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Transposons and non-coding regions drive the intrafamily differences of genome size in insects

Summary Genome size (GS) can vary considerably between phylogenetically close species, but the landscape of GS changes in insects remain largely unclear. To better understand the specific evolutionary factors that determine GS in insects, we examined flow cytometry-based published GS data from 1,326 insect species, spanning 700 genera, 155 families, and 21 orders. Model fitting showed that GS generally followed an Ornstein–Uhlenbeck adaptive evolutionary model in Insecta overall. Ancestral reconstruction indicated a likely GS of 1,069 Mb, suggesting that most insect clades appeared to undergo massive genome expansions or contractions. Quantification of genomic components in 56 species from nine families in four insect orders revealed that the proliferation of transposable elements contributed to high variation in GS between close species, such as within Coleoptera. This study sheds lights on the pattern of GS variation in insects and provides a better understanding of insect GS evolution.


INTRODUCTION
Genome size (GS) varies greatly in different organisms, with differences of 200,000-fold across all eukaryotes (Gregory 2001), 12,000-fold across plant species (Leitch et al., 2019) and an 7,000-fold difference across all animals (Gregory 2005a). GS is poorly correlated with organismal complexity or number of coding genes (the C-and G-value paradox, Gall 1981; Hahn and Wray 2002), and thus, the biological reasons for this huge variations in GS remain unclear from an evolutionary perspective. Whether the evolutionary causes and consequences of GS variation are adaptive or non-adaptive processes has been widely debated. The non-adaptive theory suggests that variation in GS are introduced through a stochastic and nearly neutral evolutionary process (Petrov 2002;Lynch and Conery 2003), since DNA insertions and deletions mainly occur through random genetic drift (Lynch and Conery 2003;Mohlhenrich and Mueller 2016), with counteracting outcomes between these two processes determining GS (Petrov 2002;Sung et al., 2016;Kapusta et al., 2017). Specifically, DNA can be rapidly added by genome duplication (Vu et al., 2015;Soltis and Soltis 2016;Marburger et al., 2018), tandem repeat amplification (Shah et al., 2020;Majid and Yuan 2021;Stelzer et al., 2021) or transposon transpositions (Kidwell 2002;Naville et al., 2019;Zhang et al., 2020;Lehmann et al., 2021;Oggenfuss et al., 2021). Extraneous and/or slightly deleterious DNA is then gradually deleted by purifying selection (Lynch and Conery 2003). Alternatively, the adaptive hypothesis emphasizes the functional effects resulting from changes in total DNA abundance (Cavalier-Smith 1978, 2005Gregory and Hebert 1999). Because DNA content directly or indirectly effects the cell volume and specific surface area, this hypothesis argues that GS variation is an adaptive process subject to natural selection for essential cellular processes (e.g., cell division rate or cell metabolism rate) (Cavalier-Smith 1978, 2005Gregory 2001;Gardner et al., 2020) and fitness-related phenotypes (e.g., body size, developmental rate) (Gregory 2002;Wyngaard et al., 2005).
Insects constitute a major proportion of invertebrates and contain the largest number of reported species, and thus serve as a good system for examining GS evolution. Some intriguing insect traits have been tested for correlations with GS. For example, the reproductive fitness (Arnqvist et al., 2015) of seed beetles and host range of moths in family Noctuidae (Calatayud et al., 2016;Zhang et al., 2019) are both correlated with GS. However, traits such as parasitism by Hymenopterans (Ardila-Garcia et al., 2010), eusociality of Hymenopterans and Blattodeans (Koshikawa et al., 2008;Ardila-Garcia et al., 2010), and flight ability and strategies among dragonflies and damselflies (Odonata) (Ardila-Garcia and Gregory 2009) are all unrelated to GS. Morphological characteristics such as eye width (Liu et al., 2017), antennae length (Liu et al., 2017) Several recent studies have conducted small scale comparative phylogenetic investigations to better understand the evolutionary patterns and processes responsible for GS variation in insects. For example, GS in fireflies (family Lampyridae, Coleoptera) (Lower et al., 2017) and flies (subgenus Sophophora, Diptera)  were found to evolve neutrally through a non-adaptive mode. Ancestral reconstruction of GS among crickets (suborder Ensifera, Orthoptera)  and butterflies (family Papilionoidea, Lepidoptera) (Liu et al., 2020) suggested that genome expansion or contraction events happened frequently, resulting in a high diversity of GS among these species.
Moreover, genomic analyses in other insects have provided evidence that the abundance of transposable elements (TE) differs widely among species and is positively correlated with GS in insects (Petersen et al., 2019;Wu and Lu 2019). For example, locust (Locusta migratoria, Orthoptera) carries the largest insect genome published to date, 6.5 Gb, 60% of which is comprised of TEs (Wang et al., 2014). In other large genomes, such as that of the grasshopper Vandiemenella viatica (4.5 Gb) (Palacios-Gimenez et al., 2020) or the rice weevil Sitophilus oryzae (770 Mbp) (Parisot et al., 2021), TEs account for an even greater proportion of the genome, exceeding 70%. By contrast, TE represent less than 1% of the genomes of the Antarctic midge (Belgica antarctica, Diptera, 83.9 Mb) (Kelley et al., 2014) and the body louse (Pediculus humanus humanus, Phthiraptera, 108 Mb) (Kirkness et al., 2010). Some studies have reported that the specific accumulation of LINE retrotransposons and DNA transposons leads to GS variation among wood-white butterfly species (genus Leptidea) (Talla et al., 2017) and Asian gypsy moths (genus Lymantria) (Hebert et al., 2019). In addition, the massive horizontal transfer of some TEs, such as the Gypsy, Bel-Pao, and Copia LTR superfamilies, significantly influences GS in mosquitoes (Melo and Wallau 2020).
The majority of these above-mentioned studies were carried out in a small set of species. Because GS may be the outcome of various counteracting factors, it may not be possible to arrive at a single, universal principle that can explain the causes of GS variation through small-scale studies. The recent emergence of numerous insect genome sequencing projects has led to the availability of GS information for more than 1000 insects, either estimated by flow cytometry, K-mer analysis, or genome assembly. To broadly uncover the evolutionary factors driving GS variation in insects, we collected GS data, estimated mainly by flow cytometry, and analyzed differences within and among various insect clades. The results showed that GS varies considerably at different taxonomic levels, even among some close insect species. Comparative phylogenetic analyses showed that different models fit the observed differences in GS in different insect orders, suggesting that different evolutionary history responsible for shaping the GS variation among different insect clades. Analysis of genome content suggested that the expansion of TEs, rather than genome duplication, led the observed variation in GS between phylogenetically close species. Moreover, we found that several specific TEs appear to drive GS variations within different insect clades, suggesting that the type of TE driving changes in GS in each insect group may be the result of lineage-specific evolutionary events.

GS variation in insects
To understand the comprehensive pattern of insect GS variation at broad level, we first collected as much GS data as possible. To this end, we obtained 1,345 records of GS, estimated mainly by flow cytometry, from the Animal Genome Size Database (Gregory 2005a). After removing redundancy, 1,145 insect GS information were kept for further analysis. We also examined previous studies, published before December 2020, to supplement the above insect GS data, which yielded 181 additional records. Ultimately, the GS dataset included 1,326 insect species from 700 genera, 155 families, and 21 orders (Table S1). Among these records, dipteran and coleopteran insects comprised the highest proportion, including 307 and 295 species, respectively, followed by Hymenoptera, Lepidoptera, Odonata and Hemiptera. Among these insect species, we observed a 266-fold variation in GS from the smallest, 68.46 Mb, belonging to the midge Clunio tsushimensis in Diptera, to the largest, 18.23 Gb, in the grasshopper Bryodemella holdereri in Orthoptera. Statistical analyses indicated several significant differences in the maximum, minimum, average, range, and variance of GS across the different insect orders (Table 1 andFigure (Table S2B). Interestingly, some families carried significantly smaller genomes than those of any other family within the same order, such as Chironomidae in Diptera (average = 127.1 Mb, n = 30), Tenebrionidae in Coleoptera (average = 370.1 Mb, n = 65), Braconidae in Hymenoptera (average = 140.2 Mb, n = 14) and Nymphalidae in Lepidoptera (average = 417.5 Mb, n = 29) ( Table S2B).
The holometabolous insects (clade Holometabola) are thought to possess relatively small genome contents under the selection of the rapid cell division and metabolism rate during the developmental stages with intensive morphological differentiation (Gregory 2002(Gregory , 2005b. We tested this hypothesis by comparing the difference in GS among the ametabolous orders (including Archaeognatha and Zygentoma), the hemimetabolous orders (including Phthiraptera, Hemiptera, Thysanoptera, Blattodea, Mantodea, Phasmatodea, Embioptera, Orthoptera, Dermaptera, Zoraptera and Odonata), and the holometabolous orders (including Diptera, Mecoptera, Siphonaptera, Lepidoptera, Trichoptera, Coleoptera, Strepsiptera and Hymenoptera). We found that the holometabolous did have the smallest genomes (average GS = 536.7 Mb, n = 990) ( Figure 1A), significantly smaller than that of hemimetabolous (p = 1.31E-73, average GS =2,781.4 Mb, n = 327) and ametabolous (p = 0.003, average GS =3,003.5 Mb, n = 9) insects ( Figure 1A and Table S2B). Collectively, the similarities and differences in GS showed a great diversity of GS within different insect clades and groups, implying distinct evolutionary histories of insect GS among the studied taxa.

Evolutionary modes and ancestral states of insects GS
To further elucidate the evolutionary history responsible for the observed GS variation pattern, we modeled GS trait evolution within various insect groups and orders. To this end, we first assembled a phylogeny containing 1,256 species out of the 1,326 records in our above GS dataset through NCBI taxonomy iScience Article database (see method details). Based on this phylogeny, we tested three commonly used models, including Brownian motion (BM), Ornstein-Uhlenbeck (OU) and Early Burst (EB) model, to check which of these evolutionary models could best explain the evolution pattern of 1,256 insects GS using the R package geiger . Ignoring the phylogenetic structure (i.e. without phylogenetic signal), the white-noise model (WN), was used as the control. The BM model depicts a stochastic process, thus suggesting that changes in GS were random events. Interpretation of the OU model suggests that changes in GS are the result of selection pressure with a central tendency toward the evolutionary optima. By contrast, the EB model depicts an evolutionary process that varied over time, beginning at a relatively fast rate, then gradually slowing. When all 1,256 insects were considered as a whole, the OU model showed the best fit for log scale representation of GS (Table 2), which also generally showed the best fit for species within the holometabolous orders Coleoptera, Diptera, Hymenoptera, and Lepidoptera (Akaike weights, AW = 0.988, 0.999, 0. 995, 0.996, respectively, Table S3). In contrast, The BM model showed a better fit with the hemimetabolous orders Odonata, Orthoptera and Hemiptera (AW =0.588, 0.599, 0.462, respectively). Taken together, these results showed that GS evolution in Holometabola and Insecta followed the OU model whereas the Hemimetabola was more fit by the BM model, suggesting that adaptive evolution was responsible for GS in insects overall but has less impact on the Hemimetabola.
We next traced the ancestral states of GS in the major insect clades or orders by comparing the z0 index from model fitting, which represented the predicted GS value for the root state of the phylogeny. We found that the ancestral GS of insects estimated from 1,256 species was likely around 1,069.05 Mb (Table 2), while the ancestral GS of Neopterans (including Polyneoptera, Paraneoptera and Holometabola) was estimated to be 853.1 Mb. Estimation for Polyneopterans revealed an ancestral GS of 2,666.9 Mb, suggesting this group experienced a dramatic increase from that of their Neopteran ancestors ( Figure 1B). Conversely, the GS of Paraneoptera and Holometabola appeared to dramatically decrease from their ancestors to 511.68 and 437.52 Mb, respectively. These findings suggested that GS could evolve from the ancestral node along opposite trends within the infraclass Neoptera. At the Order level, Orthopterans were estimated to possess the largest ancestral GS of 4,764.21 Mb, thus showing extraordinary genomic expansion iScience Article compared with that of their Polyneopteran ancestors. Coleopterans, which are holometabolous, showed a relatively small increase in estimated ancestral GS (726.11 Mb) compared with that of Holometabola.
In general, the observed GS of extant insects indicated a wide range of genome expansion or contraction compared with estimated ancestral GS. We next examined what proportion of insect species experienced a significant genome expansion or contraction, defined as an increase or decrease of larger than 10%. The results indicated that 16.8% of insects underwent a genome expansion, from that of their progenitors, while 76.5% of species exhibited genome contraction. Notably, GS appeared similar with the ancestral state in only 6.6% of the insect species examined here. Among the different orders, a high number of species within Diptera, Coleoptera and Hemiptera underwent genome contraction, whereas more species in Orthoptera and Hymenoptera experienced a significant genome expansion compared with their respective ancestral GS ( Figure S1). These results implied that changes in GS occur frequently in the evolutionary history of insects, leading to high variation in the present state of GS among insects.

Large interspecies variation in GS within Coleoptera at the genus and family levels
To investigate differences in GS difference among closely related species, we next compared variation in GS between species within genera and within families. The results showed that differences in GS among species of the same genus were generally restricted to less than 2-fold (i.e., 159 out of 187 genera, or 85%, had two or more species with this low variation), suggesting that GS was largely similar within genera. However, we did find genera with large interspecies variation in GS, such as genus Chrysolina in Coleoptera. In particular, we noted that the GS of the leaf beetle Chrysolina americana was 547.7 Mb, while that of Chrysolina carnifex was 3,608.8 Mb, a 6.59-fold range in GS (Figure 2A andTable S4A). However, interspecies comparisons within families showed that GS most commonly (i.e., in 57 of 96 families, or 60%) differed by greater than 2-fold, and by greater than 5-fold in 13 of these families. The largest fold change was observed within Family Curculionidae of Coleoptera (n = 9), which varied from 208 Mb in Dendroctonus ponderosae to 4,910 Mb in Aramigus tessellatus (a 24-fold difference with a coefficient of variation (CV) of 118.9%) ( Figure 2B and Table S4B). These findings suggested that Coleopterans apparently showed greater intra genus and intrafamily variation in GS than other clades (Figure 2).

TEs and non-coding regions drive GS variation between closely related insect species
We next sought to identify the main genetic factors contributing to large changes GS in those closely related species. To this end, we quantitatively compared the genome contents of species within the same family level that showed greater than two-fold changes in GS. Since we observed significant variation in Coleopteran GS, we specifically selected 26 beetles from six Coleopteran families for further analysis, as well as 30 other species from three families across three different orders that had high quality genome  (Table S5). We found that, among these species, the fold difference in the size of assembled genomes within families ranged from 2.36 in Nymphalidae of Lepidoptera to 10.3 in Staphylinidae of Coleoptera (Table S7A). Since whole genome duplication (WGD) events have been reported to lead to rapid increases in GS for plants and some vertebrates (Ravi and Venkatesh 2008;Hallinan and Lindberg 2011;Soltis and Soltis 2016), we first searched for evidence of possible WGDs in these species. Based on Kernel density curves of Ks age, which should show significant peak(s) if paralogous genes are the product of a WGD event, we ruled out the possibility of WGD as a driver of genome expansion in these species (Figures S2 andS3).
To compare the genome contents of these close species from the same family, we downloaded the genome annotation data for these species from public databases. If annotations were unavailable, we annotated the genomes (Table S5) with an in-house pipeline (see method details). In Coleoptera, genome content analysis showed that the larger genomes contained significantly more repetitive sequences compared to that in smaller genomes within the family (e.g. from 379.1 Mb in Colorado potato beetle Leptinotarsa decemlineata to 1418.8 Mb in the western corn rootworm Diabrotica virgifera, family Chrysomelidae). Furthermore, the repeat sequences mainly consisted of various and highly abundant TEs in these beetle species (e.g., 74.5% TEs in the genome of the mealybug ladybird beetle Cryptolaemus montrouzieri (GS = 988.1 Mb) in family Coccinellidae; 71.4% TE content in the genome of the cowpea weevil Callosobruchus maculatus (GS = 1,007.8 Mb) in family Chrysomelidae; 70.5% TE content in the rice weevil S. oryzae (GS = 770.6 Mb) in family Curculionidae) ( Figure 3A and Table S7A).
The total content of non-repetitive regions, including exons and other non-coding regions, also varied substantially among these closely related beetles (e.g. from 80 Mb in the rove beetle A. bilineata to 333.3 Mb in A. taylori, family Staphylinidae), although to a lesser extent than repetitive sequences (Figure 3B). However, because exon regions showed comparatively little variance (30 Mb at average level) (Table S6), this observed variability primarily arose from non-coding regions, such as introns and intergenic non-coding DNA. Generally, both TEs and non-coding regions contributed to changes in GS in these beetle species, supported by a significant correlation between total genomic content of TEs and non-coding sequences and GS under phylogenetic correction (r = 0.95, p = 4.53E-13; r = 0.66, p = 3.57E-04, respectively) ( Figure 3C and Table S8A). This trend was also evident in correlation analyses of species in family Nymphalidae Order Lepidoptera ( Figure S4B and Table S8A). By contrast, the total content of coding sequence of exons was similar across all tested species (20-45 Mb), regardless of differences in total GS (Table S6). In particular, TEs provided the greatest contribution to variation in    iScience Article Interestingly, we found that family Chironomidae of Diptera was an exception to this overall trend in that TEs accounted for only a small fraction of their genomes, ranging from 2.5% in the marine midge Clunio marinus to 8.5% in Chironomus tentans ( Figure S9A and Table S7b). Although the total content of TEs was still positively associated with GS in this family (Pearson's test corrected by phylogeny; r = 0.996, p = 0.0045) ( Figure S9B and Table S8A), they contributed little to GS variation between Chironomidae species. Instead, we found that non-coding regions containing non-repetitive sequences ranged from 55.27 Mb in C. marinus to 134.63 Mb in C. tentans in these five midges, accounting for >60% of their genome. Further analysis showed that the total length of non-coding regions was significantly positively correlated with GS, and moreover, provided the largest contribution among different sequence types to intrafamily variation in GS among midge genomes (Pearson's test corrected by phylogeny; r = 0.98, p = 0.02). Taken together, except in some specific families, the TEs mostly dominated changes in GS among closely related species.

Intrafamilial history of TE expansion
Since GS is strongly affected by the proliferation of TEs in the vast majority of insect families examined here, we next investigated the putative history of TE expansion in various species by estimating Kimura distance between TEs and their consensus sequences to determine whether abrupt events or gradual increase resulted in the current GS. In Coleoptera, the distribution of Kimura distance of five species, such as the red palm weevil Rhynchophorus ferrugineus in Curculionidae, showed a bimodal shape of Kimura distance distribution, with a second peak around 0.2, reflecting the continuous accumulation of TEs (Figure 4). However, eight species such as the coffee berry borer Hypothenemus hampei, showed a bell curve distribution of Kimura distance, with a peak of K2P divergence around 0.15, indicating a relatively ancient burst of TE expansion. Thirteen species, including beetles with GS of more than 1Gb, had an L-shaped distribution with a peak below 0.1, indicating a recent burst of TE transposition. This trend was obvious in common buckeye J. coenia, which had the largest GS we observed in family Nymphalidae of Lepidoptera with an L-shaped distribution with a sharp peak below 0.05 ( Figure S5C), and the parasitoid wasp of apple maggot,

. TE divergency landscapes of 26 beetles in Coleoptera
Branches on the time-calibrated phylogeny are colored according to the total abundance of TEs. Species' Latin names are followed by their GS number, GS >1 Gb are in red. Each species is numbered, and each number corresponds to a small graph on the right showing the TE divergency of species. The yaxis represents genome coverage of various types of TE, and the xaxis represents Kimura substitution level % (TE divergency based on the Kimura distance of TE copies to the corresponding consensus sequences) from 0 to 50. The landscapes are categorized as L-shaped types (the peak value of TE divergency is less than 5), bell-curved types (the peak value of TE divergency is larger than 5) and bimodal-shaped types (two main peaks). See also Figures S10-S15 for zoomed graphs. iScience Article D. alloeum, in family Braconidae of Hymenoptera ( Figure S8). These results suggested that recent TE expansions commonly determine GS in most of the insect species/families examined here.

Various TEs drive GS changes in different clades of insects
We next investigated differences in the contributions of specific TE types to the observed variation in GS.
To this end, we further analyzed the diversity of DNA transposons and retrotransposons in each species, and calculated the abundance of each TE type.
Considering that the number of annotated TE families can serve as an indicator of TE diversity, we tested the relationship between TE diversity and GS. The results showed significant positive correlations between GS and TE diversity of DNA transposons, long interspersed nuclear elements (LINEs), and long terminal repeat retrotransposons (LTRs) across Coleopteran beetles (r = 0.48, p = 0.014; r = 0.4, p = 0.046; r = 0.5, p = 0.009, respectively) ( Figure 5A). Similar positive correlations were also observed in other tested clades ( Figures S4C and S7C, Table S8B). These findings indicated that the number of TE types detectable in a genome increased with GS. However, when further quantifying these TEs, the major contributions to changes in GS could be attributed to a limited set of specific TEs ( Figure 5B and Table S9A). For example, in Coleoptera, DNA/TcMar and three superfamilies of LINE elements such as Penelope, Jockey, and RTE/ BovB contributed the most to variation in GS and the landscapes of these TEs expansion history demonstrated considerable transposition activity among different families ( Figure 5C).
In the family of Coccinellidae, DNA/TcMar-Tc1 transposons accounted for more than 160 Mb in C. montrouzieri (16.5% of the genome), 130 Mb more than that in Harmonia axyridis. The Kimura distance distributions for both DNA/TcMar-Tc1 transposons and LINE/Penelope elements followed the distribution with a sharp peak around 0-0.1, suggesting a recent burst of transposition by these two types of TE.
In Family Chrysomelidae, the total content of LINE/RTE-BovB retrotransposons reached 204.3 Mb in D. virgifera, accounting for more than 8% of the whole genome, apparently increasing its GS by 200 Mb over that of its closest relative Ophraella communa after speciation 88.5 Mya. In the Curculionidae family, both DNA/TcMar-Mariner and LINE/Jockey elements accounted for 180 Mb in R. ferrugineus (23% of genome and 45.4% of total TEs). The blunt peak distribution of Kimura distance for LINE/ Jockey, bimodal distribution for LINE/RTE-BovB and DNA/TcMar-Mariner elements all suggested that these transposon types accumulated in a sustained manner over a relatively long evolutionary period in this family.
Among the 11 butterfly species we examined in the Lepidopteran family Nymphalidae, short interspersed nuclear elements (SINEs), especially SINE/tRNA elements accounted for a significant expansion (80 Mb) in both GS of Helioconius species H. himera and H. hermathena ( Figure S6 and Table S9B) since speciation from their most recent common ancestor 7.7 Mya ago. Kimura distributions of SINE/tRNA TE divergence showed a higher peak below 0.3, indicating further accumulation following the initial transposition burst. In summary, various TEs drive GS changes in different clades of insects. Moreover, either recent bursts or the gradual accumulation of specific types of TEs can explain major changes in GS.

DISCUSSION
The mechanisms driving variation in GS have posed a long-standing question for evolutionary biologists.
As one of the most species-rich animal clades, insects represent a good system for studying the mechanism of GS evolution due to wide variability among closely related species. Here, we collected GS data for 1,326  iScience Article insect species and characterized the changes and contributing factors for 1,256 species within a phylogenetic framework. We found that GS evolution of the holometabolous insects was best-fitted with the OU model, an adaptive process under the ''stabilized selection''. In contrast, the hemimetabolous favored the BM model, a neutral evolutionary process. However, since the OU process best explained GS evolution for insects overall, we speculated that selection might still be an important driving force to shape the GS in insects, even in Hemimetabola. Insect genome experienced massive expansion and contraction events under such different evolutionary processes among clades, thus resulting in considerable differences in GS between close species (i.e., within a family) as we observed.
The causes driving these changes in GS have been widely debated and can be generally summarized as the product of either DNA accumulation or DNA elimination. The main mechanisms of DNA accumulation include WGD (Van DePeer et al., 2017), or TE (Maumus et al., 2015;Gilbert et al., 2021) and satellite DNA (Shah et al., 2020;Majid and Yuan 2021;Stelzer et al., 2021) proliferation. WGDs are widely detected in plant species (Soltis et al., 2003;Vu et al., 2015;Soltis and Soltis 2016) and have been reported in animals such as the teleost fish (Ravi and Venkatesh 2008) and mollusks (Hallinan and Lindberg 2011), whereas little evidence has emerged showing WGDs in insects (Li et al., 2018;Roelofs et al., 2020). Here, we also present strong evidence that WGD events did not occur in the insects we examined with large GS, therefore leading us to conclude that it does not likely contribute to variation in GS among close insect species.
Through quantitative characterization of a large set of high-quality insect genomes, our findings reveal that the abundance of various TE types is the major source of differences in GS between close species. That is, variations in GS are predominantly the result of TE accumulation in most insects. Detailed analysis of TE composition showed that DNA transposons or LINE retrotransposons are the main contributors to GS expansion in Coleopteran families. While SINE elements comprise a large proportion of TEs and dominate GS variation in Heliconius butterflies of Family Nymphalidae in Lepidoptera.
Since the transposition of retrotransposons follows a general principle of ''copy and paste'' (Boeke et al., 1985), genomic expansion driven by increased retrotransposon activity, especially LTR elements, has been widely reported, especially in plants (Sanmiguel and Bennetzen 1998;Piegu et al., 2006;Zedek et al., 2010;Nystedt et al., 2013;Macas et al., 2015;Zhang et al., 2020). Here, we provide additional evidence that LINEs and SINEs can also provide major contributions to changes in GS. In contrast with retrotransposons, most DNA transposons, except for Helitrons and Maverick/Polinton elements (Grabundzija et al., 2016), follow a principle of ''cut and paste '' transposition (Greenblatt and Brink 1963;Rubin et al., 1982), and thus does not increase their copy numbers or GS despite increased transposition activity. Notably, a relatively unusual abundance of these elements appears responsible for GS enlargement in the dryinid wasp Gonatopus flavifemur (Yang et al., 2021), as well as some beetle genomes examined in this study ( Figure 3B). One hypothesis that could explain this effect on GS is cross-species horizontal transposon transfer (HTT) (Schaack et al., 2010;Wallau et al., 2018). Massive HTT events have been reported in insects, with one study estimating that more than 2,000 HTT events occurred over the last 10 Mya (Peccoud et al., 2017), especially among phylogenetically close organisms (Bartolome et al., 2009;El Baidouri et al., 2014). In particular, DNA transposons appear to be more frequently transferred than retrotransposons due to relatively simple structure that is more conducive to persistence in and transfer by the transmission vectors (Gilbert and Feschotte 2018).
In the genus of Aedes, TEs that are frequently exchanged through HTT contribute to more than 6% of whole genome and thus drive GS expansion in mosquitoes (Melo and Wallau 2020). More relative explorations are still needed for further understanding the causes for content differences of these active genomic elements among close species.
Beyond that, our results led us to deduce that the evolution of GS is mainly driven by the activity of TE transposition in insects. The activation of TEs is subject to host cues, especially in early stages of gametogenesis and embryo development in which epigenetic reprogramming also enables cell developmental totipotency (Surani and Hajkova 2010;Rowe and Trono 2011). In higher eukaryotes, several surveillance systems have evolved to modulate TE activity. These systems mainly involve epigenetic transcriptional regulation, such as DNA methylation and histone modifications (Karimi et al., 2011;Groh and Schotta 2017) or small RNA-based pathways, such as small interfering RNAs (siRNAs) (Kasschau et al., 2007;Fagegaltier et al., 2009) and PIWI-interacting RNAs (piRNA) (Sienski et al., 2012;Le Thomas et al., 2013;Ernst et al., 2017). In germline cells, piRNA pathways are dedicated to suppressing TE transposition activity and maintaining genomic integrity (Shen et al., 2018;Zhang et al., 2018a iScience Article from progenitor species, both vertically and horizontally transferred TEs are widely activated in germline cells, copying and inserting themselves across the insect genome. Regulators of the piRNA pathway target potentially deleterious TEs for inactivation, although a proportion of these mobile elements successfully propagate (Wang et al., 2018). Once they escape this initial repression mechanism, the rate of TE amplification is very high (Biemont and Vieira 2006;Naito et al., 2006), and these expanded TEs are then passed to successive generations in reproductive cells.
New TE insertion events also introduce mutations with a range of subsequent effects, including alterations in gene expression, that can affect both genotype and phenotype (Wells and Feschotte 2020;Almojil et al., 2021;Gilbert et al., 2021). Those mutations that lead to positive effects on competitive fitness in the host are retained through positive selection by specific environmental conditions (Wilson 1993;Nagaraju 2001;Daborn et al., 2002). These beneficial mutations become fixed in the expanded genome throughout the population, ultimately leading to new speciation events. A large proportion of the TE insertion mutations contribute relatively neutral effects and are strongly influenced by randomgenetic drift rather than positive selection (Arkhipova 2018). For these mutations, their contribution to genome expansion is eventually regulated by the effective population size of species. In addition, GS is directly correlated with cell metabolic capacity, and bloated or excessive DNA content will strain the cellular energy metabolism normally required for replication and proliferation (Cavalier-Smith 1978, 2005Gregory and Hebert 1999). Under this cell-level selection, TE sequences under less evolutionary constraint are prone to deletion to reduce GS by replication slippage or genomic rearrangement, such as through illegitimate and unequal homologous recombination (Gray 2000;Devos et al., 2002).
These collective evolutionary processes could thus explain the variation in GS we detected between close insect species. Whether mutations caused by TE insertions become fixed in the population or TE proliferation is silenced by cellular regulatory mechanisms are all subject to probability. According to this hypothesis, the process of expanding and contracting total GS is always dynamic. In general, further investigations are necessary to better understand the mechanisms by which transposons proliferate or are suppressed and deleted in germline cells, and may shed lights on the evolution of the insect genome and insect genome size.

Limitations of the study
Utilizing published GS data from flow cytometry, we provided a comprehensive analysis of insect GS variation. Nonetheless, compared with millions of insect species recognized by humans so far, the number of insects with GS data are still limited. In addition, information is still absent in some clades such as Neuropterida (snakeflies, dobsonflies and net-winged insects). Selecting more species to fill this gap would deepen the understanding of insect GS evolution. Furthermore, we modeled the evolutionary history of insect GS based on the NCBI taxonomic tree, which is a simplified phylogenetic framework. Producing an ultrametric time-scaled tree covering more species would provide more information for insect GS evolution.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

AUTHOR CONTRIBUTIONS
F.L designed the research for the whole paper; F.L and K.H coordinated the project; F.L and X.H.Y revised the paper; Y.M annotated the genomes; Y.Y.C conducted data analysis and drafted the paper.

DECLARATION OF INTERESTS
The authors declare no competing interests.