Lectures Notes Strategic Both polyploidization

Both polyploidization and transposable element activity are known to be major

drivers of plant genome evolution. Here, we exploit the Zea-Tripsacum clade to

investigate the contribution of TE activation and accumulation on genomic divergence

after a recent shared polyploidization event. Comparisons of TE evolutionary dynamics in

various Zea-Tripsacum species, along with closely related diploid species Urelytrum and

Sorghum, revealed existing variation in repeat content between all genomes included in

the study. The repeat composition of Urelytrum is more similar to Zea and Tripsacum

compared to Sorghum, irrespective of similarity in genome size with the latter. The

similarity in the proportion of copia retrotransposons and satellite DNA in the Zea,

Tripsacum, and Urelytrum genomes suggests amplification of these elements after the

maize-sorghum split but before the allopolyploidization event leading to the

ZeaTripsacum lineage. Although the genomes of all species studied were abundant with

LTR-retrotransposons, we observed an expansion of the copia superfamily exclusively in

Z. mays (maize) and T. dactyloides. Additional analyses of the genomic distribution of

copia elements in maize provided evidence of biased insertion proximal to genes involved

in various biological processes including plant development and defense. The lack of

copia insertions near the orthologous genes in cultivated S. bicolor suggests that duplicate

gene copies may offer new neutral sites for TEs to insert, thereby providing an avenue for

subfunctionalization via TE insertional mutagenesis. Therefore, TE amplification and

polyploidization may complement one another in shaping genetic architecture during

maize domestication.

Introduction

Transposable element (TE) activation and accumulation generates significant genetic

variation that can confer a range of effects on genome structure and function. As

TEs carry ‘ready-to-use’ cis-elements, their insertions can impact gene regulation on a

genome-wide scale by providing assorted regulatory elements to the adjacent genes. The

new regulatory elements offered by inserted TEs can amplify and/or redistribute

transcription factor binding sites, therefore creating new regulatory networks or even

participate in re-wiring of pre-existing networks (Hènaff et al. 2014, Lavialle et al. 2013,

Krupovic et al. 2014, Huang et al. 2016, Carmona et al. 2016, Joly-Lopez et al. 2016).

Several empirical studies have demonstrated TE-induced phenotypic changes associated

with domestication and/or diversification of cultivated plants, including rice, maize,

wheat, soybean, melon, palm etc. (Naito et al 2009, Fernandez et al 2010, Studer et al.

2011, Uchiyama et al. 2013, Sanseverino et al. 2015, Ong-Abdullah et al. 2015, Lu et al.

2017). Indeed, TE-related polymorphisms are largely responsible for phenotypic variation

in many agronomically important crops, demonstrating their importance in creating the

genetic variability that contributes to plant genome evolution.

Hybridization, polyploidy, and stress are considered the primary triggers of

transposable element movement (Steward et al. 2000, Kalendar et al. 2000, Madlung et al.

2005, Ungerer et al 2006, Ito et al. 2011, Cavrak et al 2014, Bardil et al. 2015, Guo et al.

2017). Flowering plants are known to tolerate hybridization and polyploidy, both of

which have promoted species diversification (Payseur and Rieseberg, 2016, Soltis et al.

2016, Goulet et al. 2017). These phenomena result in TE mobilization leading to local

mutations and genome size changes (Liu and Wendel 2000; Josefsson et al. 2006; Ungerer

et al. 2006; Kawakami et al. 2010; Parisod et al. 2010; Piednoël et al. 2013). Furthermore,

such bursts of TE activity result in insertional polymorphisms, often with deleterious

effects on genome function; however, these effects could be nullified or shielded via gene

duplication in polyploid genomes. Although the precise mechanism(s) that induce TE

mobility in hybrids and polyploids is unclear, it is speculated that such TE reactivation in

response to genomic stresses could be due to incompatible suppression machinery

between the two donor genomes, or that unknown mechanisms are in place that reduce

genomic methylation under general stress conditions (Ha et al. 2009, Yaakov and

Kashkush 2012, An et al. 2014, Senerchia et al. 2014, DeFraia and Slotkin 2014, Ågren et

al. 2016).

Previous studies of polyploidy in Zea have revealed evidence for a whole genome

duplication (WGD) event at or shortly after the origin of grasses, followed by another,

more recent, WGD in the Zea history that promoted the origin of the Zea-Tripsacum

clade. Being emerged from a common ancestral allotetraploid (n=20), both the Zea

(n=10) and Tripsacum (n=18) genomes differentially responded to the rediploidization

process (Swignova et al. 2004, Schnable et al. 2009, Schnable & Freeling, 2011). In

addition to these chromosomal rearrangements, there is also evidence for retrotransposon

invasion post divergence in both Zea and Tripsacum (Gaut et al. 2000). Hence, being

divergent descendants of a common allopolyploid ancestor, the Zea-Tripsacum clade is a

good model system to understand various evolutionary processes including the

contribution of TEs to polyploidy, rediploidization, and species diversification.

Here, we describe TE activation and contribution to genome diversity in the

ZeaTripsacum clade that has undergone a recent shared polyploidization event. We

included a close diploid progenitor, Urelytrum digitatum, which provides an opportunity

to explore TE-associated evolutionary events induced by hybridization and genome

doubling. By using clustering analysis, we have characterized the repetitive landscape in

six Zea-Tripsacum species (post allopolyploidization) compared to the diploid sister taxa

Urelytrum and Sorghum (pre allopolyploidization). Our findings suggest post-divergence

and recent activity of TEs in Zea and Tripsacum with an expansion of copia elements in

cultivated lineages compared to wild relatives. Biased insertions in euchromatic regions

in Z. mays but not in S. bicolor suggests allopolyploidy induced retrotransposition in Z.

mays. Also, with more insertions near developmental and defense genes and as TEs carry

their own cis-elements, these elements may have influenced the evolution of the maize

genome during domestication.

Materials and methods Plant material sources and Illumina sequencing of DNA

The following eight panicoid grasses were used in this study: Zea mays, Z.

diploperennis, Z. luxurians, Tripsacum dactyloides, T. laxum, T. australe, Urelytrum

digitatum and Sorghum bicolor. Short-read sequence data for Zea mays (SRS291653),

Zea luxurians (SRR088692), Tripsacum dactyloides (SRS302460), and Sorghum bicolor

(SRS1323776) were downloaded from the NCBI short read archive (Chia et al. 2012,

Tenaillion et al. 2011, Ramachandran et al. 2016). Genome sequences of Zea

diploperennis (XXXXXX), Tripsacum laxum (MIA34792), Tripsacum australe

(MIA34499) and Urelytrum digitatum (SM3109) were obtained from Dr. Elizabeth

Kellogg, Donald Danforth Plant Science Center, St. Louis, Missouri. See Supplementary

Table 1 for more information on genome sequencing.

Identification of TE families

Sequences were quality trimmed using Trimmomatic v0.33 (Bolger et al. 2014)

using a sliding window of 4:25 and minimum length of 50 bp. Graph-based clustering of

quality-trimmed reads was performed with RepeatExplorer, a pipeline designed to

identify repeats from NGS reads (Novak et al. 2013). RepeatExplorer employs a

clustering algorithm that quantifies similarities between all sequence reads and produces a

graph that consists of nodes (sequence reads) and edges (connecting overlapping reads).

Nodes are frequently connected to one another if they pass a threshold of 90% similarity

over at least 55% of the sequence length, representing individual repetitive families.

Three million reads (approximately 0.2x to 0.5x genome coverage) were

subsampled from each dataset and processed to the format required by RepeatExplorer.

Species-specific clustering analysis provides information regarding repeat quantities by

reporting the number of reads per cluster, which can then be used to estimate the genome

space occupied by each particular repeat, i.e., (total length of each cluster (in Mb) x

genome size (in Mb)) / total length of all clusters (in Mb) (Kelly et al. 2015,

Ramachandran et al. 2016). Subsequently, all of the processed reads from all species

were concatenated into one combined dataset, and the RepeatExplorer clustering was

repeated in order to facilitate comparative analysis. All clusters were annotated using the

Viridiplantae RepeatMasker library and categorized into repeat families. A plot

representing interactions between repeat clusters among species was created using

UpSetR (Lex et al. 2014).

Quantitative analysis of TE activity using molecular clock analysis

To estimate the timing of TE activity in each lineage, species-specific LTR

sequences were extracted from each LTR-retroelement cluster. These species-specific

reads were assembled using the Geneious de novo assembler to obtain a consensus

sequence (Kearse et al. 2012). A grass-specific database was then used to extract LTRs

from each consensus contigs (blastn, e-value 1e-10, 85% identity). The best match for

each species was chosen and the corresponding hit region was extracted using BEDTools

v2.17.0 (Quinlan and Hall 2010).

To calculate LTR divergence (a rough measurement to estimate the age of a

specific retrotransposon family) the reads that were used for de novo assembly were

mapped to the consensus LTR sequence using the Geneious reference genome assembler.

The percent identity of each read mapped to its respective LTR consensus sequence was

derived from the reference alignment. Using a grass specific transposable element

substitution rate of 1.3 x 10-8 per site per year (Ma and Bennetzen, 2004), we estimated

the activity of each major TE family in each species.

Genomic distribution of copia retroelements

To test whether the copia elements that have expanded in select species

demonstrate an insertional bias, Illumina paired-end reads from Z. mays were mapped to a

library consisting of Z. mays copia clusters assembled by RepeatExplorer and to a filtered

gene set containing the protein-coding genes from the Z. mays reference genome.

Reference mapping of paired-end reads to the library was carried out using BWA version

0.7.12 (Li and Durbin, 2009) with the following parameters: aln -t 4 -l 12 -n 4 -k 2 -o 3 -e

3 -M 2 -O 6 -E 3 (Mascagni et al, 2015). The results were used to generate a “sam” file

via the BWA “sample” module, and then converted to a “bam” file using SAMtools (Li et

al, 2009). A copia element was considered proximal to a gene if one of the paired-end

read mapped to a copia element and the other to a gene. Genes proximal to copia

elements were further analyzed for their presence in gene-dense or gene-poor regions by

determining the number of TEs present within various distances (1 kb, 5 kb, and 10 kb)

both upstream and downstream of genes using BEDTools v2.17.0 (Quinlan and Hall

2010).

Phylogenetic analysis of retroelement families

To assess the evolutionary relationships of the shared gypsy and copia families,

the reverse transcriptase (RT) and integrase (INT) amino acid domains were used for

phylogenetic analysis. RepeatExplorer clusters were filtered for LTR-gypsy and copia

elements with RT and INT domain blastx hits. RT reads were extracted from each cluster

using the blastx output file and placed in separate genome-specific files. The reads were

assembled for each cluster using the Geneious de novo assembler (Kearse et al. 2012).

The resulting contigs were then confirmed to contain reverse transcriptase domains using

blastx against the Cores-RT database (Llorens et al. 2011). RT sequences were then

combined into a final query file for further analysis. The same analysis was performed for

INT reads using Cores-INT database (Llorens et al. 2011).

Rpstblastn (e-value = 1e-10) was performed for the sequence dataset against the

Conserved Domain Database (Marchler-Bauer et al. 2015) to identify and extract

conserved regions. The best hits for each sequence were extracted, and the filtered blast

output was converted to three-column bed format with matching coordinates for each hit.

BEDTools v2.170 (Quinlan and Hall 2010) was used to extract the conserved regions

(~540 bp for the RT domain and ~340 bp for the INT domain). The correct open reading

frame from each sequence was identified using ORFfinder. All amino acid sequences

were globally aligned with MUSCLE v3.8.31 (Edgar 2004). Alignments were manually

inspected and adjusted in Bioedit v7.3.5 (Hall, 1999). The optimal model of amino acid

substitution for each alignment was estimated using Prot-test v3.4.5 (Abascal et al. 2005).

In all cases except RT-copia, the best model selected was LG+G (Le and Gascuel. 2008).

Blosum62+G was chosen as the optimal model for RT-copia (Henikoff and Henikoff.

1992). Likelihood analyses with 1,000 bootstrap replicates were performed in RAxML v.8

(Stamatakis et al. 2008) using the best model for each alignment. Bayesian analysis of

alignments was performed in MrBayes v3.2.6 using rates=gamma and respective

substitution model (Ronquist and Huesenbeck. 2003). Two independent MCMC runs of

10 million generations were performed, sampling each run every 1,000 generations. All

trees were visualized using FigTree v1.4.0.

Results Repeat composition in the genomes of Zea, Tripsacum, Urelytrum, and

Sorghum

To evaluate the repeat content with respect to genome size, we performed a

separate clustering analysis for each species. Individual clustering allows the maximum

number of reads to assemble in each cluster, which increases the accuracy of the repeat

estimates. We estimated the quantities of each repeat family in the genome using the

following equation: (total length of each cluster (in Mb) x genome size (in Mb)) / total

length of all clusters (in Mb) (Macas et al. 2015). The estimated repeat compositions are

shown in Table 1.

As expected, LTR-retrotransposons are the most abundant repeat in all eight

genomes. Although all Zea species used in this study are diploid and contain the same

number of chromosomes, the genome size of Z. luxurians (~4,479 Mb) is nearly double

the size of other two Zea species (~2,600 Mb). From the clustering analysis, copia

elements were found to contribute approximately 710 Mb, 930 Mb, and 1,110 Mb to the

Z. diploperennis, Z. mays and Z. luxurians genomes, respectively. Gypsy elements

account for ~1,240 Mb and 1,420 Mb of the Z. mays and Z. diploperennis genomes,

respectively, whereas ~2,390 Mb of Z. luxurains genome is comprised of gypsy elements

(Table 1). The greater repeat abundance in Z. luxurians correlates with its larger genome

size. The Tripsacum species contain genomes of similar size (~3,200 Mb) and

chromosome number (2n=36), in which T. laxum contains the smallest genome (2,974

Mb). Copia elements occupied ~740 and 780 Mb in T. australe and T. laxum genomes, in

contrast to 1,050 Mb in the T. dactyloides genome. Approximately 1,760 Mb and 1,825

Mb of the genome is composed of gypsy elements in T. laxum and T. australe,

respectively, whereas the T. dactyloides genome contains 1,230 Mb of gypsy elements.

Gypsy elements contributed to more of the genome space (~53-59%) compared to copia

(22-28%) in all Zea-Tripsacum species except Z. mays and T. dactyloides, where both

gypsy and copia were equally distributed (Figure 1B). Urelytrum and Sorghum contain

~167 Mb and 92 Mb of copia, and 438 Mb and 536 Mb of gypsy elements, respectively.

DNA transposons were found to contribute only 2-6% to the Zea and 2-3% to the

Tripsacum genomes, in contrast to 10-11% in Urelytrum and Sorghum. Other groups of

repeat elements such as satellite repeats made up a significant fraction of the genome in

several species. Approximately 755 Mb of the Z. luxurians and 510 Mb of the T.

dactyloides genomes were occupied by satellite repeats. Although Urelytrum and

Sorghum contain genomes of similar size, the former is composed of only 1.76 Mb of

satellite DNA whereas the latter contained ~100 Mb of satellite DNA in its genome.

The most abundant repeat families and their contribution to genome size

From the individual repeat clustering analysis, we identified 24 copia and 30

gypsy families. Among the 24 copia families, Ji was the most abundant family in both the

Z. mays (444 Mb) and Z. diploperennis (363 Mb) genomes, whereas Opie was the most

abundant in Z. luxurians (535 Mb) and in all of the Tripsacum genomes (Table 1). Dijap

was estimated at 146-240 Mb in the three Tripsacum genomes, but contributed very little

to the genome size of Zea.

Among the gypsy families, Cinful-Zeon, Prem1, Flip, Gyma, Huck and Xilon-

Diguus were abundant in both the Zea and Tripsacum genomes. The Cinful-Zeon family

ranges from 224 - 583 Mb among the three Zea genomes with the greatest abundance in

the larger Zea genome; however, this family contributes only ~70 Mb to the Tripsacum

genomes. This is also true for the Xilon-Diguus family, with estimates ranging from 125 -

226 Mb in Zea and ~42 Mb in the Tripsacum genomes. The Huck family is estimated at

246 in Z. diploperennis, 321 Mb in Z. luxurians, 152 in T. laxum and 276 Mb in T.

australe; however, Huck occupies only ~15 Mb of the Z. mays genome and ~1.4 Mb of

the T. dactyloides genome. Similarly, elements such as Doke, Puck, Lata and CRM1 were

more abundant in the wild species relative to the domesticated species.

There were 13 gypsy families that were specific to Urelytrum and/or Sorghum. Athila and

Leviathan elements (~19 – 66 Mb) were identified in both Urelytrum and Sorghum. Apart

from these two families, the remaining 11 gypsy families were predominantly present in

Sorghum, but present in low copy number in Urelytrum, or absent altogether. For

example, Retrosor6 is estimated at ~180 Mb in the Sorghum genome but is completely

absent in all other species; however, there are a large number of unclassified gypsy

elements in the Urelytrum genome (See Table 1). Although we used a grass specific

database to annotate the elements, the majority of this repeat content could not be

annotated, suggesting the presence of species-specific repeats and retroelements.

Insertional biases in Z. mays

The copia superfamily was found to be more abundant in Z. mays and T. dactyloides

compared to the other species included in the study, suggesting recent proliferation of

some copia families in both genomes. Investigating the genomic distribution of this

expansion, we discovered that the frequency of Z. mays copia reads mapping to stress-

associated genes (~34%) was higher compared to other genes (on average ~6%). Copia

elements mapped in close proximity to genes involved in plant defense, leaf

morphogenesis, photoreceptors, homeobox proteins, signal transduction, and transcription

(Figure 5). Further analysis revealed that these genes were surrounded with

approximately four to five genes within 5kb windows both upstream and downstream.

Comparative analysis of Zea, Tripsacum, Urelytrum and Sorghum

We performed comparative repeat analysis by simultaneously clustering reads

from all eight species. This approach facilitated the identification of repeat families that

are shared between multiple species, and allowed us to determine their fate during

Andropogoneae evolution, especially during the divergence of Zea and Tripsacum. This

analysis resulted in four major cluster configurations, for which examples are shown in

Figure 3A-D. Figure 3A shows an example of a cluster (2: Prem1, LTR-gypsy) in which

the repeat family is common to all species. In this example, reads from both Zea and

Tripsacum are tightly clustered, and reads from Sorghum and Urelytrum are peripherally

connected, as would be expected based on their evolutionary relationships. Cluster 6

(Opie, LTR-copia) is an example of a lineage-specific repeat family, where sequences are

shared between Zea and Tripsacum but absent in Urelytrum and Sorghum (Figure 3B). In

Cluster 21 (Flip, LTR-gypsy), the graph indicates three separate groups (Z. mays and

T. dactyloides [top], Z. diploperennis and Z. luxurians [right], T. australe and T. laxum

[left]) in which Z. mays and T. dactyloides are more similar to one another than either is to

their sister species (Figure 3C). Finally, cluster 64 (Angela, LTR-copia) is an example of

a tightly knitted graph in a linear arrangement shared between all eight species,

demonstrating the conserved nature of ancient Angela elements across all included taxa

(Figure 3D).

From a total of two million reads from eight genomes, 248 significant clusters

were formed of various sizes and repeat families. On average, ~81% of the reads from

each species clustered with LTR-retrotransposons (127 LTR-gypsy and 48 LTR-copia

clusters, Figure 2A). Among the 175 LTR-RT clusters (or families) identified, 85 families

were present exclusively in the Zea-Tripsacum clade. For all species except Z. mays and

T. dactyloides, the proportion of reads from LTR-gypsy families (53%) was higher

compared to LTR-copia families (28%), whereas gypsy and copia were equally abundant

in Z. mays and T. dactyloides. Compared to the other genomes, Sorghum contained the

smallest proportion of reads from copia families.

Among the 127 gypsy clusters, four clusters were shared among all eight species,

two clusters were common to Zea, Tripsacum and Urelytrum (but absent in Sorghum), 34

clusters were exclusive to Zea and Tripsacum species, and 15 clusters were found only in

Urelytrum and Sorghum. In addition, we observed lineage-specific gypsy families: 10 in

Zea, 17 in Tripsacum, 21 in Urelytrum, and 14 in Sorghum (Figure S1. A). Of the 48

copia clusters, only two were common to all species, ten were common to Zea, Tripsacum

and Urelytrum, 19 clusters were exclusive to Zea and Tripsacum, and 3 were exclusive to

Urelytrum and Sorghum. Compared to gypsy super-families, there were fewer species-

specific copia families (Figure S1. B).

Evolutionary relationships and timing of transposition events

To assess the timing of major transposition events that occurred pre- and postdivergence

of the Zea-Tripsacum clade, we constructed maximum likelihood trees using INT and RT

(data not shown) of both gypsy and copia elements. Of the 127 shared gypsy clusters, 15

(total of 82 sequences) shared sufficient sequence identity within the integrase domain to

allow amino acid sequence alignment. Major repeat families such as Cinful-xeon, Prem1,

Flip, Gyma, Xilon-Diguus, and Huck were among these 15 clusters.

With a few exceptions, most clades formed as expected in regard to species relationships,

such as all Zea and Tripsacum species clustered together with Urelytrum and Sorghum

being more distantly related (data not shown). The gypsy families Flip and Gyma

clustered together. The Sorghum and Urelytrum sequences from the Flip family clustered

with Zea sequences of Gyma, whereas the Zea and Tripsacum sequences of Flip clustered

with Tripsacum and Sorghum of Gyma. Several families such as huck, puck, and grande

were clustered together with high support values, suggesting a recent origin of these

families. Clusters such as CL24 (unclassified), uwum (CL82) and guhis (CL132) also

clustered with high sequence similarity.

We employed comparative sequence analyses of LTRs from 15 prominent clusters

to estimate the temporal activity of retroelements both pre- and post-divergence of the

Zea-Tripsacum clade (Figure 4). The clusters chosen for this analysis are comprised of

the following repeat families: Prem1, Flip, Cinful-Xeon, Gyma, Ji, Opie, Dijap, Retrosor-

6, and several prominent unclassified elements. In Figure 4A, the peak activity of each

element per species per cluster is plotted against a TE-specific grass molecular clock (11

mya to present). The approximate timing of the Zea-Tripsacum divergence is highlighted

in yellow (5-6 mya). Zea and Tripsacum have experienced post divergence lineage-

specific activity for most repeat families. For example, Ji, Opie, and Dijap (CL7, CL12,

CL15, CL42, and CL51) were active between 0-3 mya for all species in which they are

present. The Opie element represented in CL7 is shared between Zea, Tripsacum and

Urelytrum and has been active within the last ~1-3 mya (Figure 4A & 4B) indicating that

amplification of Opie occurred in all three lineages after species divergence. In contrast,

the amplification of CL2 (prem1) occurred recently only in Z. luxurians (2-3 mya)

compared to all other species. Although T. dactyloides and T. laxum experience increased

activity of Prem1 around the time of divergence, the activity of this element in Z. mays, Z.

diploperennis, T. australe, and Sorghum dates as an older amplification event. Similarly,

the activity of CL5 (gyma) in Z. diploperennis is recent but lost in Z. luxurians. Several

families were shared only between Sorghum and the wild relatives of Zea (Z.

diploperennis, Z. luxurians) and Tripsacum (T. laxum, T. australe). Despite their presence,

the activity of these families varies between species. For example, the activity of CL11 in

Z. luxurians is recent (0-1 mya) but is dated as an old insertion in the other species

(Figure 4A and 4C). In contrast, elements in CL19 display postdivergence activity in all

species. For example, Z. diploperennis and S. bicolor experienced CL19 activity around

1-2 mya, whereas in Z. luxurians and T. dactyloides, CL19 elements were active around

2-3 mya (Figure 4A).

Discussion

The present study evaluates TE dynamics in divergent descendants

(ZeaTripsacum) of a common allopolyploid ancestor within a phylogenetic framework

that is rooted with two diploid relatives (Urelytrum and Sorghum). The comparative

analysis of repeat elements from Zea, Tripsacum, Urelytrum, and Sorghum provides

insight into the contribution of retrotransposons to genome evolution post a shared

polyploidization event. Inclusion of additional Zea and Tripsacum species provided an

opportunity to assess the genomic variability in repeat content between wild and

cultivated genotypes.

As expected, LTR-retrotransposons account for the majority of the repeat

composition in the genomes of all species included in this study. Individual clustering

analyses indicate that a diversity of LTR-retrotransposons contribute to genome size

variation in this taxonomic group. Based on our comparative and molecular clock

analyses, the majority of retrotransposon families are common to the Zea – Tripsacum

clade in comparison to their diploid relatives, suggesting an occurrence of retrotransposon

invasion after allopolyploidization but before the split between the two species (Figure

4A). Previous studies have hypothesized an occurrence of retroelement bursts just before

the divergence of Zea and Tripsacum based on maize retroelement activity (Gaut et al.

2000, Estep et al. 2013). The results for the Tripsacum species included in the current

analysis supports this hypothesis, revealing a high number of shared retrotransposon

families between the two species. For example, Ji and Opie of the copia superfamily

have been especially active (0-2 mya, Figure 4A, >300 Mb, Table 1) in both Zea and

Tripsacum; however, these families contribute little (~35 Mb) to genome composition in

Urelytrum and are absent in Sorghum. The presence and hyper activity of these families

in the Zea-Tripsacum-Urelytrum clade but not in Sorghum suggests amplification after the

maize-sorghum split but before the allopolyploidization event leading to the Zea-

Tripsacum lineage. Similarly, five gypsy families (Cinful-Xeon,