“Study of the Evolution and the Structure of the Ribosomal RNA

Final Degree Project Biochemistry

“Study of the evolution and the structure of the ribosomal RNA genes in plants: Analysis of the intergenic spacer (IGS) in a hybrid complex of species of the genus Armeria”

Sònia Valle Durán Internal supervisor: Sònia Garcia Giménez External supervisor: Teresa Garnatje Roca June-July 2016

Cover picture: Armeria maritina: http://www.flickriver.com/photos/ken-ichi/4620446579/

1 Abstract

Hybridization and introgression phenomena are common in the genus Armeria. Armeria pungens is an example and it has suffered introgression with other pure species, Armeria macrophylla, which is very close to it. They have been found in the south of the Iberian Peninsula. In this TFG we have studied this phenomenon based on the IGS region of its genome, a non-coding region and thus highly variable and subject to evolution. So we have sequenced both two “pure” species and the hybrid and we have studied several elements located in this region, as well as its size and its correlation with the genome size.

On the other hand we have analyzed another non-coding region of the genome, the ITS region, which is also highly variable and presents polymorphism in some nucleic bases depending on the taxon.

All information indicates that introgression has worked in favour of Armeria macrophylla as the similarity of the hybrid is higher in many aspects in which we have deepened throughout the project.

This research work is a part of a series of researches on the genus Armeria started in a previous TFG. The investigation has been extended with the analysis of more individuals and populations of the three taxa and the genome size.

3 INDEX

Page

1. Introduction 1-2

1.1. Genus Armeria 1

1.1.1. Armeria pungens 1-2

1.1.2. Armeria macrophylla 2

1.2. Hybridization 3

1.3. Introgression 3

1.4. Chloroplast capture 3

1.5. Introgression phenomenon in the genus Armeria 4

1.6. Ribosomal DNA 5-6

1.7. Transposable elements (TE) 6

2. Hypothesis and objectives 7

3. Materials and methods 8-15

3.1. Materials 8

3.2. Methods 9-15

3.2.1. DNA extraction with DNeasyTM Plant Minikit 9

3.2.2. DNA extraction with the CTAB method 9-10

3.2.3. PCR: Polymerase chain reaction. DNA amplification 10

3.2.4. Sequencing of the entire IGS unit 11

3.2.5. Extraction and purification of DNA 11-12

3.2.6. Cloning 12-13

3.2.7. Flow cytometry 13

3.2.8. Sequence analysis and primer design 14-15

3.2.9. Statistical methods 15

4. Results 16-26

5. Discussion 27-30

6. Conclusion 30

7. Literature 31-35

0 1. Introduction

1.1. Genus Armeria

Armeria Willd. is a genus of shrubby low-growing evergreen perennials (Figure 1). Although more than two hundred species have been described, only less than a hundred names are currently accepted. The genus was described by Carl Ludwig Willdenow, a German botanist from the XVIIIth century.

It belongs to the Plumbaginaceae, a flowering plant family with a worldwide distribution (from arctic to tropical conditions), particularly associated with salt-rich steppes, marshes, and sea coasts. Most species are perennial herbaceous plants. The family includes 24 genera and about 800 species. Armeria species are usually found on coastlines and are mostly native to the Mediterranean. Armeria species (such as A. pungens and A. macrophylla) are strongly affected by natural hybridization and introgression (see page 4) (Nieto Feliner et al. 2001; Tauleigne-Gomes & Lefèbvre 2005; Piñeiro et al. 2011).

1.1.1. Armeria pungens

Armeria pungens (Link) Hoffmanns. & Link is an herbaceous plant of genus Armeria that forms small shrubs reaching heights of about 80 cm. The stems are lignified at the base, robust and highly branched. Leaves are glabrous, linear to lanceolate, pointed. Flower heads (or capitula) are pale pink, gathered in globular inflorescences at the top of long pedicels (Figure 1).

Figure 1. Armeria pungens http://www.bioscripts.net/herbarios/h_HFUS/pl g_178.html This small shrub grows in coastal sand-dunes and beaches, at about 0–1 meter above sea level and they are found in the southwest of the Iberian Peninsula (Figure 2).

1 Figure 2. Distribution of Armeria pungens. Some populations are also found in the northern coast of Corsica http://www.floravascular.com/index.php ?spp=Armeria%20pungens

1.1.2. Armeria macrophylla

Armeria macrophylla Boiss. & Reuter is an herbaceous plant of genus Armeria. It is a perennial herb up to 70 cm. It has two types of leaves of about 10-27 cm: the light green and very narrow leaves with cespitose appearance and the gray -green narrow leaves up to 2 mm wide and covered with short and thin hairs. The pink flowers are grouped into capitula that grow at Figure 3. Armeria macrophylla the end of very long stems, up to http://www.florasilvestre.es/mediterranea/Plumb 60 cm. Each flower is wrapped in aginaceae/Armeria_macrophylla.htm white tissue that remains after the flower dries. The capitula are wrapped in straw-colored scales, hairless and membranous on the edge, and grow from the outside inward (Figure 3).

It grows in coastal areas on sandy soils, specifically in the South Atlantic coast of the Iberian Peninsula (Figure 4).

Figure 4. Distribution of Armeria macrophylla http://www.floravascular.com/index.php ?spp=Armeria%20macrophylla

2 1.2. Hybridization

Hybridization is the process of interbreeding between two genetically different species (interspecific) or genetically divergent individuals from the same species (intraspecific). It results in a new cross breed or specie with a new genotype. This process is very common between plants leading to usually fertile progeny that may have advantageous characteristics with respect to the parental species.

1.3. Introgression

It is a type of hybridization resulting from an interbreeding process known as backcrossing (Anderson & Hubricht 1938, Anderson 1949), the cross between an interspecific hybrid and one of its parental species. Introgression is more complex than simple hybridization because it results in a non-uniform mixture of the two parental genomes. This process increases the genetic variation of one or both of the parental species (Anderson 1949; Anamthawat-Jónsson 2001) and can contribute to their adaptation and speciation (Grant et al. 2005).

1.4. Chloroplast capture

This is an evolutionary phenomenon tied to introgression that, after hybridization and subsequent backcrossings, results in a hybrid plant with a new combination: a chloroplast genome whose origin is from one parent and a nuclear genome whose origin is from the other parent (Grant et al. 2005). Chloroplast capture may explain the inconsistencies found in gene trees based on nuclear and chloroplastic markers in plants.

Armeria pungens exhibits evidence for an ancient or ongoing introgression from sympatric congeners such as the sharing of ITS polymorphisms or the close relationship between plastid haplotypes (Piñeiro et al. 2011), suggesting chloroplast capture.

3 1.5. Introgression phenomenon in the genus Armeria

As mentioned before, genus Armeria is subject to introgression as it is the case of Armeria pungens which has experienced this phenomenon with Armeria macrophylla resulting in a hybrid termed here as introgressed Armeria pungens (Figure 5).

Armeria macrophylla Armeria pungens

Introgressed Armeria pungens

Figure 5. Introgression phenomenon in genus Armeria. The leaves of the introgressed taxa are only slightly shorter than the leaves of Armeria macrophylla but not as short as the leaves of Armeria pungens so the morphological distinction is difficult

4 1.6. Ribosomal DNA

Ribosomal DNAs (rDNA) are the genes which code for ribosomal RNA that, along with proteins, form the ribosomes. Eukaryotic rDNA consists of a tandem repeat of a “large” operon composed of 18S, 5.8S and 28S (26S) rRNA genes and intergenic spacers (Figure 6). These regions are known as the nucleolus organizer regions, forming the nucleolus, expanded chromosomal loops where rDNA is transcribed.

Figure 6. Eukaryotic rDNA https://en.wikipedia.org/wiki/Riboso mal_DNA#/media/File:Eucaryot_rd na.png

There is another gene that codes for 5S rRNA also organized in tandem repeats in the genome in most eukaryotes, although it can be linked to the large rDNA operon as a single repetitive unit in certain groups (Garcia et al. 2009).

Genes encoding ribosomal RNA and spacers are highly conserved among species and occur in tandem repeats that are thousands of copies long. Each one is separated by regions of non-transcribed DNA, termed intergenic spacer (IGS) or non-transcribed spacer (NTS) which are more variable due to insertions, deletions, and point mutations high enough to generate intra- and interspecific variability (Hillis & Dixon 1991; Baldwin et al. 1995) even between closely related species because of the low evolutionary pressures.

The intergenic spacer (IGS) is the region of non-coding DNA between the rRNA genes 18S and 26S and can change very rapidly in evolution. Variability in the IGS can cause heterogeneity in rDNA repeat length among members of the same genus or species (Saghai-Maroof 1984; Rogers 1986). This variability is found in the number of subrepetitive elements within the IGS which are generally between 100 and 200 base pairs in length (Yakura 1984; McMullen 1986). These subrepetitive elements could have arisen from transposition events since they show some similarities with transposons, and it also appears that they are "hot spots" for recombination (Rogers 1986). It is also possible that subrepeats in plant intergenic spacers act as RNA processing sites or as transcription termination sites (De Winter 1986; Labhart 1986; Harrington 1987).

5 Chloroplasts also have their own rRNA genes, composed of 23S, 16S and 5S rDNA. rRNA gene sequences and some of their intergenic spacers (both nuclear and chloroplastic) are used for phylogenetic studies.

1.7. Transposable elements (TE)

Transposable elements are DNA sequences that can move and change its position in the genome (into the same chromosome or to another), inserting or removing a mutation and possibly altering the genome size (Bennetzen 2000). They are important for genome function and evolution.

There are two classes of TEs (Figure 7):

- Class I: Also known as retrotransposons, they are transcribed from DNA to RNA, and then reverse transcribed to DNA. This sequence is inserted back to the genome but in a new position. This reaction is catalyzed by reverse transcriptase encoded by the reverse transcriptase.

- Class II: Also known as DNA transposons, most of them encode the transposase protein to insert and excise (cut-and-paste mechanism) without involving RNA. This reaction is catalyzed by several transposase enzymes which make a staggered cut in the DNA resulting in a 5’ or 3’ single-strand called “sticky end”. Some of them bind to a specific sequence of the DNA and others to a non-specific site. The cut DNA transposon is ligated to a new site where the DNA polymerase fills the gaps and the DNA ligase incorporates it to the main chain. It results in a duplication of the target site, so that means short direct repeats followed by inverted repeats (Bennetzen 2000)

Figure 7. Classes of TEs http://www.cell.com/tre nds/ecology- evolution/fulltext/S0169 -5347(13)00256-5

6 2. Hypothesis and objectives

By hybridization and introgression phenomena, the crossing between Armeria pungens and Armeria macrophylla has resulted in a novel genotype combining both genomes to different degrees. By studying the sequences of the intergenic spacers (IGS) and internal transcribed spacer (ITS) of the rRNA genes and comparing the parental species with the progeny we want to see if:

(1) The IGS and ITS sequences of the hybrid are a balance between both parental sequences or if they resemble more one of the parents

(2) There is presence of transposable element sequences in the IGS and if it differs depending on the species

We will also assess genome size of the parental species and the hybrid in order to detect significant differences in the hybrid that would point more to one parental species or the other.

Therefore, to reach these goals we will:

 Sequence the IGS region of different individuals from different populations of the three taxa (parental species and hybrid)

 Study the internal structure of the IGS region by identifying several elements such as tandem repeats and transposon fragments

 Study the polymorphic positions in the ITS region

 Study the genome size and assess significant differences if any

7 3. Materials and methods

3.1. Materials Samples of two populations of each of the three taxa have been collected from the south of the Iberian Peninsula (Figure 8).

Figure 8. Distribution of the populations of the genus Armeria collected

Information about all the samples of each population collected is contained in the following table (Table 1):

Table 1. Collection data of the populations studied

Taxa Origin Collectors Collection Date number Armeria Cádiz, Caños de Meca, Cabo J.A. Rosselló & 4703GN 29/06/2014 pungens Trafalgar, 8 m, dunas, G. Nieto Feliner 36º10'58.25"N 6º01'57.97"W Cádiz, Tarifa, Punta Camarinal, S. Garcia y G. 4723GN 01/02/2016 junto a la playa del Cañuelo, 10m, Nieto Feliner dunas fijas, playa, 36º5'16.68"N 5º48'14.90"W Introgressed Cádiz, Tarifa, Punta Camarinal, J.A. Rosselló y 4702GN 28/06/2014 Armeria 21 m, matorrales abiertos sobre G. Nieto Feliner pungens suelo arenoso, en duna fósil, 36º04'51.09"N 5º47'30.84" W, Cádiz, Tarifa, Punta Camarinal, S. García y G. 4724GN 01/02/2016 junto a la playa del Cañuelo, 20m, Nieto Feliner en claros del maqui, suelo arenoso, 36º5'4.14"N 5º48'5.79"W Armeria Cádiz, Tarifa, Punta Camarinal, J.A. Rosselló & 4701GN 28/06/2014 macrohpylla 42 m, sotobosque de pinar, suelo G. Nieto Feliner arenoso, 36º05'12.82"N 5º47'30.66" W Cádiz, Tarifa, Punta Camarinal, S. Garcia y G. 4725GN 01/02/2016 junto a la playa del Cañuelo, 20m, Nieto Feliner en claros de pinar (P. pinea), suelo arenoso, 36º5'9.98"N 5º47'33.96"W

8 3.2. Methods

A brief protocol of each of the methods used is here presented.

3.2.1. DNA extraction with DNeasyTM Plant Minikit

1. Weigh 15-16 mg of tissue and place into a 2 mL Eppendorf tube. Grind the sample (Retsch, biometa MM 301) for 3 minutes at 30 rpm. 2. Add 400 μL of buffer AP1 and 4 μL of RNase A. Mix well with vortex and incubate for 10 minutes at 65ºC (invert the tube every 2-3 minutes). 3. Add 130 μL of buffer P3, mix without the vortex. Incubate for 5 minutes on ice. 4. Centrifuge for 6 minutes at 13,000 x g. Transfer the flow-through into a QIAshredder spin column placed in a 2 mL collection tube. 5. Centrifuge for 2.5 minutes at maximum speed. Transfer the flow-through obtained in the bottom tube to a 1.5 mL graduated Eppendorf tube. 6. Estimate the volume and add 1.5 volumes of buffer AW1 homogenizing. 7. Transfer 650 μL of the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube. Centrifuge for 1 minute at 6,000 x g and discard the flow- through. Add the remaining sample. 8. Centrifuge for 1 minute at 6,000 x g and place the spin column into a new 2 or 1.5 mL Eppendorf tube. Add 500 μL of buffer AW2. 9. Centrifuge for 1 minute at 6,000 x g and discard the flow-through. Add 500 μL of buffer AW2. 10. Centrifuge for 2 minutes at 6,000 x g and discard the flow-through. 11. Transfer the spin column into a new 1.5 mL Eppendorf tube, add 30 μL of buffer AE for elution and incubate 5 minutes at room temperature. Centrifuge for 1 minute at 6,000 x g. 12. Add another 30 μL more of buffer AE for elution, incubating 5 minutes at room temperature and centrifuging for 1 minute at 6,000 x g.

3.2.2. DNA extraction with CTAB method 1. Weigh around 20 mg of tissue and place it into a 2 mL Eppendorf tube. Grind the sample (Retsch, biometa MM 301) for 3 minutes at 30 rpm. 2. Add 500 μL of CTAB buffer. Mix well with vortex and incubate for 50 minutes at 55ºC (invert the tube every 10 minutes to homogenize). 3. Add 2.5 μL of 10 μg/mL RNase and incubate for 10 more minutes at 55ºC. 4. Add 500 μL of Chloroform:Isoamyl alcohol (24:1)* and mix. Centrifuge the mixture for 10 minutes at 13,300 x g. *this solution is used to clean the DNA of impurities and separate three phases (Figure 9).

Aqueous/Liquid phase: Alcohol+CTAB+DNA Solid phase Fatty phase: Chloroform Figure 9. Resulting phases of centrifugation

5. Pipette the aqueous phase (do not touch the solid phase) and transfer it to a new 1.5 mL Eppendorf tube discarding the rest. 6. Estimate the volume and add 0.08 volumes of 7.5 M ammonium acetate stored at -20ºC and 0.54 volumes of cold isopropanol. Store around 1 hour at -80ºC. 7. Remove the sample from the ultrafreezer and wait until thawed. Centrifuge for 3 minutes at 13,300 x g and discard the flow-through. 8. Add 700 μl of cold alcohol 70% and disengage the pellet with vortex. Centrifuge for 1 minute at 13,300 x g and discard the flow-through. 9. Add 700 μl of cold alcohol 95-96% disengage the pellet with vortex. Centrifuge for 1 minute at 13,300 x g and discard the flow-through. 10. Let the DNA (pellet) dry for around 30 minutes. Resuspend in 30 μL of TE.

3.2.3. PCR: Polymerase chain reaction. DNA amplification

Table 2. Reagents and volumes for PCR reaction Reagent Volumes (µL) Buffer (each polymerase has its buffer) 2.5 dNTPs (must be at the concentration needed by the polymerase) 2.5 Primer 18S R (reverse) (Figure 10) 0.5 Primer 26S F (forward) (Figure 10) 0.5 DMSO (at room temperature, prevents the formation of secondary DNA structures) 1 H2O (first thing to add to help the mixing of everything) 15.8 ExTaq polymerase (a special polymerase for large product size amplification, last thing to 0.2 add just before starting the PCR) DNA (add just before the polymerase) 2

Figure 10. 26S F and 18S R primers. They anneal at the 3′ end of the 26S rDNA gene and the 5′ end of the 18S rDNA gene, respectively (Galián et al. 2012)

Multiply the volumes (Table 2) (except the DNA and Taq) by the number of samples to make the “cocktail” and distribute to PCR tubes. The final volume in each tube is 25 µL. Amplification program: ExTaq 3

10 3.2.4. Sequencing of the entire IGS unit

Table 3. Reagents and volumes for sequencing reaction Reagent Volumes (µL) Sequencing Mix (BigDye Terminator 3.1, polymerase and fluorescently labelled 1 dNTPs are included) Primer (depending on the region to sequence) 1 Sequencing Buffer (provided by the BigDye Terminator 3.1 kit) 3 DMSO (at room temperature, prevents the formation of secondary DNA structures) 0.4 H2O (first thing to add to help the mixing of everything) 12.6 DNA (each tube contains a different sample of DNA-PCR product) 1

Multiply the volumes (Table 3) (except the DNA) by the number of samples to make the “cocktail” and distribute to PCR tubes. The final volume in each tube is 20 µL. Sequencing program: AUTO54

3.2.5. Extraction and purification of DNA

 GEL MAKING: Add 0.4 g of agarose to 50 mL of 1X TAE and put in the microwave until it boils. Stir to dissolve, continue for 3 minutes and cool for 3 minutes. Add 40 µL of Crystal Violet, mix well and decant to the gel box. Wait 30 minutes until solidified and place into electrophoresis chamber.  LOADING PCR PRODUCT IN THE GEL: Store the samples at -20ºC. Add 8 µL of 6X Crystal Violet Loading Buffer to 40 µL of PCR product. Run the gel at 80 V until the Crystal Violet has ran 25% of the gel.  CUTTING THE PCR PRODUCT: Set Termobloc at 46ºC, defrost Sodium Iodide, Binding Buffer and 4X Final Wash. 1. Put the gel in a transparent plastic layer under a direct light and photograph the gel. Cut the gel with the fragment that we are interested in. 2. Weigh the fragment in a tared Eppendorf tube assuming that 1 µL of gel is 1 mg. Add 2.5 volumes of 6.6 M Sodium Iodide. Shake with the vortex. 3. Incubate at 46ºC until the agarose is completely melted. Wait around 2-3 minutes with the tube at room temperature. Add 1.5 volumes of Binding Buffer.  ISOLATING THE PCR PRODUCT: Set Termobloc at 37ºC. Heat a TE tube. 4. Load the previous product in the SNAP column with the collection tube, centrifuge for 30 seconds at 2500 x g and put the flow-through in the column. Centrifuge again and discard the flow-through. DNA is attached to the column.

11 5. Add 400 µL of 1X Final Wash (100 µL 4X Final Wash + 300 µL alcohol) and centrifuge for 30 seconds at 2500 x g. Repeat and discard the flow-through. Centrifuge for 2 minutes at maximum speed and discard the collection tube. 6. Transfer the column to a new 1.5 mL Eppendorf tube and add 40 µL of TE buffer (previously heated at 37ºC). Incubate for 1 minute at room temperature. 7. Centrifuge for 1 minute at maximum speed, put on ice and discard the column. Prepare a gel, load 10 µL of and check the concentration (NANODROP).

3.2.6. Cloning

 PREPARATION OF LB-AGAR PLATES (2 for each sample)

1. Dissolve 35 g of LB-Agar in 1000 mL of H2O (d) (8.75 g/250 mL) and autoclave. Cool to 55ºC and add 50 µg/mL of kanamycin (antibiotic) (12.5 mg/250 mL). 2. Add 30-35 mL for each one (enough for 8/9 plates), leave it to harden for 30 minutes and put it in the freezer (4ºC).  CLONING REACTION: Switch on the heater to 37ºC. 1. Mix 2 µL of the PCR product with 0.5 µL of XL TOPO vector. 2. DNA linkage inside the plasmid: Incubate the tubes at room temperature for 30 minutes. Adding 0.5 µL of 6x TOPO Cloning Stop Solution and mix for a few seconds at room temperature. Centrifuge briefly the tubes and leave on ice. 3. Transformation reaction: Keep the tube of competent cells at -20ºC and number the tubes. Divide every tube in 2 (25 µL per tube). Add to each tube 1 µL of the cloning reaction. Incubate for 30 minutes on ice, Float the tubes in the bath at 42ºC for 30 seconds and put them on ice immediately for 2 minutes. Add 125 µL of S.O.C. medium at room temperature to each tube. Incubate the tubes at 37ºC for 1 hour and put them in the shaker inside the heater horizontally at 225 rpm. Incubate the LB plates (2 for each sample) for 1 hour upside down. 4. Plating: Mark the cover of the plates (name, date, 50/80). Add 50 µL or 80 µL of colonies respectively and spread the colonies. Sterilize in the flame with alcohol 96% when changing the sample and incubate at 37ºC upside down overnight. 5. Transformants analysis by PCR: Use primers M13F_(-20) and M13R for PCR. Increase the water volume to 17.8 µL because we are not putting 2 µL of DNA. Prick a colony with the pipette tip leaving material and pump with the pipette. 6. Positive culture of colonies in liquid LB: Mix 2 g of LB Broth into 100 mL of

H2O(d) and autoclave. Cool and add 5 mg of kanamycin. Keep on the fridge. Add 2 mL of LB+kanamycin in 2 mL tubes and touch the select colony with the

12 pipette tip and mix with the medium. Incubate the tubes at 37ºC overnight and put them in the shaker inside the heater horizontally at 250 rpm. 7. Plasmid isolation (Durziv Kit): Centrifuge 1.5 µL of the culture for 1 minute at 12,000 x g and discard the flow-through. Resuspend the pellet in 200 µL of EPO1 (in the fridge) and 2 µL of REALBLUE Lysis control reagent. Lyse the cells with 200 µL of EPO2 and invert the tube 10 times until white and viscous mixture. Add 100 µL of EPO4 and 150 µL of EPO3 and invert the tube gently 10 times. Centrifuge for 5 minutes at maximum speed and transfer the flow- through to a new 1.5 mL tube. Add 450 µL of isopropanol at room temperature and mix. Centrifuge for 3 minutes at maximum speed and discard the flow- through by suction. Add 100 µL of EPO5 and mix by tapping. Centrifuge for 3 minutes at maximum speed, discard the flow-through by using the pipette and add 70 µL of ethanol 70%. Centrifuge for 2 minutes at maximum speed and discard the flow-through. Repeat these two last steps and let dry the tubes opened and upside down for 20-30 minutes. Resuspend the DNA in 50 µL of TE at 37ºC and check the D.O. and concentration with NANODROP. 8. Sequencing: Use primers M13F_(-20) and M13R to make the sequencing mix. Increase the water volume to 14 µL because we are not putting 1 µL of DNA and 0.4 µL of DMSO (final volume=200-250 ng). Use AUTO54.

3.2.7. Flow cytometry

1. Place a calibration standard and leaf tissue from the studied species (double) together in a plastic Petri dish in around 1000 μL of nuclei isolation buffer, chopped up and supplemented with 100 μg/mL of ribonuclease A. 2. Filter 600 μL of the suspension of nuclei in isolation buffer (usually LB01) through a nylon mesh (pore size=70 μm) and stain on ice for 20 min with 36 μL of propidium iodide (1 mg/mL) to a final concentration of 60 μg/mL. 3. Calculate the total nuclear DNA content: Multiply the known DNA content of the standard by the quotient between the 2C peak positions of the target species and the standard in the histogram of fluorescence intensities (there is a linear correlation between the fluorescence signals from stained nuclei of the unknown specimen and the known internal standard and the DNA amount). 4. Analysis of five individuals each population, two samples each individual: The mean half peak coefficient of variation for the target plant and for the internal standard, mean values and standard deviations are calculated.

13 3.2.8. Sequence analysis and primer design

- BioEdit: Biological sequence alignment editor used to introduce the sequences obtained by “primer walking” strategy and build the matrices with several sequence manipulation, analysis options and links to external analysis programs. BioEdit’s features include a basic automated ClustalW alignment.

- Standard Nucleotide BLAST (GenBank): BLASTN programs search nucleotide databases using a nucleotide query. This program has been used to identify and locate the 26S and 18S genes in our sequences. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch

- MAFFT version 7: Multiple alignment online program for amino acid or nucleotide sequences. http://mafft.cbrc.jp/alignment/server/

- WebLims: Online service to register samples obtained by sequencing and to download the results (sequences) two or three days after the samples have been given to the “Genomics” Department in the “Parc Centífic de la Universitat de Barcelona”. http://lims.sct.ub.es/index.jsp

- IDT: Integrated DNA Technologies is a company that sells DNA oligonucleotides. It is an online service that enables to design primers, order and receive them in the laboratory after two or three days. https://eu.idtdna.com/site/order/menu

- YASS: Genomic similarity search tool to compare DNA or RNA sequences in FASTA or plain text format (it produces local pairwise alignments). It has been used for primer design to avoid repetitive DNA regions. http://bioinfo.lifl.fr/yass/

- Primer3Plus: Using the “Task: Sequencing” this tool pick a series of primers on both strands from a DNA sequence for sequencing. Optionally the regions of interest can be marked using targets. It has been used for primer design. http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

- Emboss etandem: It calculates a consensus sequence for a putative repeat region and scores potential repeats based on the number of matches/mismatches to the consensus. The repeat must be within the specified minimum and maximum size and must score higher than the specified threshold. http://emboss.bioinformatics.nl/cgi- bin/emboss/help/etandem

14 - Ident and Sim: Accepts a group of aligned sequences and calculates the identity and similarity1 of each sequence pair. Their values are often used to assess whether or not two sequences share a common ancestor or function. http://www.biomol.unb.br/sms2/ident_sim.html

- CpG islands: Identify and plot CpG islands in nucleotide sequence(s). http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/

- CENSOR: Software tool which screens query sequences against a reference collection of repeats and masks homologous portions with masking symbols, as well as generates a report classifying all repeats found. http://www.girinst.org/censor/index.php

3.2.9. Statistical methods

- Rstudio: Statistical program to assess genome size, IGS size and TE size differences between taxa we performed analyses of linear regression, one-way ANOVA and LSD test with RStudio, v.0.98.1078, a user interface for R.

1Identity: Amount of characters which match exactly between two different sequences. Gaps are not counted and the measurement is relational to the shorter of the two sequences. It is not transitive. Similarity: Degree of resemblance between two sequences when they are compared. This is dependent on their identity. It shows the extent to which residues aligned, counting the gaps. Similar sequences have similar properties.

15 4. Results

First of all, an electrophoretic gel (Figure 11) was loaded with the products obtained by PCR amplification of the different taxa and populations to estimate the sizes of the DNA molecules, which ranged from 4700bp to 6000bp, both belonging to the same taxa (“pure” Armeria pungens) and population (4703). The DNA fragments include genic regions of 26S and 18S genes (824bp approximately). The image shows the bands of each clone of each population studied and their approximated length.

Figure 11. Visualization of the electrophoretic gel with the different bands corresponding to each clone, population and an estimation of their length

Given their approximated length, we have selected for sequencing the longer clones for each of these populations of the three taxa:

- 5 APNI - 28 API - 5 AM - D10 APNI - A2 API - L3 AM - 2 APPI - 2 AM

Of each of the clones, we have sequenced and aligned the IGS region. The matrix in Supplementary Material 1 contains the sequences including a fragment of 26S gene at the beginning and a fragment of 18S gene at the end, both indicated in the file.

16 Once sequenced the IGS region for the selected clones and analyzing the sequence, some specific functional elements, putative functional sequences and other sequence characteristics of the intergenic spacers (Table 4) have been found in all of the sequences.

A quite similar, almost equal, length is observed between introgressed Armeria pungens and Armeria macrophylla which present a very long IGS region with respect to the “pure” Armeria pungens.

Concerning to CpG islands, the introgressed Armeria pungens clones presents just one while the “pure” species contain more, except Armeria macrophylla clone L3 AM (population 4701) which has only the first one (83-455 (373bp)). Probably the second one is located in the part that lacks to be sequenced.

About the GC content we observe a lower percentage in Armeria pungens clones 5 and D10 APNI (population 4703) with respect to the other two taxa. But we find an exception; the introgressed Armeria pungens clone 2 APPI presents the lowest percentage (which may be explained by the fact that its sequence is not complete).

17 Table 4. Functional elements and putative function of IGS

Functional element Putative function Armeria pungens Introgressed Armeria pungens Armeria macrophylla Population 4703 4702 4724 4701 4725 clone D10 Clone clone 5 APNI clone 28 API clone A2 API clone 2 APPI clone 5 AM clone L3 AM clone 2 AM APNI TTS (CCCTCCC) 454-460 454-460 454-460 454-460 454-460 454-460 454-460 454-460 Conserved transcription 2268-2274 2268-2274 2268-2274 2268-2274 2268-2274 2268-2274 termination site 3471-3477 3471-3477 3471-3477 3471-3477 3471-3477

TCTTTTACTTCCCTAATT Pyrimidine rich 462-483 462-483 462-483 462-483 462-483 462-483 462-483 462-483 AACT sequence TTS (AACTTCG) 6415-6422 6415-6425 6415-6427 6415-6428 6415-6429

2706-2712 TTS in stem-loop 3578-3584 3578-3587 3578-3589 3578-3590 3578-3591

TIS (Transcription TACTATAGGGGGGGTG Initiation Site) A as first 3504-3519 3504-3519 3504-3519 3504-3519 3504-3519

transcribed nucleotide TACTATAGGGGGGT TIS (Transcription 2420-2433 2420-2433 Initiation Site) A as first 3621-3634 3621-3634 3621-3634 3621-3634 3621-3634 transcribed nucleotide CpG Islands* DNA regulatory regions 85-426 (342bp) 88-454 (367bp) 85-457 (373bp) 86-458 (373bp) 50-462 (413bp) 85-457 (373bp) 83-455 (373bp) 85-457 (373bp) of cytosines followed by guanine and where the 2779-3035 1994-2255 4418-4623 3603-3865 cytosines can be (257bp) (262bp) (206bp) (263bp) methylated Size of the IGS sequence 3929 3448 4794 4822 4853 4854 4811 4835 (genic regions excluded) GC content (%) 35.34 32.49 44.51 41.38 21.45 40.82 33.61 33.51

*Additional information about CpG islands can be found in Supplementary Material 2.

18 Once found the elements that probably demonstrate the functionality of this region we proceed to study its structure. The YASS genomic similarity search tool has been used to show their repetitive structure, their length and the location of repeats in the IGS.

The structure of “pure” Armeria pungens clones 5 and D10 APNI (population 4703) seem to have a distinct profile because they differ in the organization of the repetitive regions. The differences in the first repetitive regions (red square, Figures 12 and 13) could be an artifact of incomplete sequencing in clone D10 because of problems with primer design in repetitive areas. The second repetitive area, with longer repeats (blue square, Figures 12 and 13) is more complex in the second clone.

Figure 12. Armeria pungens clone 5 Figure 13. Armeria pungens clone D10 APNI (population 4703) repetitive APNI (population 4703) repetitive structure for IGS region structure for IGS region

The sequence of the introgressed Armeria pungens clone A2 API (population 4702) (Figure 14) is incomplete, so that missing sections (red squares) match with the corresponding parts of the repetitive regions in the introgressed Armeria pungens clone 28 API (population 4702) (Figure 15).

The introgressed Armeria pungens clone 2 APPI (population 4724) ha been mostly sequenced by the 18S side and it is almost incomplete at the beginning where the repetitive region should be found in 26S side (Figure 16). As previously, problems related with primer walking in a highly repetitive region have prevented us to sequence the full length of this clone.

19 Figure 14. Introgressed Figure 15. Introgressed Figure 16. Introgressed Armeria pungens clone A2 Armeria pungens clone 28 Armeria pungens clone 2 API (population 4702) API (population 4702) APPI (population 4724) repetitive structure for IGS repetitive structure for IGS repetitive structure for IGS region region region

The repetitive regions (red squares) in Armeria macrophylla clones 5 AM (Figure 17) and L3 AM (Figure 18) (population 4701) are missing in Armeria macrophylla clone 2 AM (population 4725) (Figure 19) since this last one has not been completely sequenced.

Figure 17. Armeria Figure 18. Armeria Figure 19. Armeria macrophylla clone 5 AM macrophylla clone L3 AM macrophylla clone 2 AM (population 4701) repetitive (population 4701) repetitive (population 4725) repetitive structure for IGS region structure for IGS region structure for IGS region

*Additional information about tandem repeats (repetitive sequences, localization, length, number of repeats) of these taxa and other species can be found in Supplementary Materials 3 and 4.

20 Having all the information about the elements that IGS region contains and in order to study the relatedness existing between the three taxa, we have calculated the identity and similarity of the sequences using the online service Ident and Sim (Table 5).

Armeria pungens clones 5 and D10 APNI (population 4703) are not similar between them but both two with introgressed Armeria pungens clone 28 API (population 4702). This is due to many mismatches can be noticed in the matrix such as distinct pattern of gaps and nucleotide sequence in several regions.

One of the highest similarities are reached by the introgressed Armeria pungens clones 28 API (population 4702) and 2 APPI (population 4724) which belong to the same taxa but not to the same population. However, it should be considered that this similarity has been calculated without the part of the sequence of the introgressed Armeria pungens clone 2 APPI (population 4724) which contains no information.

The introgressed Armeria pungens clone A2 API (population 4702) shows a high similarity with the introgressed Armeria pungens clone 28 API (population 4702), a coherent and expected result since they belong to the same taxon and population.

Also, the introgressed Armeria pungens clones 28 API (population 4702) and 2 APPI (population 4724) (although the last one is partly complete) have a high percentage of similarity with Armeria macrophylla clones 5 AM (population 4701) and 2 AM (population 4725), respectively.

The largest similarity is found between Armeria macrophylla clones L3 AM (population 4701) and 2 AM (population 4725) both belonging to the same taxon although different (but closely located) populations.

Table 5. Most remarkable results of similarity

HIGHEST SIMILARITIES SIMILARITY Armeria pungens clone 5 APNI (population 4703) - Introgressed Armeria pungens 28 61.51 % API (population 4702) Armeria pungens clone D10 APNI (population 4703) - Introgressed Armeria pungens 63.14 % clone 28 API (population 4702) Introgressed Armeria pungens clone 28 API (population 4702) - Introgressed Armeria 94.20 % pungens clone 2 APPI (population 4724) Introgressed Armeria pungens clone A2 API (population 4702) - Introgressed Armeria 86.47 % pungens clone 28 API (population 4702) Introgressed Armeria pungens clone 28 API (population 4702) - Armeria macrophylla 86.43 % clone 5 AM (population 4701) Introgressed Armeria pungens clone 2 APPI (population 4724) - Armeria macrophylla 71.58 % clone 2 AM (population 4725) Armeria macrophylla clone 2 AM (population 4725) - Armeria macrophylla clone L3 AM 94.92 % (population 4701) *Additional information about full results of identity and similarity can be found in Supplementary Material 5.

21 Having studied sequence similarities and knowing the IGS sizes (Table 4) we have proceeded with statistical analysis to compare IGS size data between the three taxa. The LSD test (Table 6) shows no significant differences in the IGS size between introgressed Armeria pungens and Armeria macrophylla, but the “pure” Armeria pungens is significantly different and inferior as well (p = 0.000785).

Table 6. LSD test (Least Significant Difference) showing IGS size differences between the studied taxa

Groups Treatments and means Average (bp) a Introgressed Armeria pungens 4823 a Armeria macrophylla 4833 b “pure” Armeria pungens 3866

Given the above data we can study now if the IGS size is correlated with the size of the complete genome. For that, DNA quantity has been obtained previously by flow cytometry (Table 7).

Table 7. Genome size and average of each population of the three taxa. *SD: standard deviation

Taxa Population Genome size SD* Species GS (GS) (pg) average (pg)

Armeria pungens 4703 8.38 0.03 8.40 4723 8.42 0.23 Introgressed Armeria pungens 4702 8.58 0.38 8.78 4724 8.97 0.21 Armeria macrophylla 4701 8.82 0.3 8.65 4725 8.48 0.23

The genome size has been analyzed and compared statistically by LSD test (Table 8). It shows no significant differences between the average introgressed Armeria pungens and Armeria macrophylla, but Armeria pungens genome size is significantly different (and smaller) (p=0.00547).

Table 8. LSD test (Least Significant Difference) of genome sizes between the different taxa

Groups Treatments and means Average (pg) a Introgressed Armeria pungens 8.78 a Armeria macrophylla 8.65 b “pure” Armeria pungens 8.40

22 We have subsequently analyzed the relationship between genome size and IGS size in these taxa by a linear regression (R2=0.465, p=0.06212).

To conclude with the analysis of the IGS region, we have done a screening of the sequences to locate repetitive elements such as transposable elements with CENSOR (Table 9).

In Armeria pungens clone D10 APNI (population 4703) no fragment of transposable element (TE) has been found. Most likely, this is because TE fragments may be located in the parts of the IGS of this species that are have not been sequenced

In all the remaining sequences, we have found TE fragments, all pf them belonging to class I (retrotransposons, see page 9):

The first fragment of TE that is present in almost all the clones is SABRINA3_TM_LTR, a retrotransposon described for the first time in Triticum monococcum. The only sequence that does not have this fragment is Armeria macrophylla clone L3 AM (population 4701) but again, it could be located in the part of the sequence that remains to be sequenced.

The next fragment, LTR-2_Mad-I (a non-autonomous retroelement first found in Malus domestica), can be found in almost all the sequences. This fragment has not been found in the introgressed Armeria pungens clone 2 APPI (population 4724), which as previously mentioned is incomplete, but neither in Armeria macrophylla clone 5 AM (population 4701) which has been almost completely sequenced.

The last fragment which some sequences have in common is Copia-10_ALY-I (a Copia-type retrotransposon found in Arabidopsis lyrata). This transposon has been found in two clones of Armeria macrophylla but not in the “pure” Armeria pungens. Also it has been found in introgressed Armeria pungens clone 28 API (population 4702).

23 Table 9. Name, location and description of transposable element fragments found in the IGS sequences of the studied Armeria taxa

From To Size (bp) Name Class Sim TE description TE size (bp) Armeria pungens clone 5 APNI (population 4703) 513 554 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 1850 1930 80 LTR-2_Mad-I LTR 0.7317 Malus domestica retrotransposon; nonautonomous 536 Introgressed Armeria pungens clone 28 API (population 4702) 513 554 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 1109 1202 93 LTR-2_Mad-I LTR 0.7283 Malus domestica retrotransposon; nonautonomous 536 1756 1842 86 Copia-10_ALY-I LTR/Copia 0.7558 Arabidopsis lyrata retrotransposon; internal portion 4429 clone A2 API (population 4702) 514 555 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 1108 1201 93 LTR-2_Mad-I LTR 0.7419 Malus domestica retrotransposon; nonautonomous 536 clone 2 APPI (population 4724) 516 557 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 Armeria macrophylla clone 5 AM (population 4701) 513 554 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 1765 1851 86 Copia-10_ALY-I LTR/Copia 0.7558 Arabidopsis lyrata retrotransposon; internal portion 4429 A family of nonautonomous transposable elements - 2848 2929 81 Interspersed_Repeat 0.7792 764 TE2-2_CR consensus sequence Amborella trichopoda caulimovirus; caulimoviridae; 5026 5086 60 Caulimovirus 0.8103 7748 Caulimovirus-9_ATr integrated virus clone L3 AM (population 4701) 1103 1196 93 LTR-2_Mad-I LTR 0.7419 Malus domestica retrotransposon; nonautonomous 536 1768 1858 90 LTR-2_Mad-I LTR 0.7419 Malus domestica retrotransposon; nonautonomous 536 clone 2 AM (population 4725) 513 554 41 SABRINA3_TM_LTR LTR 0.8372 Triticum monococcum retrotransposon 1571 1110 1203 93 LTR-2_Mad-I LTR 0.7444 Malus domestica retrotransposon; nonautonomous 536 1756 1842 86 Copia-10_ALY-I LTR/Copia 0.7558 Arabidopsis lyrata retrotransposon; internal portion 4429

*Additional information about transposable elements obtained by CENSOR can be found in Supplementary Material 6.

24 In order to relate the TE composition of the IGS clones with the IGS size we have compared (by linear regression) the TE composition (number of bp corresponding to TE fragments) with global size of the IGS in the fully sequenced clones (R2=0.5311, p=0.0403).

We have also performed an analysis of variance and the results show that the sizes of IGS between the studied taxa are significantly different, and the species with a highest content of TE fragments is the one with the largest IGS size (p=0.00547).

Table 10. LSD test (Least Significant Difference) between TE sizes in the different IGS

Groups Treatments and means TE total size (bp) a Armeria macrophylla 268 b Introgressed Armeria pungens 177 c “pure” Armeria pungens 121

To complete our research we have focused on another non-transcribed rDNA region, the ITS. Studies have determined the existence of two polymorphic positions in this region with different bases depending on the species. We have performed sequencing of ITS in the different populations and individuals studied of the three taxa in order to detect these.

On the one hand, population 4703 of “pure” Armeria pungens presents a T in the first polymorphic position and a C in the second one and population 4723 of the same taxon has these positions changed, a C in the first position and a T in the second (Figure 20).

On the other hand, population 4702 of the introgressed Armeria pungens shows N (polymorphism) in both positions since it is a hybrid between the two “pure” species. Instead, most individuals in population 4724 of the same taxon, excepting two (see Supplementary Material 7), present a C in the first polymorphic position and a T in the second (Figure 21).

Finally, the maternal genome donor Armeria macrophylla exhibits a C in the first polymorphic position and a T in the second position in both populations 4701 and 4725, (Figure 22) and in all individuals analyzed, excepting one which presents N in both positions (see Supplementary Material 7).

Figure 20. Polymorphic positions in the Figure 21. Polymorphic positions in the Figure 22. Polymorphic positions in the ITS of Armeria pungens (populations 4703 ITS of introgressed Armeria pungens ITS of Armeria macrophylla (populations and 4723) (populations 4702 and 4724) 4701 and 4725)

*Additional information about full results of polymorphic positions can be found in Supplementary Material 7.

26 5. Discussion

Starting with the selection of the best clones and given the length obtained by electrophoresis (Figure 9), we have selected the longest ones (Figure 11) since these would allegedly correspond to the functional IGS as functional elements have been found in them (Table 4) (Moss & Stefanovsky 1995; Suzuki et al. 1996; Nickrent & Patrick 1998). We have determined that the shortest DNA fragments (Figure 11) would be pseudogenes (e.g. Galián et al. 2012, 2014) or PCR artifacts.

About these functional elements, it has to be considered that not all the clones have been sequenced to completion; therefore more information about functional elements will probably appear when the sequencing has been completed, since obviously in the less sequenced IGS we have found less functional elements.

However, concerning CpG islands the introgressed taxon contains only the first one at 85bp while the “pure” species present more of these regions. The different location of the second CpG island (Table 4) between Armeria pungens and Armeria macrophylla could explain that the hybrid has not inherited neither, as found previously in introgressed species of Mus Musculus and Mus domesticus (Bret et al., 2005).

On the other hand, without considering the incomplete sequenced clones, the GC content is more similar and higher between introgressed Armeria pungens and Armeria macrophylla (Table 4) so it suggests that the hybrid is closer to the Armeria macrophylla in these terms. Although several studies have detected patterns in the variation of the GC content through a genomic sequence and between organisms, the function and meaning of such variation is still unknown (Madigan & Martinko, 2003).

The same applies to the IGS structure, which usually contains tandemly repeated sequences, referred to as subrepeats (Saghai-Maroof et al. 1984). The profile of the introgressed taxa is more similar to Armeria macrophylla, the likely maternal genome donor. About Armeria macrophylla clone 2 AM (population 4725) (Figure 19) we are not sure if the missing repetitive region (with respect to the other clone of the same taxa) is a product of the incomplete sequencing or because it belongs to a different population since it shows a different profile.

Supporting the concept of the functionality of this region, the IGS structure with tandemly repeated elements in the central zone is globally comparable with the pattern found in other plant species (see Supplementary Material 4), that is:

27 - Species with 2-4 repetitive zones of high or low complexity with more or less repeats with different length (long and short) (Figure 23) as Armeria:

Figure 23. From the left to the right, the IGS structure of Lycopersicon esculentum (Komarova et al. 2004, AY366528), Lens culinaris (Fernandez, unpublished, AM040289) Vicia sativa (Macas. et al. 2003, AY234366) and Artemisia absinthium (Garcia. et al. 2009, EU649668)

- Beta vulgaris (Figure 24), which is closer phylogenetically (both belong to order Caryophyllales), presents a structure quite different to that found in Armeria:

Figure 24. The IGS structure of Beta vulgaris (Fuentes et al. 2011, HE577473)

Looking at the differences between the IGS regions (particularly at those between more or less closely related species such as Armeria and Beta, but also at the differences within a given Armeria species) we can conclude that its structure may have a poor value as a phylogenetic tool, which agrees with the fact that it is a region submitted to less evolutionary constraints than 18S and 26S genes, so more variable.

The comparison of the sequences through the identity and similarity analyses also corroborates, as does the similarity of GC content and IGS structure, that the sequence of the hybrid is closer to Armeria macrophylla than to (Armeria pungens).

Moreover, the statistical studies of IGS size (Table 6) and genome size (Tables 7 and 8) variation also support a higher similarity between the introgressed Armeria pungens and Armeria macrophylla. Besides, we have observed that both sizes are positively correlated. Although it may be just a casual finding, it is also possible that a larger IGS size (which could reflect a larger intergenic space in other genes) may be prone to

28 more insertion events (such as insertion of TE or TE fragments) that would inflate the genome, as other authors have suggested (Lamppa et al. 1984; Vinogradov, 2004). It is known that TE cannot only move through the genome but also multiplicate. TE that insert into coding regions usually turn them into non-functional (Long & Dawid 1979) but apparently this is not the case if the insertion takes places in non-coding regions.

Indeed, length variations can be caused by the insertion of mobile genetic elements (Chester et al. 2010), as well as by the variation in the number of repeats. Saghai- Maroof (1984) and Rogers (1986) found that an increased expression of transposons inserted into the IGS region increased not only its size but also genome size. So we wanted to analyze whether the correlation found between IGS size and genome size could be caused by TE presence. The statistical analyses points to this direction, since the sequence with the highest TE content (Armeria macrophylla clone 5 AM population 4701) is the longest while the shortest has the lowest TE content (“pure” Armeria pungens clone D10 APNI population 4703). Although it has been previously suggested an increased number of transpositions in the IGS region related with hybridization and introgression processes, since it evolves faster than the coding region (Appels & Honeycutt 1986), our data do not allow reaching such a conclusion since the hybrid presents an intermediate number of TE fragments with respect to the parental species.

To corroborate the greater similarity between Armeria macrophylla and the introgressed Armeria pungens suggested by the IGS and genome size profiles, biparentally inherited ITS markers, which have been useful for documenting reticulate evolution in Armeria (based on the identification of individual additive polymorphisms and a pattern of variation consistent with geography but not with taxonomy; Fuertes Aguilar et al. 1999b), have been analysed). Indeed, gene-flow has been demonstrated to be frequent in the genus, and thus can cause different ITS copies to meet within the same genome (Nieto Feliner et al. 2001).

The comparison of the ITS polymorphisms in the three taxa indicates that, in the two detected polymorphic positions, the hybrid is much more similar again to Armeria macrophylla rather than to “pure” Armeria pungens.

Population 4723 of “pure” Armeria pungens that should be equal to the other population of the “pure” species also shows the same sequence as the hybrid and the maternal genome donor taxa. The clearly polymorphic peak (Figure 20) may indicate that this population is not so pure regarding the paternal genome and it would have probably been introgressed itself either with the maternal taxa or with the hybrid, so it cannot be considered a “pure” Armeria pungens genotype.

29 Population 4702 of introgressed Armeria pungens agrees with the expectation of the hybridization and introgression phenomena. However, almost all the individuals analyzed of population 4724 introgressed Armeria pungens (Figure 21) present the Armeria macrophylla profile (Figure 22), which would indicate that Armeria macrophylla is “more” than the simple maternal genome donor as previously detected by chloroplast capture (Nieto Feliner et al. 2001) and there is indeed recombination. But recombination has to be suggested with caution since at least three other factors may be involved. First, point mutations may be responsible for sequences differing by one nucleotide. Secondly, biased homogenization affecting individual sites also cannot be excluded as a possible explanation for some of the cloned sequences. Thirdly, some clones may be the result of PCR recombination (Nieto Feliner et al. 2004).

6. Conclusion

All the data here reported go into the same direction, showing a more prominent role of Armeria macrophylla with respect to Armeria pungens in the introgression phenomenon, resulting in a hybrid that has apparently more participation of the genome of Armeria macrophylla Therefore, perhaps a better name for the hybrid taxa, rather than “introgressed Armeria macrophylla” would be Armeria macropungens, as suggested previously (Nieto Feliner G., personal communication).

The geographical factor (i.e. close proximity between the studied taxa) is the most important determining the hybridization and introgression processes, since the population of Armeria pungens located farther from the others (the one in Cape Trafalgar) has conserved the paternal genome while the population located closer to Armeria macrophylla and the hybrid(s) shows similarities in the ITS polymorphic positions with these two taxa.

An important finding of this research has been not only the positive correlation between the IGS and genome size, but also the IGS and TE fragments size. So, we can assume the importance of these fragments within the IGS as their presence determines the size of this region. Also, many of these fragments are in many of the clones of different taxa indicating the high conservation of this region despite hybridization and introgression phenomena.

We must conclude, therefore, that more population and clones are needed and, more importantly, we have to sequence all them to completion so we can determine the real variability in this case.

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