the results of our work can guide researchers
who want to use the most variable portions of the plastome,
whether they are amplified independently, in larger clusters, mul-
tiplexed, or extracted from whole plastome data sets.
That said, three regions of the plastome stand out to us as
hotspots of variability (Fig. 8). First is the area in the SSC that
runs from ccsA to ndhF. This portion of the plastome contains
two of the most variable single regions (ndhF-rpl32 and rpl32-
trnL(UAG)). The second hotspot, is a larger area of the genome
from matK to 3′trnG(GCC), which contains several fairly vari-
able regions including matK, 5′trnK(UUU)-3′rps16, 5′rps16-
trnQ(UUG), and trnS(GCU)-5′trnG(GCC), among others. The third
hotspot is from rpoB to psbD, and this larger portion of the ge-
nome contains several high-ranking regions all clustered to-
gether. Interestingly, there are a fair number of rearrangements
in these regions of the plastome as well (Appendix S1). Based
on the extensive plastome survey included in the present study,
1996 AMERICAN JOURNAL OF BOTANY [Vol. 101

Fig. 4. Potentially informative characters (y-axis) for each NC-cpDNA (x-axis) within each species pair. The eight highest-ranking regions in the
analysis of all 25 lineages combined are marked with four-pointed stars (ndhF-rpl32, rpl32-trnL, rps16-trnQ, ndhC-trnV, trnT-psbD, psbE-petL, petA-psbJ,
rpl16 intron). Mag. = Magnolia, Cymb. = Cymbidium, Phalae. = Phalaenopsis, Aeth. = Aethionema, Sol. = Solanum, Chrys. = Chrysanthemum.
November 2014] SHAW ET AL.—TORTOISE AND HARE IV 1997

Fig. 4. Continued.

we suggest that is it possible to make good predictions based on We used 25 closely related species pairs (most were conge-
either the overall trends or based on the trends observed within ners) from 10 major lineages of angiosperms, with multiple spe-
a clade of interest (Figs. 2–4). cies pairs from six of these clades, to test all NC-cpDNA regions
1998 AMERICAN JOURNAL OF BOTANY
Fig. 5. Geometric mean potentially informative characters (PICs) ±
SE per region for the top 10 NC-cpDNA regions (n = 20–25 lineages per
region). Regression-based estimates [y = log10 (total PICs per region +1), x
= log10 (length of region + 1)] were calculated for a common x value. Re-
gions are coded 1 to 10 (highest to lowest) and arranged in decreasing or-
der of mean values. Mean PICs/region are reported for three statistical
groups; group means followed by different letters are significantly differ-
ent (P < 0.05; ANCOVA, Scheffé contrasts).
plus matK. Our data predicts that the regions most likely to con-
tain the greatest number of variable sites in angiosperms are
5′rps16-trnQ(UUG), ndhC-trnV(UAC), ndhF-rpl32, trnT(GGU)-psbD,
psbE-petL, petA-psbJ, rpl32-trnL(UAG), rpl16 intron, ndhA intron,
rpoB-trnC(GCA), trnS(GCU)-5′trnG(GCC), 5′trnK(UUU)-3′rps16, psbM-
trnD(GUC), and matK (Fig. 2A, B). Primers have been developed and
are “universal” for most of these regions (see Shaw et al., 2007).
However, the coding regions of ndhF-rpl32 and rpl32-trnL(UAG)
Fig. 6. Intraspecific studies of plants from 1987 to 2013 showing the
contributions of the separate plant genomes. We performed a search in Web
of Science (May 2014) to determine which plant genome(s) was most fre-
quently used in the phylogeographic literature. Search terms included “phy-
logeography or phylogeographic” refined by articles only and the term
“*aceae” to identify studies including a plant family, with subsequent inde-
pendent refinements using the terms “chloroplast or plastid or cpDNA” or
“mitochondria or mitochondrial or mtDNA” or “nuclear or nDNA”.
[Vol. 101
Fig. 7. Summary of cpDNA sequence used in 178 plant phylogeo-
graphic studies published between 2007 and 2013. Of 178 studies surveyed
over 7 years, 69% used cpDNA sequence data. This percentage varied by
year, ranging between 50% (2007) and 81% (2012), and it has not dropped
below 68% since 2009.
(specifically, ndhF and rpl32) are highly variable, making “uni-
versal” primer design difficult for these two NC-cpDNAs. Ap-
pendix S3 contains a brief discussion on the ndhF-rpl32 and
rpl32-trnL(UAG) primers and a table that will aid in lineage-
specific primer design for future projects.
The most variable NC-cpDNA regions identified in this re-
search agree with the previous findings by Shaw et al. (2007) and
others (Byrne and Hankinson, 2012; Dong et al., 2012). It is in-
teresting that these studies are highly corroborative regarding the
best NC-cpDNAs for low-level studies, even though they sam-
pled completely different species from many different plant fam-
ilies. That is, none of the 25 species pairs sampled here were the
same as any species pair from TH2 or TH3. Both the present study
and TH3 converge on ndhF-rpl32, rpl32-trnL(UAG), 5′rps16-trnQ(UUG),
ndhC-trnV(UAC), psbJ-petA, trnT(GGU)-psbD, and psbE-psbL
being among the most informative regions of the plastome for
most groups. Even analyzed in a different way and with differ-
ent species, Dong et al. (2012) and Byrne and Hankinson (2012)
showed rpl32-trnL(UAG), 5′rps16-trnQ(UUG), ndhC-trnV(UAC),
trnT(GGU)-psbD, and rpl16 intron to be among the most variable.
Additionally, new comparative plastomic data continue to be
consistent with our findings at multiple taxonomic levels, includ-
ing Illicium (O. R. Leonard and A. B. Morris, unpublished data),
Solanaceae (Särkinen and George, 2013), and Apiales (Downie
and Jansen, in press). Finally, numerous other studies with nar-
rower taxonomic scopes have also shown these same regions to
be the most variable of the angiosperm plastome (see discussion,
below).
Like TH3, we show that when making finer level comparisons,
there is a lesser degree of predictability among the top NC-
cpDNAs. For example, trnS(GCU)-5′trnG(GCC) ranked just outside of
the top 10 regions in the overall comparison, and it ranked in the top
12 in 3/6 of the comparisons of major lineages, but it ranked first
place in the Phalaenopsis (monocot) and Populus (eurosid I) com-
parisons and third place in Olea (euasterid I) (Fig. 4). Interestingly,
November 2014] SHAW ET AL.—TORTOISE AND HARE IV 1999

Fig. 8. Map of the potentially informative characters (PICs) expected to be found around the plastome. Gene content, order, and mapping, was based
on the map of Lee et al. (2006) for Gossypium hirsutum. Bars radiating in from NC-cpDNA regions show the average percentage contribution to total PICs
for each NC-cpDNA and matK. The blue, orange, red, and pink circles indicate increasing predicted PIC values for each NC-cpDNA, as demonstrated by
the scale bar. The inverted repeats are indicated by bold black lines on the genome and on the blue, orange, red, and pink circles. Note that three areas
highlighted in light green standout as hotspots or clusters of highly variable regions of the plastome: ccsA-ndhF, matK-3′trnG, and rpoB-psbD.

both Phalaenopsis and Populus have plastome rearrangements region). In another example, matK, which ranked 12th place
that result in the partial loss of the ndhF-rpl32-trnL(UAG) region overall (Fig. 2A, 2B) and in the top 12 positions in 3/6 comparisons
(actually, further supporting our data that this is a highly variable of major lineages (Fig. 3B–D), was ranked first in Aethionema
2000 AMERICAN JOURNAL OF BOTANY
(eurosid II) and second in Vigna (eurosid I) and Olea (Fig. 4). In
rare cases, we show that a NC-cpDNA that ranked very low over-
all (e.g., rpl33-rps18), ranked very highly in one species com-
parison (Nelumbo, Proteales) (Fig. 4), and did not appear in the
top eight in the remaining 24 species comparisons.
Despite the rare cases when low ranked NC-cpDNAs turn out
to be the most informative in a given lineage or when the top NC-
cpDNA regions may change rank among the top positions, a
few regions like ndhF-rpl32, rpl32-trnL(UAG), ndhC-trnV(UAC),
and 5′rps16-trnQ(UUG) are, on average, among the most informa-
tive across angiosperms (Figs. 2–4) (Byrne and Hankinson, 2012;
Dong et al., 2012). In fact, one of these four NC-cpDNAs was the
top ranked NC-cpDNA region in 11 of 25 of the species compari-
sons (of 108 total regions) and five of six major lineage compari-
sons. Furthermore, one of these four most variable NC-cpDNAs
was among the top three regions in 19 of 25 lineages. Even in the
above example of Nelumbo, where a low-ranked region overall
was the most variable in this lineage, these four regions were all
in the top 10. This finding suggests that while there is some amount
of inconsistency among the top NC-cpDNAs, and no single NC-
cpDNA is universally best for low-level molecular studies, it is
likely that one of the top NC-cpDNAs will be among the most in-
formative in a given taxonomic group of angiosperms.
How many NC-cpDNA regions should we use and how many
should we screen?—Through the last 6 years, the average number
of cpDNA regions used in intra- and interspecific studies was two
to three, with a minimum of one to a maximum of six. Unfortu-
nately, only 28 of 178 papers reviewed here reported screening
additional loci; too often it was not stated what other NC-cpDNA
regions were screened. In the papers where it was reported, the
mean number of loci ranged from four to 15, and the authors rarely
reported information on the regions screened but excluded. There-
fore, there is insufficient data from the literature to allow us to build
a quantitative database from which to draw conclusions about how
many NC-cpDNAs should be screened prior to study, and we hope
to address this question in the future.
At this point, we know that it is the exception rather than the
rule that a single NC-cpDNA region will provide enough ge-
netic information to discriminate all of the species in a genus or
section or all populations in a phylogeographic study. Our lit-
erature review showed that at the lowest levels in which work-
ers used NC-cpDNAs, the average number of NC-cpDNA
regions used was two to three, with a minimum of one to a
maximum of six. The DNA barcoding community has provided
a wealth of information regarding the species-level discriminat-
ing power of plastome regions. For a while now it has been
understood that a multilocus approach is necessary to capture
enough genetic variability to differentiate species (Kress et al.,
2005; Newmaster et al., 2006; Chase et al., 2007; Kress and
Erickson, 2007; Lahaye et al., 2008). Fazekas et al. (2008)
showed that at least four NC-cpDNA regions are necessary to
capture the total species discriminating power of the plastome
and that greatly increasing the number of regions above four,
especially above six or seven, will not increase species-level
discrimination because of a “performance plateau”.
We draw two conclusions here. First, on average the phyloge-
netics/phylogeography community is under-sampling the plastome
by using only two to three regions per study and therefore not using
cpDNA sequence data to the fullest extent of its discriminating
power. Second, while there are certainly cases where only se-
quencing the entire plastome will find genomic differ-
ences between study taxa (Whittall et al., 2010), whole plastome
[Vol. 101
sequences may not be necessary to resolve many questions in low-
level studies. The top eight regions, ~8500 bp (~5.6% of the cp-
DNA plastome), account for roughly one fifth of the NC-cpDNA
PICs present in a given lineage (based on percentage contribution
of each region averaged across the 25 species pairs) and the top 12
regions, ~12 900 bp (8.6% of the cpDNA plastome), account for
more than one third of the total NC-cpDNA PICs. Since about one
fifth of the genetic differences of the noncoding portions of the
plastome can be captured by sequencing the eight most variable
regions and there is a performance plateau with respect to species-
level discrimination beyond six or seven regions, we posit that for
many inter- and intraspecific studies whole plastome sequencing is
probably unnecessary. Depending on the lineage of interest, at
least four and up to eight of the most variable NC-cpDNA regions
will likely access the majority of the low-level discriminating
power of the plastome.
Discriminating power of the cpDNA genome—In the late
1980s and early 1990s, the cpDNA genome was believed to be ill-
fit for intraspecific studies because of its very slow rate of mutation
(Palmer, 1987). This belief was largely based on a few intraspecific
studies using cpDNA restriction digests (e.g., Banks and Birky,
1985; Sytsma and Schaal, 1985). Soon after, Palmer et al. (1988)
and Olmstead and Palmer (1994) suggested that the plastome
might be useful for intraspecific studies in some cases, but some-
how our community held firm to the idea that the plastome was
only useful for interspecific studies and above. Toward the late
1990s and early to mid-2000s, some researchers successfully used
NC-cpDNA RFLP (Demesure et al., 1996; Dumolin-Lapegue
et al., 1997a; Soltis et al., 1997; King and Ferris, 1998; Taberlet
et al., 1998; Newton et al., 1999; Petit et al., 2002) or sequence data
(Ohsako and Ohnishi, 2000; Chiang et al., 2001; Lu et al., 2001;
Okaura and Harada, 2002; Holderegger and Abbott, 2003; Yamane
et al., 2003) to infer intraspecific relationships of plants and Wid-
mer and Baltisberger (1999) even touted “extensive intraspecific
chloroplast variation.” These earliest studies to have used NC-
cpDNA sequence data all predated TH2, and most used what are
today considered intermediately variable regions, like trnL(UAA)-
trnF(GAA) and psbA-trnH(GUG), although some used rpoB-trnC(GCA)
(Yamane et al., 2003) or trnD(GUC)-trnT(GGU) (Lu et al., 2001),
which, on average, are fairly high-ranking. Other fairly high-
ranking regions like trnS(GCU)-5′trnG(GCC) and trnD(GUC)-trnT(GGU)
were shown to contain small amounts of intraspecific variation in
three unrelated species from the Alps (Schönswetter et al., 2006b,
a). Wang et al. (2009) even used regions that ranked relatively low
here, the rpl20-rps12, trnV intron, and psbA-trnH(GUG) regions, to
reveal nine haplotypes from 23 populations of Aconitum gymnan-
drum (basal eudicot). To our knowledge, some of the earliest intra-
specific studies to use a top region were Yamane and Kawahara
(2005) who used ndhF-rpl32 in combination with rpoB-trnC(GCA),
trnF(GAA)-ndhJ, and atpI-atpH to identify 2–8 haplotypes per spe-
cies in 13 Triticum-Aegilops species (monocot) and Huang et al.
(2004) who used petA-psbJ along with petG-trnP(UGG) to reveal
nine haplotypes among 24 populations of Trochodendron aralioi-
des (basal eudicot).
More recently, numerous papers have used top-performing
NC-cpDNAs to uncover intraspecific variability. Aguirre-Liguori
et al. (2014) used ndhF-rpl32, rpl32-trnL(UAG), and psbJ-petA to
identify nine haplotypes and geographic structure within Fouqui-
eria shrevei (basal asterid). James and McDougall (2014) used
5′rps16-trnQ(UUG) to reveal eight haplotypes and geographic
structure within Grevillea renwickiana (basal eudicot), and Chen
et al. (2013) used this same region to reveal 15 haplotypes among
November 2014] SHAW ET AL.—TORTOISE AND HARE IV
17 populations of Prunus pseudocerasus (eurosid I). Liu et al.
(2012) uncovered 12 haplotypes using rpl32-trnL(UAG) and
showed it to be as informative as the nuclear-encoded PAL in a
phylogeographic inquiry of Camellia taliensis (basal asterid),
and Cires et al. (2012) used rpl32-trnL(UAG) and 5′rps16-trnQ(UUG)
to reveal intraspecific variability in Ranunculus parnassifolius
(basal eudicot). In fact, Cires et al. showed each of these regions
to contain three times more variable characters than in the nuclear
ribosomal ITS. Dunbar-Co et al. (2008) used ndhF-rpl32 and
rpl32-trnL(UAG) in an intra- and interspecific study of Hawaiian
Plantago (asterid I) and showed this concatenated region to con-
tain twice the variable characters present in the ITS. Yuan et al.
(2008) used 5′rps16-trnQ(UUG) and psbA-trnH(GUG) to highlight
11 haplotypes from 16 populations of Dipentodon (eurosid II).
Yuan et al. (2011) used 5′rps16-trnQ(UUG) in addition to petB-
petD and psbA-trnH(GUG) in an intraspecific study of Paeonia
rockii (basal eurosid) and found 16 haplotypes among 335 indi-
viduals (interestingly, petB-petD outperformed 5′rps16-trnQ(UUG)
and was 1458 bp long, which, according to our average of 248 bp,
is abnormally large for this region, Fig. 2E).
We are not suggesting that NC-cpDNA can always be used to
elucidate intraspecific relationships, but rather that we should
not assume that it will not. In a study of 21 closely related spe-
cies of Kaempferia and related genera (Zingiberales), Techap-
rasan et al. (2010) showed that about half of the species (11)
displayed intraspecific variability using psbA-trnH(GUG) and
petA-psbJ sequences. There are also instances where these re-
gions are unlikely to be helpful at intraspecific levels (consider
the four species pairs that we excluded from our plastome anal-
yses due to a lack of variation: Acorus, Nicotiana, Oryza, and
Phyllostachys).
In part, the ability of our community to quantitatively com-
pare NC-cpDNA region utility has been hampered by a paucity
of detail reported in phylogeographic and phylogenetic studies.
It is problematic that for the published haplotype networks or
phylogenies, many current studies fail to report the relative
contributions of (1) the different kinds of data (e.g., NC-cpDNA
vs. microsatellite vs. AFLP) and (2) the different NC-cpDNA
regions, both those that were included and arguably more im-
portantly those that were screened and excluded. Or, even when
such data are reported, they are not always clearly presented in
an easily accessible manner. At the outset of this study, we had
hoped to mine the phylogeographic literature for data regard-
ing (1) how many gene regions were screened before choosing
ones for study and (2) how many haplotypes were uncovered
with each type of molecular marker and with each different
NC-cpDNA region. However, most studies provide only the
summary of results from concatenated data, with no indication
of the relative contribution of each sequenced region to the sum
of the whole. Within the 178 papers surveyed in our literature
review, only 45 included such information, and the information
was not parallel enough to elucidate patterns.
Conclusions—At this point, several things are clear. First, NC-
cpDNA sequences are still a tool of choice among systematists/
phylogeographers focused at the shallowest taxonomic levels. Sec-
ond, there is a strong need for us to be able to predict which mark-
ers will be the most variable within a given lineage because (1)
NC-cpDNAs are important for inter- and intraspecific studies, (2)
generating whole plastome sequences as a starting point for inter-
and intraspecific studies is still beyond the reach of most research-
ers and not necessarily needed for all low-level studies, and (3)
there is no single most variable NC-cpDNA.
2001
Data from our literature review were insufficient for us to
make recommendations on how many individuals to screen;
additionally, the screening process or strategy will likely vary
depending the taxonomic and geographic scope of the project.
Because the top regions of this study are likely to be among the
most variable in most angiosperm species, we suggest including
all of them in initial screenings. These include ndhF-rpl32, rpl32-
trnL(UAG), 5′rps16-trnQ(UUG), ndhC-trnV(UAC), trnT(GGU)-psbD,
psbE-petL, petA-psbJ, and rpl16 intron. To increase the likeli-
hood of finding the most variable regions in a specific group of
interest, we suggest (1) potentially screening some of the other
top ranking regions as well, such as ndhA intron, rpoB-trnC(GCA),
psbM-trnD(GUC), trnS(GCU)-5′trnG(GCC), matK, 5′trnK(UUU)-
3′rps16, petN-psbM, and perhaps trnT(UGU)-trnL(UAA), or (2) some
of the most variable regions from the major lineage closest to the
study group of interest (Figs. 3, 4), or (3) some NC-cpDNAs that
other studies have shown to be highly informative in taxa closely
related to your group of study. Primers and protocols for all of
these regions are referenced in Shaw et al. (2005, 2007). Further-
more, the DNA barcoding community has shown that 4–6 NC-
cpDNAs will likely capture all of the species-discriminating
power of the plastome, so for now we suggest screening up to
10–12 regions to determine which 4–6 to include in low-level
phylogenetic or phylogeographic studies.
Lastly, we ask our community to include detailed data tables in
their publications to clarify the relative contribution of each se-
quenced region and specifically include information on screened
but omitted NC-cpDNA regions (even if abandoned early be-
cause of poly A/T regions near the ends). At this point, we sus-
pect there to be a wealth of unpublished information that will
likely be the cause of repeated effort and wasted resources in the
future. At a minimum, these tables should include NC-cpDNA
name, length, nucleotide substitutions, indels, inversions, vari-
able characters, informative characters, and haplotypes uncov-
ered by the different regions, all presented within the context of
sample size by population. If each study includes this informa-
tion for each region employed, within a year’s time, there will be
a substantial amount of data that may be of use to other research-
ers that do not have plastome resources for their target groups.
As we have learned how to use it more effectively, the plastome
has become an extraordinary tool in our understanding of the
evolutionary history of plants. The literature establishes that the
plastome has allowed us to investigate the deepest to shallowest
relationships of plants, and we continue to push the envelope fur-
ther to better understand recent evolutionary patterns within and
among populations. The success we have experienced so far has
largely been without the aid of the most variable regions of the
plastome. It appears as though we are embarking on new oppor-
tunities once again, through this organelle that was once thought
to contain so little species-level information.
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