Copy number increases of transposable elements and protein‐coding genes in an invasive fish of hybrid origin

Abstract Evolutionary dynamics of structural genetic variation in lineages of hybrid origin is not well explored, although structural mutations may increase in controlled hybrid crosses. We therefore tested whether structural variants accumulate in a fish of recent hybrid origin, invasive Cottus, relative to both parental species Cottus rhenanus and Cottus perifretum. Copy‐number variation in exons of 10,979 genes was assessed using comparative genome hybridization arrays. Twelve genes showed significantly higher copy numbers in invasive Cottus compared to both parents. This coincided with increased expression for three genes related to vision, detoxification and muscle development, suggesting possible gene dosage effects. Copy number increases of putative transposons were assessed by comparative mapping of genomic DNA reads against a de novo assembly of 1,005 repetitive elements. In contrast to exons, copy number increases of repetitive elements were common (20.7%) in invasive Cottus, whereas decrease was very rare (0.01%). Among the increased repetitive elements, 53.8% occurred at higher numbers in C. perifretum compared to C. rhenanus, while only 1.4% were more abundant in C. rhenanus. This implies a biased mutational process that amplifies genetic material from one ancestor. To assess the frequency of de novo mutations through hybridization, we screened 64 laboratory‐bred F2 offspring between the parental species for copy‐number changes at five candidate loci. We found no evidence for new structural variants, indicating that they are too rare to be detected given our sampling scheme. Instead, they must have accumulated over more generations than we observed in a controlled cross.

that can increase reproductive isolation and hamper the evolution of admixed lineages (Maheshwari & Barbash, 2011). On the other hand, hybridization can lead to new combinations of allelic variants and therefore be a source of evolutionary novelty and ecological niche divergence (Lexer, Lai, & Rieseberg, 2003). A common explanation is given by the process of transgressive segregation, which is caused by novel interactions of alleles that evolved in isolation and subsequently segregate in the hybrid offspring (Rieseberg, 1997;Rieseberg, Archer, & Wayne, 1999). This is plausible because hybridization introduces a wealth of genetic diversity that can affect evolutionary processes during the initial and highly dynamic phase of hybrid speciation.
In addition to effects caused by parental alleles, an increased rate of de novo structural mutations in hybrids appears possible, similar to the finding of increased point mutations in heterozygotes ("heterozygosity instability"; Amos, 2010;Xie et al., 2016). Whether this actually occurs and leaves detectable genetic signatures has received comparatively little attention in studies on hybrid speciation. However, it has already been found that hybrid crosses may exhibit de novo copy-number variation that is frequent enough to be observed among siblings of the same litter of mice (Scavetta & Tautz, 2010). Although copy-number variations (CNVs) may infer fitness costs associated with gene dosage alteration (Orozco et al., 2009;Papp, Pal, & Hurst, 2003), they may also contribute to the acquisition of new adaptive functions and promote species diversification (Kondrashov, 2012;Machado et al., 2014). Hybridization can also increase mutation rates through the activation of transposable elements (TEs), which have been reported to undergo "bursts of transposition" in hybrids of plants (McClintock, 1984;Renaut, Rowe, Ungerer, & Rieseberg, 2014;Ungerer, Strakosh, & Stimpson, 2009) and animals (Garc ıa Guerreiro, 2015; Labrador & Fontdevila, 1994;O'Neill, O'Neill, & Graves, 1998;Renaut, Nolte, & Bernatchez, 2010).
The expected number of gene and transposon copies in a lineage of hybrid origin should correspond with the average of the copy number in the parental species (e.g., Kawakami, Dhakal, Katterhenry, Heatherington, & Ungerer, 2011;Scavetta & Tautz, 2010). Deviations from this average would be conceivable if the genetic contributions from both parent species to the hybrid gene pool are not even. Copy numbers may, however, not exceed observed parental averages due to admixture alone. Instead, copy-numbers outside the parental values could indicate the evolution of new copies and be a distinguishing feature of hybrid lineages. If rapidly evolving structural variants were also subject to positive selection, they could be among the first to contribute to adaptive evolution of hybrid lineages. Although the underlying mutational mechanisms are known from controlled hybrid crosses (e.g., Garc ıa Guerreiro, 2014;McClintock, 1984;Scavetta & Tautz, 2010), few empirical studies have documented the evolutionary dynamics of copy-number variation and transposition in natural admixed lineages. For example, studies on homoploid sunflower hybrid species revealed a massive accumulation of Ty3/gypsy-like TEs during the past 0.5-1 million years (Renaut et al., 2014;Ungerer et al., 2009). On the other hand, transposition bursts have not been observed in F 1 hybrid sunflowers or contemporary admixture zones between their parental species (Kawakami et al., 2011;Renaut et al., 2014). Consequently, the relative importance of short-term compared to long-term evolutionary processes that drive the proliferation of TEs remains unclear when hybrid lineages are ancient.
Here, we analysed whether CNVs have increased in a small freshwater fish of hybrid origin that has emerged postglacially, possibly within the last 200 years (Nolte, Freyhof, Stemshorn, & Tautz, 2005).
The so-called invasive Cottus provides a unique natural setting to explore evolutionary mechanisms that may initiate the evolution of a homoploid hybrid species (Nolte & Tautz, 2010). Invasive Cottus is a lineage of hybrid origin that has colonized lower reaches of rivers and streams of intermediate size, whereas the parental species Cottus perifretum and Cottus rhenanus live in parapatry, apparently confined to the headwaters of smaller tributaries (Nolte et al., 2005;Stemshorn, Reed, Nolte, & Tautz, 2011). A likely explanation for the success in lower reaches of rivers is that invasive Cottus has evolved new beneficial traits through hybridization that are driving divergence between invasive Cottus and its parental species. Besides barriers to gene flow in hybrid zones (Nolte, Freyhof, & Tautz, 2006;Nolte, Gompert, & Buerkle, 2009), this manifests in patterns of gene expression that are unique to invasive Cottus (Czypionka, Cheng, Pozhitkov, & Nolte, 2012). In this study, we tested whether rapid copy-number evolution of protein-coding genes and TEs occurs in natural populations of invasive Cottus. We expected to find overall more gene duplications than deletions (e.g., Katju & Bergthorsson, 2013), as well as an increased variance of copy numbers compared to the parental species (Scavetta & Tautz, 2010). To further explore the possibility that hybridization causes rapid evolution of de novo structural variants, we tested laboratory-raised F 2 (selfed F 1 ) C. rhenanus 9 C. perifretum crosses for copy-number changes of candidate genes and transposons that have increased in copy number in natural populations of invasive Cottus.
Finally, we searched for associations between changes in gene copy number and gene expression in invasive Cottus to explore possible contributions of CNVs to phenotypic change.  (Grabherr et al., 2011). For the transcriptome assembly, total RNA was extracted from different tissues (gills/heart, liver, fins/skin, digestive tract, head without gills, white muscle) of a C. rhenanus male from Fockenbach using a TRIzol protocol (Invitrogen, Carlsbad, CA, USA).

| MATERIALS AND
Libraries were prepared with the Illumina Truseq Kit and sequenced from two sides (100-bp read length) on an Illumina GAIIx sequencer.
We only targeted genes that could be annotated to the threespine stickleback genome, aiming for a coverage of one probe per exon ("exon-array CGH"; Dhami et al., 2005). Additionally, we included probes for genes that were identified to be either upregulated or downregulated in invasive Cottus in a previous gene expression microarray study (Czypionka et al., 2012). We first conducted a BLAT search (BLAT version 35x1; Kent, 2002; e-value < 10 À3 ) of Cottus ESTs against the stickleback genome and subsequently used best matching sequences larger than 60 bp for a second BLAT search against stickleback coding sequences (retrieved from the BIOMART database version 0.7; www.ensembl.org/biomart). The sense direction of all probe sequences was inferred from the directionality given in the BLAT T A B L E 1 Individual samples used for comparative genomic hybridization array (aCGH) microarrays and genome mapping. Indicated are number, gender and geographic coordinates for each population Population aCGH Genome mapping Lat./Long.
results. Sequences identified as negative strands were reverse-transcribed, and sequences with contrasting directionality information between the ensemble database and the BLAT results were removed.
To optimize probe selection and to validate the performance of probes, we initially performed a microarray calibration experiment (Czypionka et al., 2012;Pozhitkov, Noble, Bryk, & Tautz, 2014 Table S3). The final probe set was sent to SureDesign for the construction of an experimental 8x60k array with five replicates per probe, and a 15-bp linker added to each probe as recommended by Agilent (SureDesign-ID: 070467).
The experimental array was used for a common reference aCGH experiment. Three array slides were used to cohybridize 24 test samples (eight individuals from each of C. rhenanus, C. perifretum, and invasive Cottus; all Cy5 labelled) with a common reference sample (a female individual of C. perifretum from Larse Beek; Cy3 labelled) (

| Quality filtering, normalization
Quality control metrics provided in the FEATURE EXTRACTION output files were carefully inspected for the spatial distribution of outliers on the arrays. A pipetting error before hybridization created a noticeable bubble in the centre of nine experimental arrays, resulting in decreased signal intensity values and clustering of outliers in these regions. We therefore decided to remove all data affected by such bubble artefacts (~12% of 1,452,600 probe replicates present on 24 arrays). For this purpose, we first retrieved coordinates for individual probe replicates ("features") from the FEATURE EXTRACTION output files and plotted the spatial distribution of log-signal intensities for each array (using the "levelplot" function in the R package LATTICEEXTRA). We used the R package "SPLANCS" (version 2.01-36; Bivand & Gebhardt, 2000) to extract the coordinates of features located within bubble artefacts by drawing polygons around the bubbles, which were clearly visible through reduced log-signal intensities. Coordinates of all features located within bubble areas were then used to remove affected data from the complete data set. Next, we excluded outliers among probe replicates as determined by signal intensities exceed- To account for probe-specific binding coefficients in the observed signal intensities, we applied a calibration on average signal intensities as recommended by Pozhitkov et al. (2014). For this purpose, we used the probe-specific linear regression parameters (slope, intercept) from the calibration array, to estimate a calibrated starting DNA value (DNAcal) for each channel (Cy3, Cy5), calculated as DNAcal = (observed signal À intercept)/slope. Although this procedure reduces artefacts introduced through probe-specific differences in binding behaviour, it does not remove intensity effects, which are visible in a "MA"-plot as increasing log2-(Cy5/Cy3)-ratios ("M") with increasing average (log-) signal intensities ("A"). To remove such intensity effects and to normalize log2-ratios to an average of zero, we performed a LOESS normalization (span = 0.3, iterations = 4) without background correction (Zahurak et al., 2007), as implemented in the R package LIMMA (version 3.22.4, Ritchie et al., 2015).
This method fits a smoothing curve based on locally weighted leastsquares regression and uses the residuals of the fitted curve as normalized log2-ratios.

| Array-CGH data analyses and comparison to gene expression
Genes showing increased or decreased copy-numbers in invasive  (Kofler et al., 2011). To facilitate the assembly process, sequence reads were first error-corrected using the software LIGHTER, with a k-mer length of 31, a genome length of 1 Gbp, and a subsampling fraction of 0.1 (Song, Florea, & Langmead, 2014 (Wicker et al., 2007). Remaining unclassified sequences were further analysed using BLASTX searches in BLAST2GO (Conesa et al., 2005). Beek, respectively).
To estimate copy numbers, we used multiplex reactions of the target sequence together with a single-copy reference gene (McDermott et al., 2013;Miotke, Lau, Rumma, & Ji, 2014). As a reference single-copy gene, we chose rpl13a, which has been used as a singlecopy housekeeping gene in threespine stickleback (Hibbeler, Scharsack, & Becker, 2008), and showed no signs of CNV in our own CGH-array experiments. All primers were designed using PRIMER3

| Exon-Array CGH copy-number variation
We compared CNV in eight invasive Cottus individuals with eight individuals of each of the parental species, C. rhenanus and C. perifretum to detect copy-number changes of genes in invasive Cottus. Among 10,979 genes (exons) and additional 446 probes derived from a previous gene expression study (Czypionka et al., 2012), we identified 12 genes with increased as well as 13 genes with decreased copy numbers in invasive Cottus compared to both parental species (two-tailed Wilcoxon rank-sum test; FDR corrected; p < .05) (Figure 1; Table S2).
Between the parental species C. perifretum and C. rhenanus, we found 1,920 genes (~15.9%) to differ in copy number (two-tailed Wilcoxon test; FDR corrected; p < .05). Copy numbers did not differ significantly between males and females (results not shown).
In accordance with the general expectation to find more gene duplications than deletions, copy-number variable genes in invasive Cottus showed a stronger signal of copy number increase than decrease (D'Agostini test: skew = 0.8918; z = 34.6861; p < .001).
This trend of invasive Cottus to show more increases of copy numbers was also visible using arbitrary log2-thresholds: Increased copy numbers were inferred for 409 genes that exceeded a log2-ratio threshold of 0.5 (putative duplications), whereas decreased copy numbers were inferred for 314 genes falling below a log2-ratio threshold of À0.5 (putative deletions) in at least one individual.
Among those genes, 125 included individuals with signs of both deletions and duplications, leaving a total of 598 copy-number variable genes (4.9%). When applying a less stringent log2-ratio threshold of (AE) 0.4, a total of 1346 (11.1%) of all genes were estimated to be copy number variable in at least one individual, which included 916 putative duplications and 733 putative deletions.
In contrast to our expectation, we found no increased variance of copy numbers in invasive Cottus compared to the parental spe-

| De novo assembly and copy-number variation of repetitive elements
To assess a possible accumulation of TEs in invasive Cottus, we used a genome mapping approach to compare the relative number of reads mapped to a de novo assembly of repetitive elements. The de novo assemblies in TEDNA resulted in a total of 7,548 sequences, which were assembled in SEQMAN PRO into 1,005 contigs (hereafter referred to as "repetitive elements"; size range: 500-50k bp; n50: 2,978 bp; Table S4). Using PASTECLASSIFIER, we classified 452 repetitive elements (45%) according to Wicker's classification system of TEs. To assess whether invasive Cottus individuals (n = 10) showed higher copy numbers compared to both parental species (n = 20), we applied Wilcoxon rank-sum tests to compare individual count data for each of 1,005 repetitive elements (one-tailed; FDR corrected). Among all 1,005 repetitive elements, we found significant copy-number increases in 208 (20.7%) repetitive elements with an average copy-number increase of 30.5% compared to the parental mean (Figure 2; marked by red sidebar). In contrast, significant decreases in copy number were rare, affecting only 12 (0.01%) repetitive elements (nine unclassified elements, two putative retrotransposons, one potential host gene). Overall, increased repetitive elements showed a strong bias towards those being present in higher copy numbers in C. perifretum compared to C. rhenanus (Figure 2). This was also evident when comparing copy numbers between C. perifretum and C. rhenanus for the 208 candidate ele-

| Validation of CNV candidates and screen for de novo CNVs in F2 crosses
We used ddPCR to validate increased copy numbers in invasive Cottus for three selected CNV candidate genes (MIEF1, NUGGC, CYP27C1) and three repetitive elements (one LINE element, two uncharacterized elements). These CNV candidates showed some of the strongest signals of copy-number increases in invasive Cottus and were therefore chosen as a proof of principle to confirm our alternative approaches to detecting copy-number variants. In the parental species, ddPCR revealed CYP27C1 as a single-copy gene, whereas MIEF1 occurred mostly in two copies and NUGGC was present as a more variable multicopy gene with most individuals showing five to seven copies (Figure 3). Candidates with strong signals in our analyses were considered useful to test for de novo mutations in controlled crosses. To explore a possible role of rapidly evolving de novo structural variants, we screened 32 individuals from each of two F 2 families (C. rhenanus 9 C. perifretum) for CNV of validated candidate genes (MIEF1, NUGGC, CYP27C1) and repetitive elements (one LINE element, one uncharacterized element) (Figure 3). None of the examined CNV candidates showed signs of de novo copy-number increases compared to the parental species (Figure 3).

| DISCUSSION
Copy-number variation has long been recognized to play a role in the evolution of phenotypic novelty. However, the possible contribution of structural variations to the evolution of admixed lineages remains largely unexplored in natural systems. Here, we report copy-number increases of candidate genes (exons) as well as a proliferation of repetitive elements in a homoploid hybrid lineage of Cottus. The origin of invasive Cottus is most likely postglacial and the biogeographic settings suggest an age of <200 generations (Nolte et al., 2005). Hence, copy number increases that are specific to invasive Cottus could have evolved within a relatively short time frame following admixture.
Herein, we found no evidence for de novo structural mutations in three genes and two repetitive elements in two families of laboratory F 2 crosses between the parental species. However, this limited set of 64 F 2 offspring only represents an upper bound for the frequency of such mutations in early generation hybrid crosses. It is still possible that de novo mutations occurred in natural hybrid populations at a frequency that could not be detected here. On the other hand, our ability to distinguish whether CNVs in admixed lineages are recruited from standing genetic variation or from de novo mutations is limited because of uncertainty about the variation present in natural populations. We discuss evolutionary processes that may be relevant to explain copy-number changes in invasive Cottus and how they could be involved in adaptive evolution.

| Exon copy-number variation in admixed lineages
Although copy-number variation between the parental species occurs frequently, we found only a very low proportion of copy number variable exons that may have increased in invasive Cottus.

This is not surprising given the presumably recent origin of invasive
Cottus and considering that gene duplication rates are typically slow F I G U R E 2 Heatmap showing mapping results of genomic reads from 30 Cottus genomes against 1,005 repetitive elements. Colour-coding represents the percentage of sum of reads (number of mapped reads per Mio reads) for each repetitive element. Coloured sidebars on the left indicate 208 repetitive elements with significantly higher numbers of mapped reads in invasive Cottus compared to the parental species (red), 12 elements with significantly lower numbers in invasive Cottus than in the parental species (orange), as well as repetitive elements that are significantly higher in C. rhenanus (light grey), and significantly lower in C. rhenanus (dark grey) compared to combined invasive Cottus and C. perifretum DENNENMOSER ET AL.
| 4719 (Katju & Bergthorsson, 2013;Lynch & Conery, 2000). It is also consistent with the general assumption that new gene duplications are quickly removed from the genome by purifying selection (Katju & Bergthorsson, 2013;Lynch & Conery, 2000;Ohno, 1970 (Cheng, Czypionka, & Nolte, 2013) infers si:ch211-256m1.8 as the most likely neighbouring gene, which shows a joint increase of copy number in invasive Cottus. Increased copy numbers could also originate from standing genetic variation already present in populations or individuals of the parental species not sampled here. This seems plausible for genes such as MIEF1 or NUGGC, for which ddPCR identified single individuals of the parental species with increased copy numbers.
Hence, our study did not yield conclusive evidence that genomic admixture caused de novo copy number increases of coding genes in invasive Cottus.
While we cannot rule out genetic drift, the occurrence of highfrequency gene duplicates that are close to fixation in invasive Cottus but not the parental species indicates a possible role of selection.
A recent effort to disentangle the relative role of drift and selection suggested that about half of the high-frequency gene duplicates in Cottus would translate into a Ne of 50 that seems unrealistically low.
Drift is therefore unlikely to explain all gene duplicates, and instead, some of the gene duplicates may have risen to high frequencies through selection (e.g., Feulner et al., 2013;Kondrashov, 2012).
Selection could favour new alternative splice variants following duplications of exons (Abascal, Tress, & Valencia, 2015) or act on gene dosage effects that are thought to underlie most of the reported adaptive gene duplicates (Kondrashov, 2012). strongest candidates for increased copy numbers in our aCGH analyses and were overexpressed in juvenile stages of invasive Cottus in a previous gene expression study (Czypionka et al., 2012). Functions associated with cytochrome P450 genes are highly diverse (Danielson, 2002) and have been related for example to insecticide resistance (Wondji et al., 2009), adaptation to desert environments (Jirimutu et al., 2012) or immune responses to bacterial and virus infections (Iizuka et al., 2004). In teleost fishes, CYP2K1 appears to be mostly expressed in liver and kidney and involved in detoxification of environmental pollutants (e.g., Buhler, Zhao, Yang, Miranda, & Buhler, 1997;Uno, Ishizuka, & Itakura, 2012). Enright et al. (2015) reported that CYP27C1 may red-shift the spectral sensitivity in zebrafish by converting vitamin A 1 into A 2 , which translates into an enhanced behavioural response to long-wavelength light. If CYP27C1 has a similar function in Cottus, increased gene dosage could be beneficial for enhanced vision in more turbid environments (Fain, 2015) such as the larger streams inhabited by invasive Cottus. Finally, GTF2IRD2 might affect the development of skeletal muscle during the growth period of juvenile stages, with increased gene dosage leading to a higher proportion of slow-type muscle fibres (Palmer et al., 2012). Changed proportions of muscle fibre types could be involved in the acclimation to different temperature regimes by invasive Cottus, resembling what has been reported for other teleost fishes (e.g., Woytanowski & Coughlin, 2013). Altogether, we found some gene duplicates with potentially adaptive gene dosage effects in an evolutionary young homoploid hybrid species, but their low number and the possibility that they were recruited from standing genetic variation of the parental species does not suggest a major role of increased de novo evolution of structural variants.

| Increases of repetitive elements
We detected a significant increase of copy numbers in 20.7% of repetitive elements including putative transposons in invasive Cottus. The frequency of these copy-number increases exceeded what we observed with protein-coding genes by far. Their proliferation could have been be induced by a "genomic shock" following exposure to environmental stress or hybridization (Belyayev, 2014;Fontdevila, 2005;McClintock, 1984;Senerchia, Felber, & Parisod, 2015). A weak bias towards non-LTR retrotransposons among increased TEs is in line with such a burst of transposition, but this excess is not significant and other repetitive elements have increased almost equally. For example, we find increased copy numbers of putative DNA transposons that use a cut-and-paste mechanism and should only rarely increase in copy number. Similarly, a range of uncharacterized repetitive elements that are not likely to multiply via transposition also increased. Hence, the accumulation of repetitive elements in invasive Cottus cannot be fully explained through transposition. Resembling our results, transposition bursts are not evident in F 1 hybrid sunflowers or contemporary hybrid zones between their parental species (Kawakami et al., 2011;Renaut et al., 2014). This has led to argue that a combination of hybridization and environmental stress during colonization of extreme habitats (deserts, salt marshes) could have initiated bursts of transposition in hybrid sunflower species (Kawakami et al., 2011), although subsequent experimental tests failed to induce TE proliferation under various stress conditions (Ungerer & Kawakami, 2013). The absence of transposition bursts has also been reported from retrotransposons in polyploid plant hybrids, which instead emphasized the importance of TE silencing through reorganization of methylation states and deletions of TE loci (e.g., Senerchia et al., 2015Senerchia et al., , 2016. Accordingly, an accumulation of TEs through transposition activities may be rare and is presumably limited to only a small fraction of TEs that escape silencing mechanisms without provoking strong deleterious effects (Parisod et al., 2010).
As an underexplored alternative, increased copy numbers of repetitive elements could be explained through multiple segmental duplications in centromeric or pericentromeric regions, which typically harbour large arrays of satellite repeats and TEs (Horvath et al., 2005;Ma & Jackson, 2006). Centromeres have been suggested as a primary target for genomic destabilization following interspecific hybridization in marsupial hybrids, which was shown to cause amplification of satellite and TEs (Metcalfe et al., 2007). Likewise, a massive accumulation of retro TEs has occurred in pericentromeric regions of homoploid hybrid species of sunflowers (Staton, Ungerer, & Moore, 2009). Interestingly, most of the repetitive elements increased in invasive Cottus have higher copy numbers in C. perifretum compared to C. rhenanus ( Figure 3). Conversely, repetitive elements that occur in higher copy numbers in C. rhenanus compared to C. perifretum have rarely expanded in the invasive Cottus genome. This asymmetric pattern of repetitive element amplification correlates with an overrepresentation of C. perifretum ancestry in the genome of invasive Cottus, which con-tributes~60% to the hybrid gene pool (Stemshorn et al., 2011). Thus, a nonrandom mechanism of repetitive element amplification with respect to ancestry appears possible. Centromere drive could quickly create such an asymmetry though a transmission advantage of longer centromeres during female meiosis, mediated by a higher number of microtubule binding sites during the segregation of chromosomes (Henikoff, Ahmad, & Malik, 2001). in invasive Cottus is in line with such a scenario but appears to be relatively rare in this study. We speculate that centromere drive followed by segmental duplications might contribute to the conspicuous accumulation of repetitive elements. While this appears to be attributable to hybridization and genomic admixture, it also suggests that it may be particularly pronounced in gene poor regions around the centromeres.
On the one hand, this means that the massive copy number increase is unlikely to affect a large number of coding genes, while on the other hand, it makes it plausible that the large number of new repetitive element copies was not removed by selection. This warrants more studies that distinguish whether increased transposition rates or transposition-independent mechanisms cause the accumulation of copy numbers in hybrid lineages and, more broadly, whether this may lead to rapid evolution of hybrid species.