Introduction:
Cytosolic phosphoglucose isomerase (pgiC, E.C. 5.3.1.9) is an enzyme essential to the
glycolysis, a cellular-respiration pathway known in almost all species of living things. pgiC acts
uring the second step of glycolysis, converting Glucose-6-Phosphate to its isomeric form,
Fructose-6-Phosphate. An array of regions within the exons of pgiC have remained relatively
conserved across kingdoms (Claes et al. 1994, Thomas et al.1990), while other regions of the
exons and introns are highly variable (Thomas et al. 1993, Kawabe et al. 2000, Koenemann et al.
2011). Variation in PGIC is known to yield allozyme polymorphisms in both flora and fauna
(The butterfly Colias, Watt 1977; blue mussel Mytillus, Hall 1985; Arabidopsis thaliana,
Kawabe et al. 2000; Wild Yam Dioscorea tokoro, Terauchi 1997).
These polymorphisms have in some cases been shown to be under natural selection
(Gillespie 1991; Riddoch 1993). Both selection for based kinetic performance and structural
stability at different temperatures have been documented. In the blue mussel, Mytilus edulis, two
PGIC alleles replace one another across a latitudinal cline; they differ in their efficiency at
physiologically higher temperatures (Hall 1985). The sierra willow leaf beetle, Chrysomela
aeneicollis, also has multiple pgiC alleles that vary along a cline that follows latitude and
elevation (Dahlhoff and Rank 2000, Rank and Dahlhoff 2002). In C. aeneicollis specific pgiC
alleles can be associated with cold or warm habitats; these alleles differ in their thermal stability
as well as biochemical performance at different temperatures (Rank and Dahaloff 2002).
Directional selection of PGIC is observed along latitudinal clines in both C. aeneicollis and M.
edulis. Another example of temperature-based selection of PGIC is the selection for
heterozygous genotypes of PGIC frequently observed in the sulfur butterflies (Colias, Watt
1977). The heterozygote dominance is inferred to be the result long-term balancing selection
favoring a group of charge-changing nonsynonymous substitutions, which are believed to alter
the function of the pgiC enzyme (Wheat 2006). Together, these findings suggest an important
and complex relationship between pgiC activity and environmental conditions, especially
temperature (Dahaloff et al 2000, Rank et al. 2002, Wheat et al. 2006, Wheat 2010). It is likely
that similar temperature-based selection can be found in species other than arthropods and
mollusks, given the high level of conservation of PgiC across major evolutionary lineages.
Intraspecific nucleotide variation in pgiC has been reported for a number of angiosperms
species, often resulting in amino acid substitutions (Arabidopsis thaliana Kawabe et al. 2000,
Dioscorea tokoro Terauchi et al. 1997, and Leavenworthia stylosa Filatov and Charlesworth
1999). In some cases it is suggested that natural selection is acting upon the different alleles of
pgiC in these plants. In a study of wild populations of A. thaliana, a low level of populationlevel DNA polymorphisms and a high level of non-synonymous nucleotide replacement suggests
that the genetic variants are under natural selection (Kawabe et al. 2000). Variation of pgiC has
also been studied in ferns, focused on exons 14–16 and their two included introns (Dryopteris
nipponensis (Ishikawa et al., 2002) as well as in the fiddlehead fern Matteuccia struthiopteris
(Koenemann 2011). In addition, isozyme studies of Central American Polystichum species have
shown that they host at least five different PGIC alleles (including two intraspecific variants),
implying different amino acid sequences (Barrington 1990). Beyond this work, essentially
nothing is known about pgiC variation in ferns, especially in relation to habitat.
An excellent study system for exploring genetic diversity related to environmental
variables in pgiC is the genus Polystichum, which comprises approximately 380 species
worldwide (Barrington, unpublished data). This genus includes a recently evolved lineage
endemic to the Andes comprising an array of species found at diverse elevations and hence
temperature regimes (McHenry and Barrington 2014). Recent phylogenetic work in the
Barrington lab has revealed that during the history of Andean Polystichum, there have been at
least three major elevational transitions, both up and down the Andes (McHenry and Barrington
2014). As pgiC is variable in allozyme polymorphisms in Central American Polystichum species
(Barrington 1990) and in arthropods (Dahaloff et al. 2000, Rank et al. 2002, Wheat et al. 2010),
it is plausible to expect that the Andean Polystichum species may exhibit PgiC variation across
an elevation gradient due to environmentally induced selection on alleles of this gene.
Alternatively, PgiC variation may be due to selectively neutral, historically informative
transformations.
Understanding how the PgiC gene varies in Polystichum can help broaden our
understanding of how the protein functions in general. Just as the Andes have seen climatic
change during their uplift, different regions of the world are constantly undergoing ecological
changes. Given the abundant evidence for climate change, insights into the response of
temperature-dependent enzymes such as PgiC have immediate relevance to improving our
understanding of plants’ responses to global warming.
*Enzymatic variation PGIC, DNA variation pgiC.
MATERIALS AND METHODS
Taxonomic Sampling—A total of nine Polystichum species were included in the study group,
including eight rom the Andes. Species were chosen to include closely related taxa from a range
of elevations in the central and northern Andes. A single species (P. pallidum)—still from the
same clade— was collected from Brazil. The lowest accession sampled (P. pallidum) was
collected at 480 m, the highest (P. orbiculatum) at 4650m . The collections for this analysis
were largely made during the investigation reported in McHenry and Barrington, 2014. For a
complete list of sampled taxa, including accession numbers and voucher information, see the
appendix.
DNA Isolation, Primer design, Amplification, and Sequencing—Leaf material was stored in
silica desiccant gel at -80°C prior to extraction. Total genomic DNA was extracted from the
silica-dried frond material using a modified CTAB extraction protocol (Doyle and Doyle 1987).
PgiC Primers: Primer sets (Table 1) provided the basis for retrieving sequences from a
total of six markers representing ##% (3200bp of a estimated total of #### bp) of the pgiC gene
from Andean polystichums. The first four markers, pgiC exons 3-7, 6-8, 7-10, and 9-13, were
from the roughly 2000 base pair region from pgiC exons 3-13. The remaining two markers, pgic
exons 14-17 and 16-19, were from the 1200 base pair region from pgiC exons 14-19. The region
between exons 13 and 14, as well as exons 1-3 and 19-22, failed to amplify despite numerous
attempts with a number of different primers, PCR reagents, and conditions.
The first pgiC primers (to amplify exons 14–16) for Polystichum were adapted from those used
by Ishikawa et al. (2002) to amplify Dryopteris. Primer sets for pgiC exons 16–19 were designed
by Barrington lab members. My design of the remaining primers was considerably aided by the
contribution of pgiC cDNA for Polystichum achrostichoides by Carl Rothfels (see Rothfels et al.
2013). This same cDNA was also used to modify previously designed primers in order to
improve amplification.
Amplification: Amplification by the polymerase chain reaction was performed in a TC-
3000, TC-3000X or a 3Prime thermal cycler (Techne, Burlington, New Jersey, USA) in 25 μL
aliquots with the following components: 150 ng of genomic template; 0.1 μM of each primer; 1
× Denville PCR Buffer (Denville Scientific Inc., South Plainfield, New Jersey, USA); 200 μM/L
of each dNTP; and 0.625 U ChoiceTaq DNA Polymerase (Denville Scientific Inc.). All
sequences were amplified as follows: (1) initial denaturation at 94 °C for 2 min; (2) followed by
35 cycles (94 °C for 30 s, 55-58 °C (annealing temperatures were adjusted for each marker set
see Table1) for 30 s, 72 °C for 1 min); and (3) a final extension at 72 °C for 10 min. PCR
products were purified using ExoSAP-IT (USB Corporation, Cleveland, Ohio, USA). Purified
PCR products were then further cleaned and labeled using ABI reagents (Applied
Biosystems/Life Technologies, Foster City, California, USA).
Sequencing: Sequences were resolved on an ABI Prism 3730xl DNA analyzer (High
Throughput Genomics Center, Seattle, WA, USA). The raw chromatograms from each amplified
PCR product sequenced were aligned using both the forward and the reverse sequences, and
consensus sequences were assembled for each region of pgiC using Geneious Pro version 6.1.5
(Drummond et al. 2007 ).
Phylogenetic Analysis—Bayesian Inference (BI) was carried out using Mr. Bayes v. 3.2
(Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The Bayesian-analysis
model selection was done using Modeltest v. 3.7 under the Akaike Information Criterion
(Posada and Crandall 1998; Posada and Buckley 2004; Posada 2006). A mixed-model Markov
Chain Monte Carlo analysis was performed with each marker allowed to evolve under its own
best-fit model, with eight independent Markhov chains for 10 million generations and trees
sampled every 1,000 generations. Stationarity was determined using the log-likelihood scores for
each run plotted against generation in the program Tracer (Rambault & Drummond, 2007). 10
% of the trees were discarded as the burn-in phase and a 50% majority rule consensus tree was
calculated for the remaining trees.
Results
Characteristics of the Dataset—The aligned dataset totaled 3376 characters, of which 148
(4.38%) were variable and 56 (1.66%) were informative. 1134 (33.59%) of the total are exonic;
25 (2.20% of the 1134) imply amino acid substitutions . Of these, 18 are synonymous
substitutions (six informative, three charge-changing), and seven are nonsynonymous (five
informative). The remaining 2242 characters (66.41%) were intronic; they included 129
substitutions (51 informative). The introns also included 11 informative indels and 21
autapomorphic indels varying in size from one base pair to 188 base pairs.
The amplification of pgiC in Andean Polystichum resulted in the sequences for two
central regions of the gene in nine accessions. One region spans 2113 base pairs and ranges from
the end of exon 3 to the beginning of exon 13. The second section is 1263 base pairs long
starting at the end of exon 14 and ends halfway through exon 19. Of the pgiC regions amplified
in Andean Polystichum both exon and intron lengths were found to be relatively variable. Exons
range from 43 base pairs (exon 7) to 156 base pairs (exon 5) with and average length of 79 base
pairs. Introns are more variable, ranging from 67 base pairs (intron 9) to 533 base pairs (intron
7).
Three introns (7, 15, and 18) contain much more variability than any of the other introns.
Well known for its variability, intron 15 is frequently used for phylogenetic analysis in plants,
including ferns (Ishikawa et al. 2002, Koenemann et al. 2011). In Andean Polystichum, pgiC
intron 15 ranges from 207 to 391 base pairs long and contains 14 variable characters, five of
which are informative. Intron 15 also contains eight autapomorphic indels and four
synapomorphic indels, one of which is 188 base pairs long—it is the major source for the
variation in the the size of this intron. Despite intron 15’s documented variation, the most
variable region I retrieved was in intron 7, which contains 28 variable characters: 12 of these are
informative. Intron 7 is also the largest intron sequenced for Andean Polystichum pgiC; it ranges
from 503 to 533 base pairs in length. Intron 7 also contains a number of different microsatellites
that vary in size and sequence and have resulted in several indels and single nucleotide
substitutions. The third unusually variable region is intron 18, which contains 13 variable
characters (eight informative). A single autapomorphic indel in intron 18 accounts for a variable
length ranging from 138 to 183 base pairs. As a whole these three introns contain 55 (42.64%)
of the 129 variable characters and 25 (49.02%) of the 51 informative characters found within the
introns.
Phylogeny with All Characters—Bayesian inference analysis was used to construct a tree for all
amplified regions of Andean Polystichum pgiC (exons 3-13 and 14-19 and their respective
introns). Four monophyletic groups were resolved. The first group comprises a single species,
P. stuebelii, a mid-elevation species found in pre-montane forests along the western slopes of the
Andean Equatorial Region. The particular accession of P. stuebelii used in this analysis was
collected at 2400 meters. As group one contains the earliest-divergent species in cpDNA
analyses (McHenry and Barrington 2014), P. stuebelii was chosen to root the phylogeny. The
remaining three groups form a polytomy. The largest clade in this phylogeny, group 2, consists
of P. orbiculatum, P. gelidum, P. pallidum, and P. polyphyllum. Polystichum orbiculatum is a
widespread species (Mexico to Bolivia) found at upper elevations and is prominent in the páramo
(collected at 4650 and 4669m). Polystichum gelidum is found throughout the northern and
central Andes at upper elevations in subpáramo and Polylepis (Rosaceae) forests (collected at
3200m). P. pallidum is found only in southeastern Brazil at low elevations in wet-premontane
forests (collected at 480m). P. polyphyllum is an alpine species found growing in the páramo at
high elevations in the northern to central Andes (collected at 3870m). Group 3 contains P.
platyphyllum and the hybrid P. platyphyllum X P. bulbiferum both species are found on
premontane to montane forested slopes (collected at 1729m and 1321m respectively). Finally,
group four comprises the single species P. dubium, a relatively widespread species (Costa Rica
to Bolivia) found in wet-montane forests along stream banks (collected at 2655m).
The chloroplast-based phylogenetic tree constructed for Andean Polystichum contains
three major clades, clade one contains P. stuebelii, clade two contains P. polyphyllum, P.
orbiculatum, and P. gelidum, and clade three contains P, bulbiferum, P. platyphyllum, P.
pallidum, and P. dubium(McHenry & Barrington 2014). Phylogenetic analysis of Andean pgiC
is congruent in part with the chloroplast phylogeny. With the exception of P. pallidum, group 2
contains the same species as clade two in the chloroplast phylogeny. However the relationships
between these species are different as in the chloroplast tree P. polyphyllum is sister to P.
orbiculatum but in the pgiC tree P. gelidum is sister to P. orbiculatum. In addition 16 characters,
including 3 nonsynonymous substitutions, unite P. gelidum and P. orbiculatum, whereas only a
single character (2972) unites P. polyphyllum with P. gelidum and P. orbiculatum. Polystichum
pallidum’s inclusion in group two is supported by two characters (2972 and 2297), i.e. relatively
weak support. Polystichum pallidum’s weak relationship to group two is not unexpected given
that it is nested within clade three of the chloroplast tree. With the exception of the union of P.
platyphyllum and P. platyphyllum X bulbiferum, clade three of the chloroplast tree is poorly
resolved in pgiC.
A number of synonymous and nonsynonymous substitutions are charted throughout the
phylogeny. Four of the seven nonsynonymous substitutions are found in group two, none occur
in P. polyphyllum and only one is found in P. pallidum (Character 2297) while all four are found
in P. gelidum and both species of P. orbiculatum. Three of the substitutions change between two
nonpolar amino acids (character 95 valine to alanine, character 541 valine to isoleucine, and
character 2297 phenylalanine to leucine). The fourth nonsynonymous substitution that occurs in
this group results in a change in charge resulting in an amino acid substitution of proline to
glutamine, a nonpolar to a polar amino acid (character 3339) occurring in P. gelidum and P.
orbiculatum. The only remaining nonsynonymous substitution that is informative is character
2756, a non-charge changing substitution that occurs in both species of group 3. The two
remaining nonsynonymous are autapomorphic occurring only in P. dubium, both of which result
in a charge changing substitution. The first amino acid substitution in P. dubium is character 118
and results in an amino acid substitution of acidic glutamic acid to basic lysine. The other
substitution is character 416 and results in the substitution of polar alanine to nonpolar threonine.
Fragments of pgiC were also sequenced for an additional accession of P. dubium (not included in
this analysis due to incomplete data), interestingly this accession only shares the substitution at
character 416 and does not share the character 118 substitution. However this additional P.
dubium has a different nonsynonymous substitution that lies seven base pairs downstream from
character 118 occurring within the same exon, and results in a non-charge changing substitution
of glutamic acid for aspartic acid.
Of the 18 synonymous substitutions occurring in the exons of Andean Polystichum seven
are informative. Four of the informative synonymous substitutions are found uniting group 3
while the other three are found uniting taxa in group two, at various nodes. Nine of the 11
autapomorphic nonsynonymous substitutions can be found in P. stubellii (four), P. polyphyllum
(two), and P. dubium (three), the remaining two substitutions are found in P. pallidum and P.
platyphyllum with one each. All remaining substitutions are intronic occurring outside of the
coding region, of these there are 51 synapomorphic characters and 78 autapomorphic characters.
All of these synapomorphic characters are found uniting closely related taxa such as the 30
characters that unit P. platyphyllum and P. platyphyllum X bulbiferum. The two species of P.
orbiculatum are united by 10 of these intron based synapomorphies and the remaining 11
characters unit P. orbiculatum and P. gelidum.
As a nuclear gene, pgiC can give great insights on the hybridity of species. Direct
sequencing of a hybrid species often results in double peaks in the chromatogram due to
differences between the copies of pgiC, resulting in heterozygous nucleotides (?). A number of
these nucleotides are found in the sequences for P. platyphyllum X bulbiferum and it’s very clear
that P. platyphyllum is indeed a progenitor of this hybrid species as three of the heterozygous
nucleotides are the results of single nucleotide polymorphisms occurring in P. platyphyllum. The
tetraploid P. gelidum hybrid included in this study also contains a number of heterozygous
nucleotides and while a single substitution in P. orbiculatum and a single substitution in P.
polyphyllum match some of these nucleotides, there are still three other sites that are not
accounted for by any of the species included in this study.
Fragments of pgiC were sequenced for a number of different accessions of P.
orbiculatum during the early stages of this project. Due to difficulty in sequencing several of
these accessions were not used for the final dataset. Sequence data from these accessions
suggest that there are both diploid and tetraploid varieties of P. orbiculatum and that P.
polyphyllum is one of the progenitors of the tetraploid P. orbiculatum. Previous work on
Polystichum hybrids has determined that P. orbiculatum and P. talamancanum (another
tetraploid hybrid found in Central America) share a progenitor that is noted by a 200 base pair
deletion in intron 15. As P. polyphyllum also shares the 200 bases pair deletion as well as
several nucleotide polymorphisms it is likely the previously unknown progenitor of P.
talamancanum as well.
Comparison of Exon and Intron Trees—Intronic and exonic based trees are relatively similar
with only a few exceptions. Both and intron and exon trees retain groups one, three and four as
described for the character state tree for all regions of pgiC making the structure of group the
major difference. In both trees P. polyphyllum has lost resolution to group two, which is likely
due to a lack of support as it was only united to this group based on a single synonymous
substitution( character 2972). In addition to this the intron trees has also lost the resolution of P.
pallidum. In addition the clade containing both species of P. orbiculatum has collapsed resulting
in a polytomy of three branches, this also indicates a high level of similarity in the exonic
sequences of P. orbiculatum and P. gelidum. Other than a loss of resolution there are no
significant structural differences between the intron and exon trees.
Discussion
Variation of pgiC in Andean Polystichum—Of all of the regions sequenced for Andean
Polystichum pgiC single nucleotide polymorphisms are found occurring more frequently in the
introns opposed to the exons, indicating a greater level of conservation of the coding
region. Conservation of the exons is often a consistent theme of many nuclear genes involved in
critical regulatory processes such as glycolysis. Of the substitutions that did occur in the exons
the majority were synonymous and therefore did not result in any type of amino acid change. In
addition of the seven amino acid substitutions that did occur only three resulted in amino acid
substitutions that changed charge, two of which occurred in a single accession. Therefore the
majority of the coding region of pgiC remains relatively conserved across a number Andean
polystichum accessions.
More than half of the nucleotide polymorphisms that were observed in Andean
Polystichum pgiC resulted in autapomorphic characters, and a number of the informative
characters occurred in many closely related taxa. Very few informative characters showing
relationships between more distantly related lineages were present in either the exons or introns
of the pgiC regions sequenced in this study. The lack of more ancestral characters led to a loss
of resolution and the formation of a three branch polytomy in the phylogeny constructed with all
regions of pgiC. The fact that many of the characters are more recently derived gives strong
evidence that pgiC is rapidly evolving in Andean Polystichum.
Elevational variation of pgiC in Andean Polystichum—Major similarities were found between
the chloroplast based phylogeny of Andean Polystichum and both the pgiC intron and exon
phylogenies. These similarities indicate that the variation in both the introns and exons of pgiC
are likely the result of a phylogenetic history, rather than temperature based selection of
pgiC. The only major dissimilarities that arose were the relationships of P. pallidum and P.
polyphyllum, the remaining differences are mostly polytomies that can be attributed to a lack of
informative ancestral characters. P. pallidum’s placement in the exon based phylogeny is
particularly strange. First it’s placement within the chloroplast tree is in clade three where it is
sister to P. platyphyllum and P. platyphyllum X bulbiferum yet in the exon tree is very closely
related to P. orbiculatum and P. gelidum, taxa that belong to clade 2 of the chloroplast tree. In
the exon phylogeny P. pallidum is linked with accession from very high elevations (P.
orbiculatum, P. gelidum, and P. polyphyllum), all of which were collected above 3000 meters,
while P. pallidum was collected at 480 meters. Therefore it is likely that the two exonic
characters linking P. pallidum to these high elevations accessions are homoplastic characters that
evolved separately of one another. P. polyphyllum is species that is sister to P. orbiculatum in
the chloroplast phylogeny, yet P. polyphyllum is unresolved in both intron and exon
phylogenies. It is revealed in the character state tree that P. polyphyllum is linked to P.
orbiculatum and P. gelidum by a single character (character 2972, fig. 2). However in the
chloroplast tree P. orbiculatum is sister to P. polyphyllum yet they only share a single
synonymous substitution in pgiC, yet P. orbiculatum and P. gelidum share four nonsynonymous
substitutions as well as a number of characters in the introns. While this difference in
relationships is odd it is not likely that it is not due to elevational differences as all accessions for
these species were collected above 3000 meters.
While it is possible that the divergence of pgiC follows a pathway similar to the
phylogenetic history of Andean Polystichum, patterns of amino acid substitution may give
insight on the adaptation of these plants to higher elevations. Six of the seven amino acid
substitutions that occur in Andean Polystichum pgiC can be localized to two regions within the
phylogeny, a portion of group two(P. gelidum and P. orbiculatum) and group four (P.
dubium). Four of the amino acid substitutions occur in P. gelidum and both accessions of P.
orbiculatum, three of these substitutions result in no change in charge. The remaining
substitution results in change from the nonpolar amino acid proline to the polar amino acid
glutamine. It is possible that these substitutions particularly the charge changing substitution, in
some way alter the structure of pgiC in this group of plants allowing for a more robust
performance of glycolysis at higher elevations and thus lower temperatures. However P.
polyphyllum, an additional upper elevation species that is included in this study, does not share
any of the four substitutions as P. orbiculatum and P. gelidum. As P. polyphyllum is a rare
species and P. gelidum and P. orbiculatum are more prominent and widespread, it is possible that
these species simply outperform P. polyphyllum as P. polyphyllum has and inferior pgiC
allozyme. In P. dubium two amino acid substitutions occur solely in the single accession
included in this study, both of which result in charge changing amino acid
substitutions. However it is known that intraspecies variation resulting in differences in amino
acid substitution occurs in P. dubium. As P. dubium is a widespread species it is likely that
varying pgiC allozymes exist between and potentially among different populations of P.
dubium. While limited the amino acid variation that is found in the exons of Andean Polystich
pgiC could potentially give insight on the adaptation of andean polystichum to new elevations
and environmental presures.
Future Focuses—While variation of pgiC in this subset of Andean Polystichum would seem to
indicate that elevation and thus temperature does not play a role in allozyme formation this does
necessarily mean that it is not selected for. A number of Andean Polystichum species were not
included in this study, these species may give further insight into the role of pgiC and how it is or
is not affected by temperature in ferns. It would be beneficial to continue this study with a
greater sample size including more species as well as accessions. In addition it would be
beneficial to look at pgiC in a single species of Polystichum that ranges across the Andes, P.
dubium would be a good potential candidate for this study as it is already known to have at least
two pgiC allozymes.
While pgiC is used in a number of phylogenetic analyses, it may not always be the best
nuclear marker. It is clear that pgiC is very rapidly evolving in Andean Polystichum, particularly
by the large number of autapomorphic characters and lack of ancestral synapomorphic
characters. In some cases, such as Andean Polystichum, it may be difficult to resolve the lineages
of particular species due to pgiC’s rapid evolution. However, in other cases it is the rapid
evolution of pgiC that would make it an ideal marker for resolving the identity of very closely
related or recently divergent species. In addition microsatellites found in intron 7 would make
excellent markers for molecular based studies of Polystichum (or other closely related groups)
populations. In addition the variability of pgiC makes it a good marker for resolving hybridity in
ferns.
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Figure 1: Gene structure for the regions of pgiC amplified for Andean Polystichum. Solid black
boxes represent fully sequenced exons, rectangles with outlined in red indicate missing or
partially sequenced exons. Black lines represent fully sequenced introns, the redline joining
exons 13 and 14 represents intron 13 which we were unable to amplify. Exon and Intron
numbers are based off of an alignment of Polystichum acrostichoides pgiC CDNA with
Arabidopsis thaliani pgiC, therefore we are assuming that Polystichum pgiC introns 1, 2, 13, 19,
20, and 21 exist. We are also assuming that exons 1, 2, 3, 13, 14, 19, 20, 21 and 22 are not
joined or further divided from one another.
Figure 2: Phylogeny of Andean Polystichum for all amplified regions of pgiC (exons 3-13 and
14-19 as well as their respective introns). Each number represents a variable character and its
position in the final alignment. Green numbers represent homoplastic characters while black
numbers represent single change characters. Characters that are highlighted are those that occur
within the exons blue represents synonymous substitutions and red represents nonsynonymous
changes. All nonsynonymous substitutions are marked by black letters and arrows representing
the specific amino acid substitutions that occur at these sites, those that are highlighted in orange
represent amino acids substitutions that result in a change in charge while those that are not
retain the same charge. Letters are based off of the 1-letter IUPAC amino acid code. Characters
that are not highlighted occur in the introns. Numbers next to taxa represent the meters above
sea level that each taxa was sampled from.
Figure 3: Phylogeny of pgiC exonic regions. Bayesian percent probabilities are located above
branches, support was given when values were greater 50%. The scale bar represents 0.05
expected substitutions per site.
Figure 4: Phylogeny of pgiC intronic regions. Bayesian percent probabilities are located above
branches, support was given when values were greater 50%. The scale bar represents 0.003
expected substitutions per site.
Figure 5: Trimmed chloroplast tree of Andean Polystichum (McHenry and Barrington 2014)
Primer Name Primer Sequence
Annealing Temp (°C)
3F
AGTGACAGCAGAAATCATGGAA
55
7R
AAGCCGACGTCCTTTACAAG
55
6F
AACTGGGAAACCATTGACTGATG
58
8R
TCAATAGGATCCACATTTGCCA
58
7F
TTGTAAAGGACGTCGGCTTC
57
10R
ACGGCATCTGGACTGCAAAT
57
9F
TGGATCACATCAGTCCTTGG
55
13R
GGAGCTGTTTTGAAATGGTTGT
55
14F
GGGTCTTTTGAGTGTTTGG
55
17R
CCAATGAAATCACATGGAATAACAC
G
55
16F CTTAGTATGGAAAGCAATGGAAAGG
55
19R
55
TTATGGGGCACCAAATGTTCC
Table 1: Primers developed in this study for the amplification of pgiC exons 3 - 13 and 14-19.
Variable
Informative Nonsynonymous Synonymous
Exon Length (base pairs) Characters characters Substitutions
Substitutions
3*
16*
0
0
0
0
4
77
2
1
2
0
5
156
2
1
1
1
6
97
3
1
1
2
7
43
1
0
0
1
8
68
0
0
0
0
9
85
3
2
0
3
10
49
3
1
0
3
11
73
1
0
0
1
12
61
2
1
0
2
13*
22*
0
0
0
0
14*
35*
0
0
0
0
15
54
2
1
1
1
16
129
2
1
1
1
17
64
2
1
0
2
18
74
1
0
0
1
19*
31*
1
1
1
0
Total
1134
25
10
7
18
Table 2: Exon size and nucleotide variation occurring in the pgiC gene in Andean Polystichum.
Length (base Variable
Informative
pairs)
characters Characters
Intron
3
77
10
4
4
113-124
10
0
5
86
7
1
6
84-85
6
3
7
503-533
28
12
8
92-96
7
2
9
67-68
5
2
10
134-146
3
2
11
88
4
1
12
91-92
5
2
14
92
3
2
15
207-391
14
5
16
80
5
0
17
78
3
2
18
138-183
13
8
Total
1930-2219
123
46
Table 3: Intron size and nucleotide variation occurring in the pgiC gene in Andean Polystichum.
Accession
41 2386 2391 2523 3077 210 1140 2065
2709 2769
M1076 orbiculatum
G
C
T
A
C
G
A
T
A
T
M1075 orbiculatum
?
C
T
A
C
?
A
T
A
T
M1087 gelidum
G
C
T
A
C
K
R
W
D
Y
M1051 polyphyllum
G
C
T
-
C
G
G
T
A
C
M1055 platyphyllum
A
T
G
A
C
G
G
T
A
T
M1037 platyXbulb
R
Y
K
W
Y
G
G
T
A
T
M1123 dubium
G
C
T
A
C
G
G
T
A
T
JC662 pallidum
G
C
T
A
C
G
G
T
A
T
M1117 stuebelii
G
C
T
-
C
G
G
T
A
T
Table 4: Heterozygotic nucleotides occurring in accessions of Andean Polystichum in the gene
pgiC.