Parallel evolution of morphological and genomic selfing syndromes accompany the breakdown of heterostyly.

Evolutionary transitions from outcrossing to selfing in flowering plants have convergent morphological and genomic signatures and can involve parallel evolution within related lineages. Adaptive evolution of morphological traits is often assumed to evolve faster than nonadaptive features of the genomic selfing syndrome. We investigated phenotypic and genomic changes associated with transitions from distyly to homostyly in the Primula oreodoxa complex. We determined whether the transition to selfing occurred more than once and investigated stages in the evolution of morphological and genomic selfing syndromes using 22 floral traits and both nuclear and plastid genomic data from 25 populations. Two independent transitions were detected representing an earlier and a more recently derived selfing lineage. The older lineage exhibited classic features of the morphological and genomic selfing syndrome. Although features of both selfing syndromes were less developed in the younger selfing lineage, they exhibited parallel development with the older selfing lineage. This finding contrasts with the prediction that some genomic changes should lag behind adaptive changes to morphological traits. Our findings highlight the value of comparative studies on the timing and extent of transitions from outcrossing to selfing between related lineages for investigating the tempo of morphological and molecular evolution.

Purging due to self-fertilization does not prevent accumulation of expansion load.

As species expand their geographic ranges, colonizing populations face novel ecological conditions, such as new environments and limited mates, and suffer from evolutionary consequences of demographic change through bottlenecks and mutation load accumulation. Self-fertilization is often observed at species range edges and, in addition to countering the lack of mates, is hypothesized as an evolutionary advantage against load accumulation through increased homozygosity and purging. We study how selfing impacts the accumulation of genetic load during range expansion via purging and/or speed of colonization. Using simulations, we disentangle inbreeding effects due to demography versus due to selfing and find that selfers expand faster, but still accumulate load, regardless of mating system. The severity of variants contributing to this load, however, differs across mating system: higher selfing rates purge large-effect recessive variants leaving a burden of smaller-effect alleles. We compare these predictions to the mixed-mating plant Arabis alpina, using whole-genome sequences from refugial outcrossing populations versus expanded selfing populations. Empirical results indicate accumulation of expansion load along with evidence of purging in selfing populations, concordant with our simulations, suggesting that while purging is a benefit of selfing evolving during range expansions, it is not sufficient to prevent load accumulation due to range expansion.

No detectable changes in reproductive behaviour of Caenorhabditis elegans males after 97 generations under obligatory outcrossing.

In Caenorhabditis elegans, a species reproducing mostly via self-fertilization, numerous signatures of selfing syndrome are observed, including differences in reproductive behaviour compared to related obligatory outcrossing species. In this study we investigated the effect of nearly 100 generations of obligatory outcrossing on several characteristics of male reproductive behaviour. A genetically uniform ancestral population carrying a mutation changing the reproductive system to obligatory outcrossing was split into four independent populations. We predicted that the transition from the natural reproductive system, where males were extremely rare, to obligatory outcrossing, where males comprise 50% of the population and are necessary for reproduction, will increase the selection pressure on higher effectiveness of mating behaviour. Several characteristics of male mating behaviour during a 15 min interaction as well as copulation success were compared between the ancestral and evolved populations. No significant differences in male mating behaviour or fertilization success were detected between generations 1 and 97 of obligatory outcrossing populations. We found, however, that longer contact with females increased chances of successful copulation, although this effect did not differ between populations. We conclude that either selection acting on male mating behaviour has not been strong enough, or mutational input of new adaptive variants has not been sufficient to cause noticeable behavioural differences after 97 generations of evolution starting from genetically uniform population.

Genetic and demographic signatures accompanying the evolution of the selfing syndrome in Daphne kiusiana, an evergreen shrub.

BACKGROUND AND AIMS: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood. METHODS: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets. KEY RESULTS: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period. CONCLUSIONS: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.

The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

Balancing selection in self-fertilizing populations.

Self-fertilization commonly occurs in hermaphroditic species, either occasionally or as the main reproductive mode. It strongly affects the genetic functioning of a population by increasing homozygosity and genetic drift and reducing the effectiveness of recombination. Balancing selection is a form of selection that maintains polymorphism, which has been extensively studied in outcrossing species. Yet, despite recent developments, the analysis of balancing selection in partially selfing species is limited to specific cases and a general treatment is still lacking. In particular, it is unclear whether selfing globally reduced the efficacy of balancing selection as in the well-known case of overdominance. I provide a unifying framework, quantify how selfing affects the maintenance of polymorphism and the efficacy of the different form of balancing selection, and show that they can be classified into two main categories: overdominance-like selection (including true overdominance, selection variable in space and time, and antagonistic selection), which is strongly affected by selfing, and negative frequency dependent selection, which is barely affected by selfing, even at multiple loci. I also provide simple analytical results for all cases under the assumption of weak selection. This framework provides theoretical background to analyze the genomic signature of balancing selection in partially selfing species. It also sheds new light on the evolution of selfing species, including the evolution of selfing syndrome, the interaction with pathogens, and the evolutionary fate of selfing lineages.

Genomic characterization of selfing in the cyclic parthenogen Daphnia magna.

Inbreeding refers to the fusion of related individuals' gametes, with self-fertilization (selfing) being an extreme form of inbreeding-involving gametes produced by the same individual. Selfing is expected to reduce heterozygosity by an average of 50% in one generation; however, little is known about the empirical variation on a genome level surrounding this figure and the factors that affect variation. We selfed genotypes of the cyclic parthenogen Daphnia magna and analysed whole genomes of mothers and selfed offspring, observing the predicted 50% heterozygosity reduction on average. We also saw substantial variation around this value and significant differences among mother-offspring pairs. Crossover analysis confirmed the known trend of recombination occurring more often towards the telomeres. This effect was shown, through simulations, to increase the variance of heterozygosity reduction compared to when a uniform distribution of crossovers was used. Similarly, we simulated inbred line production after several generations of selfing and we observed higher variance in achieved homozygosity when we consider a higher recombination rate towards the telomeres. Our empirical and simulation study highlights that the expected mean values of heterozygosity reduction show remarkable variation, which can help understand, for example, differences among inbred individuals.

Flowering phenology in a Eucalyptus loxophleba seed orchard, heritability and genetic correlation with biomass production and cineole: breeding strategy implications.

Reproductive synchronicity within a seed orchard facilitates gene exchange and reduces self-fertilisation. Here we assessed key flowering traits, biomass and foliar 1,8-cineole concentrations of Eucalyptus loxophleba (subsp. lissophloia and gratiae) in an open-pollinated seed orchard. Monthly flowering observations were made on 1142 trees from 60 families and nine provenances across 2 years. The percentage of trees flowering in both years was similar at 87%. There were differences between provenances and families within provenances for flowering traits, biomass and 1,8-cineole and interactions between provenances and year for flowering traits. Heritability of start and end flowering, and 1,8-cineole were high to moderate ([Formula: see text] = 0.75-0.45) and duration of flowering, propensity to flower and biomass estimates were moderate to low ([Formula: see text] = 0.31-0.10). Genetic and phenotypic correlations between flowering traits were high (rg = 0.96-0.63 and rp = 0.93-0.34) except between duration and end of flowering. The correlations were weaker between flowering traits and biomass or 1,8-cineole. 'Dual flowering', when trees underwent two reproductive cycles in a year, was responsible for out-of-phase flowering and those with low biomass and 1,8-cineole concentration should be removed from the breeding programme to hasten selection for desirable traits.

Spatial and temporal expression pattern of sex-related genes in ovo-testis of the self-fertilizing mangrove killifish (Kryptolebias marmoratus).

In vertebrates, sex determination and differentiation comprehend a fine balance between female and male factors, leading the bipotential anlage to develop towards ovary or testis, respectively. Nevertheless, the mangrove killifish, (Kryptolebias marmoratus) a simultaneous hermaphroditic species, could overcome those antagonistic pathways and evolved to develop and maintain reproductively active ovarian and testicular tissues in the same organ. Morphological and mRNA localization analyzes of developing and adult gonads demonstrate that genes related to testis (dmrt1 and amh) and ovary differentiation (foxl2 and sox9a) follow the same expression pattern observed in gonochoristic species, thus functioning as two independent organs. In addition, Amh expression patterns make it a strong candidate for initiation of the formation and maintenance of the testicular tissue in the hermaphroditic gonad. Differently from described so far, foxl3 seems to have an important role in oogenesis as well as spermatogenesis and gonadal structure.

A Similar Genetic Architecture Underlies the Convergent Evolution of the Selfing Syndrome in Capsella.

Whether, and to what extent, phenotypic evolution follows predictable genetic paths remains an important question in evolutionary biology. Convergent evolution of similar characters provides a unique opportunity to address this question. The transition to selfing and the associated changes in flower morphology are among the most prominent examples of repeated evolution in plants. In this study, we take advantage of the independent transitions to self-fertilization in the genus Capsella to compare the similarities between parallel modifications of floral traits and test for genetic and developmental constraints imposed on flower evolution in the context of the selfing syndrome. Capsella rubella and Capsella orientalis emerged independently but evolved almost identical flower characters. Not only is the evolutionary outcome identical but the same developmental strategies underlie the convergent reduction of flower size. This has been associated with convergent evolution of gene expression changes. The transcriptomic changes common to both selfing lineages are enriched in genes with low network connectivity and with organ-specific expression patterns. Comparative genetic mapping also suggests that, at least in the case of petal size evolution, these similarities have a similar genetic basis. Based on these results, we hypothesize that the limited availability of low-pleiotropy paths predetermines closely related species to similar evolutionary outcomes.

Comparative studies on population genetic structure of two closely related selfing and outcrossing Zingiber species in Hainan Island.

How mating system impacts the genetic diversity of plants has long fascinated and puzzled evolutionary biologists. Numerous studies have shown that self-fertilising plants have less genetic diversity at both the population and species levels than outcrossers. However, the phylogenetic relationships between species and correlated ecological traits have not been accounted for in these previous studies. Here, we conduct a comparative population genetic study of two closely related selfing and outcrossing Zingiber species, with sympatric distribution in Hainan Island, and obtain a result contrary to previous studies. The results indicate that selfing Z. corallinum can maintain high genetic diversity through differentiation intensified by local adaptation in populations across the species' range. In contrast, outcrossing Z. nudicarpum preserves high genetic diversity through gene exchange by frequent export of pollen within or among populations. Contrary to expectations, the major portion of genetic variation of outcrossing Z. nudicarpum may exist among populations, depending on the dispersal ability of pollen and seed. Our results also reveal that the main factor affecting population structure of selfing Z. corallinum is mountain ranges, followed by a moist climate, while that of outcrossing Z. nudicarpum is likely moisture, but not mountain ranges, due to gene flow via pollen.

Sibling competition does not magnify inbreeding depression in North American Arabidopsis lyrata.

About half of all angiosperms have some form of molecular self-incompatibility to promote outcrossing. If self-incompatibility breaks down, inbreeding depression (δ) is the main barrier to the evolution of self-fertilisation (selfing). If inbreeding depression is lower than 50% (δ < 0.5), the inherent transmission advantage of selfers should theoretically drive the evolution of selfing. However, this does not always happen in practice. For example, despite frequent breakdowns of self-incompatibility in North American Arabidopsis lyrata, selfing has only evolved in few populations. This is surprising given that previous inbreeding-depression estimates were well below the 0.5 threshold. Here, we test whether this could be due to underestimation of true inbreeding depression in competition-free environments. Specifically, we tested whether direct competition between crossed and selfed siblings magnified inbreeding-depression estimates in A. lyrata. We found that this was neither the case for belowground nor for aboveground biomass. For reproductive traits, there was hardly any significant inbreeding depression regardless of competition. Combined with previous findings that drought stress and inducing defence also did not magnify inbreeding depression, our results suggest that the relatively low estimates of inbreeding depression for biomass are indeed realistic estimates of the true inbreeding depression in North American A. lyrata.

Evolution of sex ratio through gene loss.

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved a mating system in which selfing hermaphrodites and males coexist. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species. Using a transgene to restore mss function to the androdioecious Caenorhabditis briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, when mss+ and mss-null (i.e., wild type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. However, in genetically homogenous androdioecious populations, mss+ both increases male frequency and depresses population growth. We propose that the lack of inbreeding depression and the strong subdivision that characterize natural Caenorhabditis populations impose selection on sex ratio that makes loss of mss adaptive after self-fertility evolves.

Phylogenomic analysis reveals multiple evolutionary origins of selfing from outcrossing in a lineage of heterostylous plants.

Evolutionary transitions from outcrossing to selfing often occur in heterostylous plants. Selfing homostyles originate within distylous populations and frequently evolve to become reproductively isolated species. We investigated this process in 10 species of Primula section Obconicolisteri using phylogenomic approaches and inferred how often homostyly originated from distyly and its consequences for population genetic diversity and floral trait evolution. We estimated phylogenetic relationships and reconstructed character evolution using the whole plastome comprised of 76 protein-coding genes. To investigate mating patterns and genetic diversity we screened 15 microsatellite loci in 40 populations. We compared floral traits among distylous and homostylous populations to determine how phenotypically differentiated homostyles were from their distylous ancestors. Section Obconicolisteri was monophyletic and we estimated multiple independent transitions from distyly to homostyly. High selfing rates characterised homostylous populations and this was associated with reduced genetic diversity. Flower size and pollen production were reduced in homostylous populations, but pollen size was significantly larger in some homostyles than in distylous morphs. Repeated transitions to selfing in section Obconicolisteri are likely to have been fostered by the complex montane environments that species occupy. Unsatisfactory pollinator service is likely to have promoted reproductive assurance in homostyles leading to subsequent population divergence through isolation.

Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude.

Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the Qinghai-Tibet Plateau (QTP) about 4,000 m above sea level and represents an attractive model system for studying speciation and ecological adaptation in extreme environments. We assembled a draft genome sequence of 234.72 Mb encoding 27,019 genes and investigated its origin and adaptive evolutionary mechanisms. Phylogenomic analyses based on 4,586 single-copy genes revealed that C. himalaica is most closely related to Capsella (estimated divergence 8.8 to 12.2 Mya), whereas both species form a sister clade to Arabidopsis thaliana and Arabidopsis lyrata, from which they diverged between 12.7 and 17.2 Mya. LTR retrotransposons in C. himalaica proliferated shortly after the dramatic uplift and climatic change of the Himalayas from the Late Pliocene to Pleistocene. Compared with closely related species, C. himalaica showed significant contraction and pseudogenization in gene families associated with disease resistance and also significant expansion in gene families associated with ubiquitin-mediated proteolysis and DNA repair. We identified hundreds of genes involved in DNA repair, ubiquitin-mediated proteolysis, and reproductive processes with signs of positive selection. Gene families showing dramatic changes in size and genes showing signs of positive selection are likely candidates for C. himalaica's adaptation to intense radiation, low temperature, and pathogen-depauperate environments in the QTP. Loss of function at the S-locus, the reason for the transition to self-fertilization of C. himalaica, might have enabled its QTP occupation. Overall, the genome sequence of C. himalaica provides insights into the mechanisms of plant adaptation to extreme environments.

Postglacial ecotype formation under outcrossing and self-fertilization in Arabidopsis lyrata.

The formation of ecotypes has been invoked as an important driver of postglacial biodiversity, because many species colonized heterogeneous habitats and experienced divergent selection. Ecotype formation has been predominantly studied in outcrossing taxa, while far less attention has been paid to the implications of mating system shifts. Here, we addressed whether substrate-related ecotypes exist in selfing and outcrossing populations of Arabidopsis lyrata subsp. lyrata and whether the genomic footprint differs between mating systems. The North American subspecies colonized both rocky and sandy habitats during postglacial range expansion and shifted the mating system from predominantly outcrossing to predominantly selfing in a number of regions. We performed an association study on pooled whole-genome sequence data of 20 selfing or outcrossing populations, which suggested genes involved in adaptation to substrate. Motivated by enriched gene ontology terms, we compared root growth between plants from the two substrates in a common environment and found that plants originating from sand grew roots faster and produced more side roots, independent of mating system. Furthermore, single nucleotide polymorphisms associated with substrate-related ecotypes were more clustered among selfing populations. Our study provides evidence for substrate-related ecotypes in A. lyrata and divergence in the genomic footprint between mating systems. The latter is the likely result of selfing populations having experienced divergent selection on larger genomic regions due to higher genome-wide linkage disequilibrium.

Does the evolution of self-fertilization rescue populations or increase the risk of extinction?

Background and Aims: As a major evolutionary transition in seed plants, the evolution of plant mating systems has been much debated in evolutionary ecology. Over the last 10 years, well-established patterns of evolution have emerged. On the one hand, experimental studies have shown that self-fertilization is likely to evolve in a few generations (microevolution) as a response to rapid environmental change (e.g. pollinator decline), eventually rescuing a population. On the other, phylogenetic studies have demonstrated that repeated evolution towards self-fertilization (macroevolution) leads to a higher risk of lineage extinction and is thus likely to be disadvantageous in the long term. Scope: In either case - the short-term or long-term evolution of self-fertilization (selfing) - these findings indicate that a mating system is not neutral with respect to population or lineage persistence. They also suggest that selfing can have contrasting effects depending on time scale. This raises the question of whether mating system evolution can rescue populations facing environmental change. In this review, empirical and theoretical evidence of the direct and indirect effects of mating systems on population demography and lineage persistence were analysed. A simple theoretical evolutionary rescue model was also developed to investigate the potential for evolutionary rescue through selfing. Key Findings: Demographic studies consistently show a short-term advantage of selfing provided by reproductive assurance, but a long-term disadvantage for selfing lineages, suggesting indirect genomic consequences of selfing (e.g. mutation load and lower adaptability). However, our theoretical evolutionary rescue model found that even in the short term, while mating system evolution can lead to evolutionary rescue, it can also lead to evolutionary suicide, due to the inherent frequency-dependent selection of mating system traits. Conclusions: These findings point to the importance of analysing the demographic consequences of self-fertilization in order to predict the effect of selfing on population persistence as well as take into account the indirect genomic consequences of selfing. The pace at which processes such as inbreeding depression, purging, reproductive assurance and genomic rearrangements occur after the selfing transition is the key to clarifying whether or not selfing will result in evolutionary rescue.

Phylotranscriptomics of Pristionchus Nematodes Reveals Parallel Gene Loss in Six Hermaphroditic Lineages.

Mutation and recombination are main drivers of phenotypic diversity, but the ability to create new allelic combinations is strongly dependent on the mode of reproduction. While most animals are dioecious (i.e., separated male and female sexes), in a number of evolutionary lineages females have gained the ability to self-fertilize [1, 2], with drastic consequences on effective recombination rate, genetic diversity, and the efficacy of selection [3]. In the genus Caenorhabditis, such hermaphroditic or androdioecious lineages, including C. briggsae and C. tropicalis, display a genome shrinkage relative to their dioecious sister species C. nigoni and C. brenneri, respectively [4, 5]. However, common consequences of reproductive modes on nematode genomes remain unknown, because most taxa contain single or few androdioecious species. One exception is the genus Pristionchus, with seven androdioecious species. Pristionchus worms are found in association with scarab beetles in worldwide samplings, resulting in deep taxon sampling and currently 39 culturable and available species. Here, we use phylotranscriptomics of all 39 Pristionchus species to provide a robust phylogeny based on an alignment of more than 2,000 orthologous clusters, which indicates that the seven androdioecious species represent six independent lineages. We show that gene loss is more prevalent in all hermaphroditic lineages than in dioecious relatives and that the majority of lost genes evolved recently in the Pristionchus genus. Further, we provide evidence that genes with male-biased expression are preferentially lost in hermaphroditic lineages. This supports a contribution of adaptive gene loss to shaping nematode genomes following the evolution of hermaphroditism.

Generation of self-compatible diploid potato by knockout of S-RNase.

Re-domestication of potato into an inbred line-based diploid crop propagated by seed represents a promising alternative to traditional clonal propagation of tetraploid potato, but self-incompatibility has hindered the development of inbred lines. To address this problem, we created self-compatible diploid potatoes by knocking out the self-incompatibility gene S-RNase using the CRISPR-Cas9 system. This strategy opens new avenues for diploid potato breeding and will also be useful for studying other self-incompatible crops.