Julia Harenčár,Oscar M Vargas,Merly Escalona,Douglas W Schemske,Kathleen M Kay
Genome assemblies and comparison of two Neotropical spiral gingers: Costus pulverulentus and C. lasius
genome resources,herbaceous,monocot,synteny,tropical plant
Abstract The spiral gingers (Costus L.) are a pantropical genus of herbaceous perennial monocots; the Neotropical clade of Costus radiated rapidly in the past few million years into over 60 species. The Neotropical spiral gingers have a rich history of evolutionary and ecological research that can motivate and inform modern genetic investigations. Here, we present the first 2 chromosome-level genome assemblies in the genus, for C. pulverulentus and C. lasius, and briefly compare their synteny. We assembled the C. pulverulentus genome from a combination of short-read data, Chicago and Dovetail Hi-C chromatin-proximity sequencing, and alignment with a linkage map. We annotated the genome by mapping a C. pulverulentus transcriptome and querying mapped transcripts against a protein database. We assembled the C. lasius genome with Pacific Biosciences HiFi long reads and alignment to the C. pulverulentus genome. These 2 assemblies are the first published genomes for non-cultivated tropical plants. These genomes solidify the spiral gingers as a model system and will facilitate research on the poorly understood genetic basis of tropical plant diversification.
Genome assemblies and comparison of two Neotropical spiral gingers: Costus pulverulentus and C. lasius

The spiral gingers (Costus L.) are a pantropical genus of herbaceous perennial monocots; the Neotropical clade of Costus radiated rapidly in the past few million years into over 60 species. The Neotropical spiral gingers have a rich history of evolutionary and ecological research that can motivate and inform modern genetic investigations. Here, we present the first 2 chromosome-level genome assemblies in the genus, for C. pulverulentus and C. lasius, and briefly compare their synteny. We assembled the C. pulverulentus genome from a combination of short-read data, Chicago and Dovetail Hi-C chromatin-proximity sequencing, and alignment with a linkage map. We annotated the genome by mapping a C. pulverulentus transcriptome and querying mapped transcripts against a protein database. We assembled the C. lasius genome with Pacific Biosciences HiFi long reads and alignment to the C. pulverulentus genome. These 2 assemblies are the first published genomes for non-cultivated tropical plants. These genomes solidify the spiral gingers as a model system and will facilitate research on the poorly understood genetic basis of tropical plant diversification.

The tropics encompass the highest plant species diversity (Mutke and Barthlott 2005; Cai et al. 2023), yet there are relatively few tropical plant reference genomes. Whereas high-quality genomic resources exist for some cultivated tropical plants (e.g. Argout et al. 2011—chocolate; Droc et al. 2013—banana; Dereeper et al. 2015—coffee), there are no published high-quality genomes for wild tropical plants. Without reference genomes and broader investigation of tropical plant genetics, we cannot hope to understand the ecology and evolution of tropical plant diversity. The Neotropical spiral gingers (genus Costus L.) are emerging as a model system for tropical plant evolutionary biology. Costus is a pantropical genus of perennial understory herbaceous monocots that contains over 80 species (Maas 1972, 1977). The majority (over 60) of these species diverged rapidly and recently (~3 mya) in Central and South America (Vargas et al. 2020). Despite their recent divergence, Neotropical spiral ginger species vary widely in reproductive traits, habitat affinities, and species interactions. Their variable ecology and rapid diversification make spiral gingers an excellent system for studying both speciation and the evolution of morphological, biochemical, and ecological traits. Evolutionary investigation of the genus dates back to monographic work (Maas 1972, 1977) and early research on Costus species interactions and reproductive biology (Schemske 1978, 1980, 1981, 1982, 1983; Schemske and Pautler 1984) and has continued since, building a rich foundational understanding of mechanisms of speciation, species interactions, local adaptation, floral evolution, and macroevolutionary patterns of diversification (e.g. Kay 2006; Bartlett and Specht 2010; Chen and Schemske 2015, 2019; Vargas et al. 2020; Ávila-Lovera et al. 2022). Costus provides one of the earliest empirical examples of reinforcement, a process which acts to increase reproductive isolation between 2 species occurring in sympatry (Kay and Schemske 2008; Yost and Kay 2009; Surget-Groba and Kay 2013). Costus species have specialized pollination interactions with orchid bees and hummingbirds, provide extrafloral nectar rewards to ant defenders, host specialized flower mites and leaf herbivores, and exhibit novel ecophysiological strategies (e.g. Schemske 1980; Kay and Schemske 2003; Kay et al. 2005; García-Robledo et al. 2013; Bizzarri et al. 2022; Harenčár et al. 2022; Kay and Grossenbacher 2022). This richness of ecological and evolutionary research provides an excellent foundation for genomics. Spiral ginger research also benefits from their unusual tractability compared with other tropical plants. They are generally well described and identifiable by vegetative characteristics, can be maintained in greenhouses, and can be grown from seed or cuttings for use in experiments such as reciprocal transplants. Costus species are also tractable for genetic investigation as they are generally diploid with stable ploidy (Maas 1972, 1977), have relatively small genomes (~1 Gb), have publicly available transcriptome data for 5 species (NCBI Acc. PRJNA600282), and have a well-resolved phylogeny estimated from 853 genes (Vargas et al. 2020). Further, Costus species are widely interfertile (Kay and Schemske 2008), enabling propagation of hybrids for genetic mapping and focused investigation on the evolutionary role of extrinsic barriers to gene flow. Here, we improve Neotropical Costus as a model system with 2 reference genomes spanning the deepest node in the phylogeny. We generated chromosome-level genome assemblies for C. pulverulentus C. Presl and C. lasius Loes. (Fig. 1). We first assembled a C. pulverulentus genome using a hybrid de novo assembly approach combining short-read shotgun and mate-pair libraries with Hi-C chromatin-proximity and Chicago libraries. We then used a RAD-seq genetic linkage map to pair half-chromosome scaffolds and generate a chromosome-level assembly. We assembled the C. lasius genome from PacBio HiFi long-read sequencing data and aligned it with the C. pulverulentus genome to assemble chromosome-scale scaffolds. We also estimated both genome sizes with flow cytometry. These genomes, combined with the other genomic resources available for the genus, allow us to leverage existing and ongoing field research to build a nuanced understanding of the almost entirely unexplored genomics of Neotropical plant diversification.

The C. pulverulentus individual selected for sequencing was grown at the University of California, Santa Cruz (UCSC) from field-collected seed. The seed was collected by DWS on Barro Colorado Island in Panama (9.155, −79.85). A voucher is deposited at the Michigan State University (MSU) Herbarium (Kay 0328 (MSC)). We collected stem meristem and nascent leaf tissue from the live greenhouse individual.

The C. lasius individual selected for sequencing was grown in the UCSC greenhouses from a field-collected cutting. The cutting was collected by KMK from a wild individual growing in El Valle de Antón, Panama (8.633, −80.117). A voucher is deposited at the MSU Herbarium (Kay 0321 (MSC)). We collected stem meristem, nascent leaf, and nascent bud tissue from the greenhouse individual.

We extracted high molecular weight (HMW) DNA from fresh tissue ground in a bead beater with PVP buffer. We used a modified Qiagen Puregene Gentra kit protocol (S1) for HMW DNA extraction. We assessed DNA concentration with a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA) and ran an agarose gel to assess DNA fragment integrity and size. An Illumina DNA TruSeq library was created for regular whole-genome shotgun sequencing. Mate-pair libraries were created with 6 kbp insert sizes using an Illumina Nextera mate-pair library kit. Additional flash-frozen leaf tissue was sent to Dovetail Genomics (Scotts Valley, CA) for Hi-C and Chicago library preparation and sequencing.

We extracted HMW DNA from 1 g of fresh mixed leaf and bud tissue. We isolated cell nuclei following Workman (2018) and then used the Circulomics Nanobind Plant Nuclei Big DNA kit to extract HMW DNA. We assessed DNA concentration with a Qubit Fluorometer (Thermo Fisher Scientific), purity with 260/280 and 260/230 absorbance ratios from a NanoDrop Spectrophotometer (Thermo Fisher Scientific), and ran a 7% agarose gel at 70 V for over 8 h to assess DNA fragment integrity and size.

We sequenced the whole-genome shotgun libraries on a single lane of an Illumina HiSeq 4000 in paired-end 150 bp mode. We sequenced the 6 kb mate-pair libraries on an Illumina NextSeq 500 in paired-end 150 bp mode. Dovetail Genomics (Scotts Valley, CA) conducted Hi-C and Chicago sequencing. We conducted initial assembly of the shotgun and 6 kb mate-pair sequencing data with Meraculous-2D (Goltsman et al. 2017). Genome scaffolding was conducted by Dovetail Genomics using their HiRise pipeline (Putnam et al. 2016), combining the initial assembly from shotgun and mate-pair sequencing with Hi-C, and CHiCago library data. This produced 13 large scaffolds representing presumptive full and half-chromosomes. To assemble the 13 largest scaffolds into 9 chromosomes, we mapped RAD-seq markers (Kay and Surget-Groba 2022) against the draft genome using BBmap (Bushnell 2014) and visualized the results with R. We based chromosome naming on the previously published chromosome names (Kay and Surget-Groba 2022), which were based on chromosome centimorgan length from a linkage map. To create a draft annotation for the C. pulverulentus genome, we mapped a transcriptome of C. pulverulentus (GenBank accession SRX7544604, Vargas et al. 2020) to the draft genome using PASApipeline (Haas et al. 2003). To search for potential gene functions in our draft annotation, we queried our mapped transcripts against a protein database (GO; Harris et al. 2004) using EnTAP (Hart et al. 2020). Matching protein names and functions were added as annotation notes in the .gff3 annotation file generated with PASA using a custom python script (available in Dryad repository; see “Data availability”). We then imported annotation files to Geneious Prime 2022.2.1 for visualization and, because annotations were added prior to the assembly of large scaffolds into chromosomes, to maintain annotations during final assembly. All assembly software versions and parameters are listed in Table 1. We conducted additional trimming and removal of mitochondrial sequences per NCBI recommendations upon submission.

We sent HMW DNA to the Vincent J. Coates Genomics Sequencing Lab (Berkeley, CA) for Pacific Biosciences HiFi library preparation and circular consensus sequencing (CCS) on 2 PacBio Sequel II HiFi SMRTcells. We assembled the PacBio CCS data into preliminary primary and alternate assemblies with HiFiasm (Cheng et al. 2021). To improve the assemblies, we used purge_dups (Guan et al. 2020) to remove duplicated haplotigs and sequence overlaps from the primary assembly and add them to the alternate assembly. Finally, RagTag (Alonge et al. 2019) was used to align the primary C. lasius assembly to the C. pulverulentus assembly and assign corresponding chromosome names. The assembly software versions and parameters are listed in Table 1. We conducted additional trimming and removal of mitochondrial sequences per NCBI recommendations upon submission.

For size estimation, we sent fresh leaf tissue from the sequenced individuals of both species (and 5 other species; S3) to the Flow Cytometry Lab at the Benaroya Research Institute, Seattle, WA. Four replicates were run for each sample. We also calculated the total genome assembly lengths with QUAST (Gurevich et al. 2013). We also used QUAST to generate contiguity metrics and base pair content. To further evaluate genome completeness and quality, we ran BUSCO (Manni et al. 2021) with the Embryophyta database (embryophyta_odb10; 1,614 genes). To assess base call accuracy (QV) and k-mer completeness, we first generated meryl databases with the shotgun and 6 kb mate-pair sequence data for C. pulverulentus and with the PacBio HiFi reads for C. lasius. We then ran merqury (Rhie et al. 2020) with the meryl databases and genome assemblies to obtain the QV and k-mer completeness estimates. To determine the number of gaps we first used det_gaps, a submodule from asset (https://github.com/dfguan/asset) that describes the existing gaps in the assemblies. Finally, to understand genome size discrepancies and determine synteny we compared the C. pulverulentus and C. lasius assemblies by generating a dotplot with D-GENIES (Cabanettes and Klopp 2018) using Minimap2 (version 2.24; Li 2018) as the aligner and the “some repeats” option.

The combined shotgun sequencing runs yielded 615 million PE 150 reads, and the 6 kb mate-pair sequencing yielded 136 million reads. The genome coverage reported by Dovetail Genomics after HiRise Assembly was ~1,959-fold. The final C. pulverulentus genome assembly size (~736 Mbp) is about 300 Mbp smaller than the average flow cytometry estimate (1.036 Gbp). The 9 largest contigs correspond to chromosomes following successful matching of chromosome arms with the linkage map S2. The assembly is composed of 23,271 contigs with an N50 of 63.17 Mbp and the longest contig is 97.28 Mbp. Complete assembly statistics are reported in Table 2. For the draft annotation, ~93% of our C. pulverulentus transcripts (258,934 out of 279,542) mapped to our genome, and about 14% of the mapped transcripts (37,512) matched an entry in the protein database. The transcriptome of C. pulverulentus is highly repetitive, representing different protein configurations from single genes. For example, gene 1 (see the .gff annotation files in the Dryad repository listed in “Data availability”), can produce 5 different proteins depending on the inclusion/exclusion of exons.

The combined PacBio sequencing runs yielded 6.3 million HiFi reads representing ~19-fold coverage based on flow cytometry estimated genome size (HiFi read data averaged across both runs: N50 read length 166,084 bp; mean read length 144,929 bp). The final C. lasius genome assembly size (1.28 Gbp) is similar to the average flow cytometry estimate (1.27 Gbp). The 9 largest contigs correspond to homologous chromosomes in the C. pulverulentus assembly. The assembly is composed of 278 contigs with an N50 of 119.75 Mb and the longest contig is 163.08 Mb. Complete assembly statistics are reported in Table 2.

Comparison of the C. pulverulentus and C. lasius genomes shows that the genomes are largely syntetic (Fig. 2). There is one potential transposition between chromosome 7 in the C. pulverulentus genome and chromosome 5 in the C. lasius genome. There are also several small potential inversions on chromosomes 3, 8, and 9. However, both the potential transposition and inversions need further validation. The C. lasius assembly tends to be longer at chromosome ends and centromeric regions. Flow cytometry results from a total of 6 species and a hybrid support previous chromosome count data showing that Costus are largely diploid (S3; Maas 1972, 1977). Genome sizes ranged from 924 to 1,376 Mbp; the 2 species sequenced here represent relatively small and large genomes at 1,036 Mbp (C. pulverulentus) and 1,268 Mpb (C. lasius).

The 2 genome assemblies described here are the first chromosome-level genomes for the Costus genus. The C. pulverulentus genome has already facilitated the generation of a well-supported phylogeny (Vargas et al. 2020) and genetic mapping of evolutionarily important floral traits (Kay and Surget-Groba 2022). Both genomes are being used in ongoing investigation of tropical plant evolution, including research on the population genetics of hybridizing species, the genetic basis of habitat isolation, and the extent of introgression across the clade. Comparison of the C. lasius and C. pulverulentus genomes clearly demonstrates the advantage of long-read data for genome assembly. The C. pulverulentus genome assembly is shorter than the flow cytometry estimate of its size (assembly = 736 Mbp, flow cytometry = 1,036 Mp), potentially due to collapsed assemblies in repetitive regions with short-read data. Many of the contigs from the C. pulverulentus genome that were not placed on chromosomes aligned to what are likely centromeric and chromosome end regions in C. lasius, which tend to be highly repetitive (Blackburn and Szostak 1984; Lamb et al. 2007). In contrast, long-read data improved the assembly of these regions in C. lasius. The synteny plot also hints at why the C. lasius genome is longer than the C. pulverulentus genome; the majority of breaks in the synteny plot showing where C. lasius is longer are also in centromeric and chromosome end regions. This suggests that the larger size of the C. lasius genome is due to increases in repetitive regions. The abundance of repetitive DNA is both highly variable even between closely related plants and strongly correlated with genome size (Hancock 2002; Lee and Kim 2014). Differences in repetitive regions, which mostly comprise transposable elements (TEs), can have implications for gene expression and species divergence (Heslop-Harrison 2000; Feschotte and Pritham 2007; Jurka et al. 2007; Heslop-Harrison and Schmidt 2012; Kim et al. 2012; Fedoroff 2013; Lee and Kim 2014). Comparison of the 2 genomes indicates the advantage of long-read data for understanding repetitive regions, enabling investigation of the role of TEs in species distinction. Genome comparison also reveals that C. lasius and C. pulverulentus genomes are largely conserved structurally. C. lasius and C. pulverulentus diverged ~3 mya (Vargas et al. 2020), yet display only a few relatively small potential rearrangements, remaining predominantly syntenic. Some organisms with similar divergence times show similarly conserved genomes. For example, the American bison (Bison bison bison) and cattle (Bos taurus) diverged ~3.5 mya (Kumar et al. 2017) and have largely conserved chromosomes with no large inversions or transpositions, just insertions and deletions (Oppenheimer et al. 2021). Drosophila melanogaster and D. simulans diverged ~4 mya (Kumar et al. 2017) and, while their karyotypes are also conserved, they demonstrate more structural differences (especially inversions) which could partially be due to longer divergence and shorter generation time than our focal Costus species (Ranz et al. 2007). Shorter generation time may also partially explain greater structural divergence between more closely related organisms, such as an inversion between ecotypes of Mimulus guttatus and multiple inversions between Helianthus annuus and H. argophyllus (diverged ~1.5 mya; Wellenreuther and Bernatchez 2018). Continued sequencing and comparison of genomes like that presented here will enable evaluation of how and when genomes are more versus less structurally conserved. Several genome assemblies exist for economically important tropical plants, but, unlike Costus, these genomes are not well positioned for studying tropical plant diversification. We were only able to find published tropical plant genomes for cultivated species (e.g. Prochnik et al. 2012—cassava; Ming et al. 2012—papaya; Guignon et al. 2016—various). In contrast to these cultivated species, Costus is emerging as a model system for investigating the evolution and ecology of tropical plant diversification. These 2 genome assemblies add to growing genomic resources for the genus that include transcriptome data, floral QTL, and a well-resolved phylogeny (Vargas et al. 2020; Kay and Surget-Groba 2022). Further, genomic resources will enable researchers to leverage a rich foundation of natural history, ecological, and evolutionary research in Neotropical Costus to understand the genetic basis of coexistence and speciation.

Supplementary material is available at Journal of Heredity online. S1. Kay HMW extraction protocol. S2. Linkage map generated with RAD-seq data from a C. pulverulentus × C. scaber hybrid plotted against the C. pulverulentus assembly sequence. S3. Flow cytometry results. S4. D-GENIES synteny dotplot interactive offline viewer (https://jharencar.github.io/Costus-interactive-synteny-plot/Clasius_X_Cpulverulentus_interactive_synteny_plot.html). Click here for additional data file. Click here for additional data file. Click here for additional data file.