Flanking regions of monomorphic microsatellite loci provide a new source of data

for plant species-level phylogenetics

Lars W. Chatroua, M. Pilar Escribanob, Maria A. Viruelb, Jan. W. Maasc, James E.

Richardsond, José I. Hormazab

a Nationaal Herbarium Nederland, Wageningen branch, and Wageningen UR,

Biosystematics Group, Generaal Foulkesweg 37, 6703 BL, the Netherlands. Email:

Lars.Chatrou@wur.nl

b Estación Experimental La Mayora-CSIC, Algarrobo-Costa, Málaga 29750, Spain.

Email: pilescri@eelm.csic.es, marian.viruel@eelm.csic.es, ihormaza@eelm.csic.es

c Nationaal Herbarium Nederland, Utrecht branch, Heidelberglaan 2, 3584 CS Utrecht,

the Netherlands. Email: J.W.Maas@uu.nl

d Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, United

Kingdom. Email: J.Richardson@rbge.ac.uk

Corresponding author:

Dr. Lars W. Chatrou

Nationaal Herbarium Nederland, Wageningen branch, and Wageningen UR,

Biosystematics Group

Generaal Foulkesweg 37

6703 BL Wageningen

The Netherlands

Phone: +31 – 317 – 483854

Fax: +31 – 317 – 484917

Email: Lars.Chatrou@wur.nl

1

Abstract

Well-resolved phylogenetic trees are essential for us to understand evolutionary

processes at the level of species. The degree of species-level resolution in the plant

phylogenetic literature is poor, however, largely due to the dearth of sufficiently

variable molecular markers.

Unlike the common genic approach to marker development, we generated DNA

sequences of monomorphic nuclear microsatellite flanking regions in a phylogenetic

study of Annona species (Annonaceae). The resulting data showed no evidence of

paralogy or allelic diversity that would confound attempts to reconstruct the species

tree. Microsatellite flanking regions are short, making them practical to use, yet have

astounding proportions of variable characters. They have 3.5-10-fold higher substitution

rates compared to two commonly used chloroplast markers, have no rate heterogeneity

among nucleotide positions, evolve in a clock-like fashion, and show no evidence of

saturation. These advantages are offset by the short length of the flanking regions,

resulting in similar numbers of parsimony informative characters to the chloroplast

markers.

The neutral evolution and high variability of flanking regions, together with the wide

availability of monomorphic microsatellite loci in angiosperms, are useful qualities for

species-level phylogenetics. The general methodology we present here facilitates to find

phylogenetic markers in groups where microsatellites have been developed.

Key words: microsatellite flanking regions, species-level phylogenetics, neutral

evolution.

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1. Introduction

Increased focus on species-level phylogenetics in angiosperms has encouraged the

pursuit of molecular markers that are capable of resolving phylogenetic relationships at

lower taxonomic levels, i.e. have a mutation rate that is fast enough to produce

sufficient variation (Crawford and Mort, 2004). The need for such markers resonates

within the literature (Bailey et al., 2004; Choi et al., 2006; Crawford and Mort, 2004;

Whittall et al., 2006), as only a small percentage of the published species phylogenetic

trees in plants are fully resolved (Hughes et al., 2006).

Chloroplast markers have been an important source of data for plant phylogenetics.

Apparent advantages of the application of chloroplast markers are its relative abundance

in plant total DNA and the relatively conservative mutation rates, facilitating extraction

and amplification using conserved primer binding sites. Furthermore, chloroplast

markers are essentially single-copy. This avoids the reconstruction of erroneous

organismal phylogenies due to the application of paralogous gene copies, which may be

a problem when applying nuclear markers (Bailey et al., 2003; Baker et al., 2000;

Sanderson and Shaffer, 2002).

The features that simplify the application of cpDNA markers at the species level, are

however traded off against less desirable qualities for organismal species-level

phylogenetics (Sang, 2002). Chloroplast markers generally evolve at rates that are too

slow to provide sufficient phylogenetically informative characters over recent time

spans (Richardson et al., 2001), even after considerable data collection (Perret et al.,

2003; Pirie et al., 2006). It is not to say that not fully resolved phylogenies are

meaningless. As long as critical nodes are well-supported they can serve to pinpoint

biogeographical phenomena (e.g. Erkens et al., 2007b), or to falsify current

classification based on morphological characters (e.g. Shaw and Small, 2004) Only few

3

papers with fairly large chloroplast data sets have generated reasonably well-resolved

and robustly supported species-level phylogenies (e.g. Clarkson et al., 2004).

Furthermore, chloroplast markers are uniparentally inherited, usually maternally in

angiosperms, and therefore provide only part of the evidence for the evolutionary

development of a lineage if hybridization and introgression have taken place (Chase et

al., 2003; Sunnucks, 2000; Vriesendorp and Bakker, 2005).

The search for useful markers for plant species-level systematics has predominantly

yielded markers from genic regions, or, in the case of noncoding DNA, at short

distances from genic regions. There is a growing body of literature on single- or low-

copy nuclear genes that provide sufficient informative characters and do not complicate

organismal phylogeny reconstruction with paralogous copies (Edwards et al., 2008;

Emshwiller and Doyle, 1999; Sang et al., 1997; Small et al., 2004; Whittall et al., 2006).

However, it has also become clear that rates of nucleotide substitution of these markers

may differ significantly among lineages, and even among closely related species

(Hughes et al., 2006). Therefore, attempts to extrapolate the utility of these markers for

resolving species-level relationships outside the taxonomic group for which they have

been developed may not succeed. These difficulties have led some researchers to

suggest that a universal approach should be abandoned in favour of a lineage-specific

one (Small et al., 2004).

However, an alternative to a gene-based approach to the development of variable

nuclear markers involves search strategies that focus on randomly amplified regions

throughout the genome (Bailey et al., 2004; Hughes et al., 2006). The high variability,

abundance, uniform and genome-wide distribution, and neutral evolution of one of

these, namely microsatellites (Ellegren, 2004), make them potentially useful at the

species level. However, their polymorphic nature brings about analytical problems,

4

related to the translation of allele sizes to distance-based characters that are susceptible

to incorrect homology assessment (Matsuoka et al., 2002; Primmer and Ellegren, 1998).

We avoid this drawback by only focusing on the nucleotide sequences of the flanking

regions alongside the microsatellite repeat region, not on the repeat region. A further

factor possibly complicating phylogeny reconstruction is the presence of multiple

alleles, i.e. of variation that doesn’t necessarily have a one-to-one relationship to the

organismal phylogeny, for example because of incomplete lineage sorting. The

distinction between paralogous and orthologous microsatellite copies is less of a

complicating factor. Microsatellites, including the flanking regions, usually represent

unique and therefore orthologous loci (Sunnucks, 2000), although duplication events

affecting microsatellite loci have been reported (Antunes et al., 2006; Zhang and

Rosenberg, 2007).

Thus, an optimal microsatellite flanking region marker for plant species-level

phylogenetics has a rate of substitution that allows resolving shallower relationships, is

represented by orthologous copies, and is monomorphic within species, populations and

individuals. We present examples of such markers from the plant family Annonaceae,

and outline the potential for the broader applicability of this approach in other clades of

angiosperms. We have taken orthology-by-default of as a starting point of our study,

further hypothesizing that a neutral, highly variable marker system such as

microsatellites, including the flanking regions, evolves at a fast enough rate to elucidate

relationships among species of Annona (Annonaceae). In this plant family, the

application of chloroplast sequence data has produced phylogenies that are poorly

resolved at the species level, despite the gathering of large amounts of data (Erkens et

al., 2007a; Mols et al., 2004). Annona is paraphyletic with respect to Rollinia, and the

5

two genera together comprise a clade of approximately 175 species. Species of Rollinia

were synonymised into Annona recently (Rainer, 2007). Here we provide the first

published phylogenetic support for this taxonomic decision, as the former species of

Rollinia (Annona cuspidata, A. herzogii, A. mucosa, A. neochrysocarpa) appear as a

well-supported clade within Annona from the analyses we present here. Although our

taxon sampling reflects one eighth of the species diversity in Annona, covering the

entire morphological diversity as well as the geographical distribution of the genus,

additional taxon sampling would be needed to confidently corroborate the inclusion of

Rollinia into Annona.

To assess the utility of microsatellite flanking regions, we need to address the following

issues: (1) can we produce flanking region sequences that are monomorphic within

individuals? (2) Can we confirm the assumed orthology of the flanking region

sequences? (3) What is the transferability of microsatellite regions across species of

Annona and other Annonaceae? (4) What is the strength of the phylogenetic signal at

the species level?

2. Materials and Methods

2.1 Taxon sampling

For this study we sampled 24 species: 22 species of Annona and two species of Asimina

as outgroup species (Table 1). Richardson et al. (2004) have shown that Asimina is

sister to Annona. The samples of Annona represent the complete geographical range of

the genus, as well as the considerable morphological (particularly floral) variation.

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2.2 Character sampling

Two chloroplast markers, rbcL and trnLF, were sequenced. These markers have

commonly been applied in phylogenetic analyses of Annonaceae (e.g. Couvreur et al.

(2008), Erkens et al. (2007a), Pirie et al (2006)). These markers are generally

considered to be useful at taxonomic levels above that of species. In a family-wide

analysis the relationships among nine species of Annona were fairly well resolved but

generally poorly supported, based on these two chloroplast markers only (Richardson et

al., 2004). The microsatellite loci were selected based on a screening with the first 15

microsatellite loci that were developed in cherimoya (Annona cherimola) (Escribano et

al., 2004). Seven of them (LMCH4, 5, 6, 9, 10, 11, and 14) produced amplification

bands in the eight species studied initially (Annona sp. nov., A. glabra, A. montana, A.

muricata, A. oligocarpa, A. reticulata, A. senegalensis, and Rollina cuspidata [now

Annona cuspidata, (Rainer, 2007)]. No amplification was obtained for two loci

(LMCH1 and LMCH13). For two additional loci (LMCH7 and LMCH8) amplification

was obtained only with A. montana and A. glabra. LMCH9 and LMCH10 were selected

for this study because they showed clear and monomorphic single-allele amplification

bands in all the species tested.

2.3 DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted following a protocol adapted from the CTAB

method (Doyle and Doyle, 1987), as described in Pirie et al. (2006). PCR conditions

and primers for the chloroplast markers were standard, and are identical to Pirie et al.

(2006). PCR products were purified using QIAquick PCR purification kits (Qiagen),

7

and sequenced with the PCR primers. PCR conditions and primers for the

microsatellites LMCH9 and LMCH10 are according to Escribano et al. (2004).

PCR products were resolved in 3% high resolution agarose (Metaphor, FMC

Bioproducts, Rockland, ME) gel electrophoresis in SB buffer at 5V/cm.

Sequencing reactions had a total volume of 10 µl contained 0.5 µl DYEnamic ET

Terminator (Amersham Pharmacia Biotech), 3.5 µl ET Terminator dilution buffer

(Amersham Pharmacia Biotech), and 2-4 µl of DNA template, depending on the

concentration. Template concentration was assessed by gel electrophoresis through a

1.5% agarose gel using a molecular weight marker (Smart-Ladder, Eurogentec, Seraing,

Belgium). Sequencing products were purified in a Sephadex G-50, DNA grade

(Sigma-Aldrich, St. Louis, MO, USA), and analyzed on an automatic sequencer ABI

3730XL (Applied Biosystems).

2.4 Phylogenetic analysis

DNA sequences were edited in SeqMan 4.0 (DNAStar Inc., Madison, Wisconsin), and

aligned manually. After exclusion of ambiguous positions, the resulting alignment of

rbcL consisted of 1364 positions, trnLF 845 positions, LMCH9 117 positions, and

LMCH10 233 positions. Indels were coded following Simmons and Ochotorena (2000),

and resulted in 15 further characters (trnLF: 4, LMCH9: 3, LMCH10: 8).

Maximum parsimony analyses [Fitch parsimony (Fitch, 1971)] for each marker

separately were done applying heuristic searches, with 100,000 random addition

sequence replicates, saving maximally 100 trees per replicate, using TBR branch

swapping. The program PAUP* 4.0b10 (Swofford, 2000) was used for the phylogenetic

analyses. The concatenated data matrix of all markers was analyzed using the branch

8

and bound method, with furthest addition sequence, and the MulTrees option in effect.

Bootstrap resampling of the data matrix was used to assess support, with 1000 bootstrap

replicates for each bootstrap analysis. For each marker individually, full heuristic

searches were done of 100 random addition sequences, TBR, saving 100 trees each

time. For the concatenated matrix, each bootstrap resampled matrix was analyzed using

the branch and bound algorithm, with settings as above.

DNA substitution models for each data partition separately were identified using

ModelTest 3.04 (Posada and Crandall, 1998). Individual data partitions were optimized

onto the combined topology. Based on the model identified by ModelTest, a likelihood

ratio test (Felsenstein, 1988) was applied to test whether each data partition evolves

along all branches within the combined topology at a homogenous rate (molecular

clock). The difference in likelihoods of the tree topologies, with and without clock

constraint, was used to calculate the likelihood ratio test statistic , which is reported in

Table 2. Likelihood values were produced with PAUP* 4.0b10.

To test for congruence between the chloroplast data partition and the flanking region

data partition, we applied the incongruence length difference (ILD) test (Farris et al.,

1995a, b), implemented in PAUP* 4.0b10, using informative characters only, with 5000

replicates, and heuristic searches as described above. The chloroplast markers rbcL and

trnLF were combined into a single data partition, and we tested incongruence of the

plastid data partition with both flanking region markers separately. Incongruence

between the two flanking regions was tested as well. Statistics of the incongruence

length difference tests are given in Table 3.

Saturation plots were made by plotting corrected vs. uncorrected distances for all

possible species pairs, both of which are produced by PAUP*. Distances were corrected

9

applying the models of molecular evolution for each marker separately, as found with

Modeltest 3.06 (Posada and Crandall, 1998). These models are given in Table 2.

Substitution rates were estimated using the program r8s (Sanderson, 2004), using

penalized likelihood as reconstruction method. To estimate branch lengths as accurately

as possible, sequences of each individual markers were optimized onto the combined

topology. Rates were calculated in absolute time (10-9 substitutions / site / year) by

calibrating the crown node of Annona at 19.1 myr. Richardson et al. (2004) estimated

the crown node of Annona at 25.6 3.8 myr. Unpublished results (Pirie et al., in

prep.), analysing more sequence data and calibrating with more fossils compared to

Richardson et al. (2004) have pushed this age up to 19.1 2.0 myr. Reliable fossil data

for Annona are unavailable. Given the broad taxon and character sampling of the study

from which we derive this age, the quality of the fossils, and the small confidence

intervals, we consider this secondary calibration reliable.

3. Results and discussion

3.1 Monomorphism and allelic diversity

Monomorphic microsatellites, i.e. those with only a single allele for a locus, are

routinely discovered during the screening of microsatellite loci in plants (Squirrell et al.,

2003). Two out of 15 microsatellites developed for Annona cherimola (Escribano et al.,

2004), LMCH9 and LMCH10, meet this criterion, as they produced clear single

amplification bands after PCR. We produced LMCH9 and LMCH10 nucleotide

sequences for 22 species of Annona (Table 1). The two microsatellite loci both contain a

dinucleotide repeat region, as well as short 5’ and 3’ flanking regions.

10

PCR of these regions generally produced homogeneous bands, supporting our

assumption of orthology of the included flanking region sequences (Small et al., 2004).

Standard PCR conditions were adequate for obtaining amplification products, and no

cloning was required. The small size of the fragments enhances the ease of

amplification, which makes them especially useful when working with degraded DNA.

In the unusual case of double bands, fragment size similarity amongst different species

was easy to assess, and fragments were cut out from the high-resolution separation gel.

Sequencing of PCR products typically produced chromatograms indicative of

monomorphic loci, which could be interpreted unequivocally. Single nucleotide

polymorphisms (SNPs), i.e. identical polymorphisms that were present in both the

forward and reverse sequence, were hardly ever encountered. Three LMCH9 sequences

(Annona dumetorum, A. mucosa, and A. urbaniana) and two LMCH10 sequences (A.

bicolor and A. hypoglauca) contained 1-3 SNPs, causing a polymorphism frequency

between 0.4% and 1.7% for these five sequences. There was no overlap in positions at

which the polymorphism occurred between any of these species. These SNPs might

indicate the presence of multiple alleles. However, given the very low frequency of

SNPs, possible alleles were highly similar and their effect on the results of the

phylogenetic analyses was non-existent. Even if the SNPs would point at allelic

variation, it would only cause problems for species-level phylogeny reconstruction in

case the coalescence of alleles at deeper phylogenetic levels, ancestral to the species

sampled here. And so, despite the use of a highly variable marker, the careful selection

of monomorphic loci precluded the gathering of intra-specific polymorphisms that

would have rendered reconstruction of the species phylogeny problematic.

3.2 Orthology

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Alignment of the LMCH9 and LMCH10 flanking regions was straightforward. The 5’

flanking region of LMCH9 was only 30 bps and therefore only the 3’ flanking region

was included in the analyses. Both the 5’ and 3’ flanking region of LMCH10 were

included. The dinucleotide repeat regions were excluded from the analysis. The aligned

flanking regions of LMCH9 and LMCH10 comprise 117 and 233 characters,

respectively. Additionally, 11 indel characters were scored and included in the analyses

(Table 2).

Orthology of the flanking region sequences was supported by the similarity of

phylogenetic signal in the flanking regions and in an independent data source, viz.

chloroplast rbcL and trnL-F sequences. Manual observation of bootstrap support values

for each marker separately (Fig. 1) revealed the absence of any well-supported

conflicting clades (bootstrap support 85 %). The sister group relationship of the A.

glabra / A. senegalensis clade with a clade containing the former Rollinia species, as

reconstructed with LMCH9 sequences, is in conflict with the position of the former

clade after analysis of the other markers. However, the bootstrap support of 75% is only

moderate, and insufficient to consider the signal of LMCH9 to be different.

Furthermore, congruence of the combined chloroplast markers and each flanking region

was demonstrated using the parsimony-based incongruence length difference (ILD) test

(Table 3). Both LMCH9 and LMCH10 were not significantly incongruent with the

chloroplast data partition at the 95% confidence level. Finally, incongruence of the two

flanking regions was clearly refuted.

3.3 Transferability

12

We were unable to amplify either of the two microsatellite loci for any species outside

Annona, not even for its sister genus Asimina (Richardson et al., 2004). It should be

noted that the clade to which Annona and Asimina belong is characterized by long

branches subtending generally species-rich clades, causing sister genera to be relatively

distant (Richardson et al., 2004). However, similar patterns of good amplification

success within a target group and poor success in non-congeneric species have been

reported in other plant and animal clades too (Fraser et al., 2005; Peakall et al., 1998;

Wilson et al., 2004) In Annona the limited transferability only poses problems with

regard to the rooting of the tree, as the flanking regions sequences could be produced for

the entire ingroup. We predict that this would also be the case in other similar studies in

angiosperms. Datasets for phylogenetic analyses typically comprise multiple markers,

and can easily be designed to contain flanking region sequences as well as markers that

can be sequenced for a broader range of taxa. The latter sequences would ensure

appropriate rooting of phylogenetic trees and the former would provide many

informative characters at nodes within the ingroup. The potential for utilizing

microsatellite flanking regions for species-level phylogenetics in plants is fairly large, as

published monomorphic microsatellite sequences are available for species-rich tropical

groups, such as Begonia or Melaleuca, as well as temperate groups such as Pinus and

Primula (Squirrell et al., 2003), and could readily be scrutinized for their phylogenetic

utility. At the same time our results as well as the other reports on transferability

suggest that the usefulness of the methodology we describe here is limited to resolving

the shallow branches of the tree of life and will not contribute to taxonomically large

data sets (Chase et al., 2006; Driskell et al., 2004).

3.4 Phylogenetic utility

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The addition of flanking region sequence data has a positive effect on the resolution of

the phylogenetic tree of Annona. The number of well-supported nodes increases

compared to the application of the chloroplast sequences only (Table 4), and the

simultaneous analysis of the four markers produced a single most-parsimonious tree,

generally with high bootstrap support for the nodes (Fig. 2).

The flanking region sequences are much more variable than the chloroplast markers, as

expressed by the higher percentage of both variable and parsimony informative

characters (Table 2). Also, mean substitution rates are higher for the flanking regions,

approximately 3.5 to 10-fold the rate of the chloroplast markers, although it should be

noted that the standard deviations of the substitution rates are large (Table 2). The

models of evolution of the flanking regions evolve are simpler than of the chloroplast

markers. All sites of each of the flanking regions, respectively, evolve at the same rates

as the model estimates showed the absence of rate substitution heterogeneity among the

positions ( = ). Moreover, substitutions accumulate linearly over time in the flanking

regions. For LMCH9, the molecular clock hypothesis was not rejected by the likelihood

ratio test (LRT) at any significance level (Table 2). For LMCH10, the molecular clock

hypothesis is just rejected at the 5% level, though not at the 2.5 % level. For both

chloroplast markers the molecular clock hypothesis was rejected (p < 0.001).

For each marker we plotted uncorrected pairwise distances against distances corrected

using models of molecular evolution (Table 2) as identified using ModelTest (Posada

and Crandall, 1998), to assess the occurrence of saturation (Fig. 3). The chloroplast

markers show initial saturation as the graphs deflect from linearity but don’t reach a

saturation plateau yet. In contrast, the flanking regions that show no evidence of

multiple substitutions at nucleotide positions despite the higher overall substitution

14

rates. The first signs of saturation in the chloroplast markers might be attributable to the

sampling of only 22 out of 175 species of Annona. Increased taxon sampling would

likely reduce the phylogenetic distances among sequences, and consequently could

minimize the appearance of saturation. However, saturation in the four markers is

compared against the background of the same taxon sampling. The tentative conclusion

that the microsatellite flanking regions are less saturated than the chloroplast markers is

therefore warranted.

Due to the higher percentage of variable characters and the higher rate of substitution, it

would be reasonable to expect higher levels of homoplasy in the flanking regions,

simply because of the availability of four character states only for each nucleotide

position. First, superimposed substitutions at a nucleotide position would be supposed to

occur more readily, causing the saturation plot to deflect from linearity. In addition to

this hidden homoplasy, we would expect to see higher levels of ‘visible’ homoplasy, i.e.

the independent multiple origin of identical character states, as reflected in lower values

of the consistency index (CI). However, both the saturation plot (Fig. 3) and the CI

values (Table 2) show results differing from these expectations; there is not as much

saturation in the flanking regions as in the chloroplast markers, and CI values are

similar. The explanation must be sought in the characteristics of molecular evolution of

the flanking regions, notably the clock-like accumulation of substitutions, and the

absence of rate heterogeneity. Both characteristics are assumed by neutral molecular

evolution: a constant substitution rate over evolutionary lineages and over sites in DNA

sequences (Bromham and Penny, 2003). The even distribution of substitutions over the

nucleotide positions in the flanking regions allows the substitution rates to be higher

than for the chloroplast markers, while at the same time making the flanking regions

15

less prone to saturation. In addition, the K80 model of molecular evolution estimated for

LMCH9 is congruent with these indications of neutrality.

These characteristics of the flanking regions are noteworthy as they are in contrast to

findings in the literature on homoplasy in chloroplast markers. The correlation between

levels of homoplasy on the one hand, and substitution rates and/or levels of sequence

divergence on the other hand is often assumed without further testing, for instance to

rule out the possible deleterious effect of saturation in data partitions with low

substitution rates (e.g. Cronn et al., 2002; Zgurski et al., 2008). Such a positive

relationship between substitution rate and level of homoplasy, as expressed by the

consistency index, has been demonstrated for nucleotide substitution rates in chloroplast

genes (Graham and Olmstead, 2000), and even for rates of chloroplast indel characters

(Ingvarsson et al., 2003). Wortley et al. (2005) found that a simulating an increase of

substitution rate of rbcL, matK and ndhF soon resulted in the decrease of phylogenetic

resolution, probably because of saturation. This is likely to be related to the fact that the

evolution of these chloroplast genes is governed by mild functional constraints

(Savolainen et al., 2002). The absence of comparable correlations in microsatellite

flanking regions between substitution rate and homoplasy mirrors the neutral evolution

of microsatellites (Ellegren, 2004), which apparently is present at the nucleotide level in

the flanking regions too.

The clock-like behaviour of the flanking regions makes them a helpful tool for the

dating of divergences. An additional advantage is the availability of a nuclear data

partition, providing a more complete picture of species level phylogenies than based on

chloroplast markers only.

These advantages are traded off by the drawback of the limited size of the flanking

regions. Despite the high percentage of variable and parsimony informative characters,

16

the absolute number of these characters is comparable to the chloroplast markers, the

latter being a more relevant characteristic for determining phylogenetic utility. Wortley

and Scotland (2006) fine-tuned this criterion by describing the minimum number of

parsimony informative character-state changes, as a measure of utility. For the markers

in this paper the difference between this measure and the number of parsimony

informative characters was negligible, as there was only a difference of 1 between the

two measures for trnLF and LMCH10.

3.5 Conclusion

To our knowledge, microsatellite flanking regions have only once been demonstrated to

be congruent with phylogenies based on other data, and subsequently been used in

species-level phylogenetics, viz. in cichlid fishes (Zardoya et al., 1996). Here we

present the first example of the utility of flanking regions for angiosperm phylogenetics.

Our data strongly suggest that the suitability of the flanking regions for resolving

species-level relationships is related to the neutral molecular evolution of these regions,

as exemplified by the substitution rate constancy, similar substitution rates over

nucleotide positions, and the lack of saturation. Given the large number of microsatellite

libraries that have been created for a broad range of species in a large number of

angiosperm genera, the potential for using a similar approach to that employed here for

discovering phylogenetically useful markers in problematic groups is high. In a survey

of the utilization of microsatellites for population genetic studies on individual species

Squirrel et al. (Squirrell et al., 2003) highlighted 66 examples in angiosperms. Of the

studies considered in Table 3 of that publication an average of 17.7% of microsatellite

loci producing PCR products were monomorphic. Our study demonstrates that these

17

loci may represent a great untapped source of phylogenetically informative nuclear

neutral markers for plant species-level systematics. Obviously, on the basis of our

results we cannot accurately predict the phylogenetic utility of these markers in other

plant groups, as little is known about the molecular evolution of microsatellite flanking

regions in general. Nevertheless, it is likely that these molecular evolutionary patterns

will resemble that of Annona, given the frequency and distribution of the bulk of

microsatellites in the genome, and given the neutral evolution of the repeat regions

(Ellegren, 2004). Given the universal utility of our approach microsatellite flanking

regions have the potential to become useful tools for resolving relationships amongst

recently diverged taxa.

18

Acknowledgments

The authors acknowledge financial support from the Spanish Ministry of Education

(Project Grants AGL2004-02290/AGR and AGL2007-60130/AGR). M.P.E. was

supported by a FPI grant of the Spanish Ministry of Education.

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26

Table 1. Species, voucher information and GenBank, NCBI accession numbers.

Species Geography Voucher rbcL trnLF LMCH9 LMCH10Asimina angustifolia A.Gray USA Weerasooriya, A.

s.n. (U) DQ124939 b AY841677 a – –Asimina triloba(L.) Dunal USA Chatrou, L.W.

276 (U) AY743441 c AY743460 c – –Annona amazonica R.E.Fr. Bolivia Chatrou, L.W.

462 (U) EU420853 a EU420836 a EU420768 a EU420790 a

Annona bicolorUrb. Mexico Maas, P.J.M.

8381 (U) EU420854 a EU420837 a EU420769 a EU420791 a

Annona cornifoliaA.St.-Hil. Bolivia Chatrou, L.W.

343 (U) EU420855 a – EU420770 a EU420792 a

Annona cuspidata(Mart.) H.Rainer Guyana Jansen-Jacobs, M.J.

5957 (U) EU420869 a EU420851 a EU420787 a EU420809 a

Annona deminutaR.E.Fr. Peru Rainer, H.

271 (WU) EU420857 a EU420839 a EU420772 a EU420794 a

Annona dumetorum R.E.Fr.

Dominican Republic

Maas, P.J.M.8374 (U) EU420856 a EU420838 a EU420771 a EU420793 a

Annona glabraL.

Neotropical / African

Chatrou, L.W.467 (U) AY841596 a AY841673 a EU420773 a EU420795 a

Annona herzogii(R.E.Fr.) H.Rainer Bolivia Chatrou, L.W.

347 (U) AY841656 a AY841734 a EU420788 a EU420810 a

Annona holosericeaSaff. Honduras Maas, P.J.M.

8445 (U) EU420858 a EU420840 a EU420774 a EU420796 a

Annona hypoglaucaMart. Bolivia Chatrou, L.W.

444 (U) EU420859 a EU420841 a EU420775 a EU420797 a

Annona montanaMacfad. Neotropical Chatrou, L.W.

484 (U) EU420860 a EU420842 a EU420776 a EU420798 a

Annona mucosaJacq. Peru Chatrou, L.W.

247 (U) EU420870 a EU420852 a EU420789 a EU420811 a

Annona muricataL. Neotropical Chatrou, L.W.

468 (U) AY743440 c AY743459 c EU420777 a EU420799 a

Annona neochrysocarpa H.Rainer Peru Pirie, M.D.

43 (U) EU420868 a EU420850 a EU420786 a EU420808 a

Annona oligocarpa R.E.Fr. Ecuador Maas, P.J.M.

8522 (U) EU420861 a EU420843 a EU420778 a EU420800 a

Annona pruinosa G.E.Schatz Costa Rica Chatrou, L.W.

77 (U) EU420862 a EU420844 a EU420779 a EU420801 a

Annona reticulataL. Bolivia Chatrou, L.W.

290 (U) EU420863 a EU420845 a EU420780 a EU420802 a

Annona scandensDiels Bolivia Chatrou, L.W.

365 (U) EU420864 a EU420846 a EU420781 a EU420803 a

Annona senegalensisPers. West African Chatrou, L.W.

469 (U) AY841597 a AY841674 a EU420782 a EU420804 a

Annona squamosaL. Curação van Proosdij, A.S.J.

1133 (U) EU420865 a EU420847 a EU420783 a EU420805 a

Annona symphyocarpa Sandw. Guyana Ek, R.C.

1270 (U) EU420866 a EU420848 a EU420784 a EU420806 a

Annona urbanianaR.E.Fr.

Dominican Republic

Maas, P.J.M.8392 (U) EU420867 a EU420849 a EU420785 a EU420807 a

a This studyb Erkens et al. (2007b)c Pirie et al. (2005)

27

Table 2. Statistics per marker on features of data and molecular evolution.

# nucl.

chars.

#

indel

chars.

# and %

variable

chars.

# and %

pars. inf.

chars.

# most

pars. trees

tree

length

CI RI

rbcL 1364 0 72 / 5.3 % 42 / 3.1 % 24 103 0.874 0.913

trnL-F 845 4 101 / 12.0 % 47 / 5.6 % 66 142 0.887 0.898

LMCH 9 117 3 41 / 35.0 % 26 / 22.2 % 31 55 0.873 0.932

LMCH 10 233 8 69 / 29.6 % 42 / 18.0 % 189 98 0.898 0.906

model among-

site rate

variation

LRT statistic

(2)

rate (10-9

substit. /

site / year)

rbcL GTR + Γ =

0.1504

58.95 a 0.3127 ±

0.2225

trnL-F TIM + Γ =

0.7028

102.89 a 0.8682 ±

0.5258

LMCH9 K80 ∞ 16.37 b 2.962 ±

1.999

LMCH10 HKY ∞ 33.26 c 3.065 ±

1.693

a p < 0.0001

b 0.6 < p < 0.7

c 0.03 < p < 0.04

28

Table 3. Statistics of incongruence length difference tests.

Partitions ILD p-value

chloroplast markers vs. LMCH9 0.0746

chloroplast markers vs. LMCH10 0.0856

LMCH9 vs. LMCH10 0.2160

Table 4. Effect of combining data partitions on the number of clades with bootstrap

support ≥ 85 %.

rbcL 10

trnLF 6

rbcL / trnLF 12

LMCH9 2

LMCH10 6

LMCH9 / LMCH10 9

rbcL / trnLF / LMCH 9 13

trnLF / LMCH9 / LMCH10 12

rbcL / trnLF / LMCH9 / LMCH10 16

29

30

Figure legends

Figure 1. Maximum parsimony phylogram for each of the four markers. The total

number of most parsimonious trees from which the trees shown here were arbitrarily

chosen is given in Table 2. Thick grey branches indicate bootstrap support 85%, thick

black branches bootstrap support between 70% and 84%. Lack of flanking region

sequences for Asimina precluded outgroup rooting. For ease of comparison, trees of

LMCH9 and LMCH10 have been drawn rooted at the midpoint between the clade with

Annona muricata and the remainder of the ingroup species (as found in the plastid trees

rooted with Asimina). Horizontal bars equal indicate branch lengths of five steps.

Figure 2. Single most-parsimonious phylogram resulting from maximum parsimony

analysis of all data (rbcL, trnLF, LMCH9, and LMCH10) combined. Thick grey

branches indicate bootstrap support 85%, thick black branches bootstrap support

between 70% and 84%.

Figure 3. Saturation plots, displaying corrected versus uncorrected pairwise distances.

Distances were corrected using the models of molecular evolution given in Table 2.

31