Eulimnadia texana is no ordinary crustacean. It belongs to a rare androdioecious breeding system, meaning populations consist of males and selfing hermaphrodites but no pure females. This arrangement, found in only a handful of animal and plant species, produces three mating types built from two proto-sex chromosomes: Z and W. Males are homozygous ZZ, while hermaphrodites are either ZW or, remarkably, WW. The WW hermaphrodite genome was already sequenced and assembled in a previous study, revealing a genome of approximately 120 million bases with 108 scaffolds and roughly 30 percent repetitive elements. The current study augments that resource by reporting the full sequence of the ZZ male genome, followed by de novo assembly and annotation to explore DNA composition in ZZ males.

The sequencing effort began with a single male clam shrimp derived from a laboratory line originally sampled from Jornada, New Mexico. Researchers extracted high molecular weight genomic DNA using a modified Qiagen Magattract protocol, then prepared an ultra-low input library targeting an average insert size of approximately 10 kilobases. The library was sequenced on one SMRT cell 8M on a PacBio Sequel II using Circular Consensus Sequencing mode with a 30-hour movie time. The result was 1,945,182 HiFi reads totaling over 17 gigabases at 99.9 percent accuracy, with a mean read length of 9 kilobases and a maximum of 32 kilobases. Quality assessment confirmed no remaining adapters or overrepresented sequences.

Before assembly, researchers estimated genome characteristics from the raw reads using k-mer profiling. GenomeScope 2.0 initially estimated a genome size of 103 million bases with 88.4 percent unique sequence, 97.7 percent homozygosity, and 76.5-fold coverage. However, the k-mer profile showed three peaks rather than the expected two, suggesting contamination. After contamination filtering with BlobToolKit and re-running GenomeScope on cleaned reads, the estimated genome size dropped to 87 million bases with 88.2 percent unique sequence, 97.9 percent homozygosity, and approximately 79-fold coverage, and the k-mer distribution reverted to the expected two-peak pattern. The researchers interpret these values as lower-bound approximations, noting that k-mer modeling of this highly homozygous, repeat-rich genome can underestimate assembly span.

The de novo assembly proceeded through the Vertebrate Genome Project pipeline on the Galaxy platform using HiFiasm for contig-level assembly and duplicate purging. Contigs were screened for contamination with BlobToolKit, which combines sequence similarity, GC content, and per-contig coverage to identify non-target taxa. Contigs with dominant Arthropoda assignments and GC content between 0.30 and 0.45 were retained as clean E. texana sequence. After remapping clean contigs to isolate E. texana HiFi reads and reassembling with the VGP pipeline, the final de novo ZZ assembly comprised 97 contigs totaling approximately 113 million bases with a contig N50 of 3.5 megabases, a maximum contig length of 10 megabases, and GC content of 38.77 percent. BUSCO analysis reported 98.1 percent complete arthropod orthologs, indicating a highly complete, contamination-filtered assembly.

To achieve chromosome-scale contiguity, researchers scaffolded the ZZ contigs onto the curated WW hermaphrodite reference genome. The published WW assembly was first re-evaluated with BlobToolKit using the same contamination-filtering criteria, reducing it from 120 million bases in 108 scaffolds to approximately 113 million bases in 42 scaffolds with an N50 of 10 megabases. Gaps in the curated reference were then filled with ZZ HiFi reads using TGS-GapCloser, and RagTag was used to align and orient ZZ contigs onto the gap-filled reference. RagTag placed 49 of 97 ZZ contigs onto the 11 largest WW scaffolds, with 17 contigs mapping to the putative sex chromosome scaffold C0001, which spans approximately 43 megabases in both assemblies. The remaining 48 contigs, predominantly short and representing only 1.1 percent of the assembly, were placed in an unlocalized scaffold. The scaffolded ZZ genome comprised 12 scaffolds with a total length of approximately 113 million bases, only 0.18 percent gap sequence, and a scaffold N50 of 18 megabases.

Whole-genome alignments between the ZZ and WW assemblies revealed broad collinearity across most autosomal scaffolds, but the putative sex chromosome scaffold stood out. At the 90 percent identity threshold, corresponding ZZ scaffolds covered 92.67 percent of WW scaffold C0002, 99.12 percent of C0003, and 98.14 percent of C0005. In contrast, the putative sex chromosome scaffold C0001 was covered by only 68.43 percent at 90 percent identity and 82.83 percent at 80 percent identity. Among other large scaffolds, coverage at 90 percent identity ranged from 23.18 percent for C0007 to 49.38 percent for C0004. These results indicate that most structural divergence between the ZZ male and WW hermaphrodite genomes is concentrated on the sex chromosome scaffold and several smaller scaffolds, while autosomal regions remain almost completely covered by high-identity ZZ sequence.

Repeat analysis of the ZZ male genome reported that 34.27 megabases, or 30.4 percent of the assembly, consisted of regions annotated as repetitive. The majority of these repeats belong to interspersed elements spanning 29.89 percent of the genome, including rolling-circle elements at 0.1 percent, long-terminal repeat retrotransposons at 1.44 percent, long interspersed nuclear elements at 1.49 percent, DNA transposons at 1.97 percent, and a large fraction of unclassified repeats at 24.99 percent. The researchers caution that these estimates represent a broad first-pass summary based on de novo repeat family discovery and standard masking, rather than a fully resolved transposable element landscape, and that detailed curation of E. texana-specific repeat families is in preparation.

Genome annotation with the MAKER pipeline, incorporating de novo transcript assemblies from adult males and protein homology evidence from the related branchiopod Daphnia magna, identified 21,165 genes in the scaffold-level male genome with an average gene length of 3,842 bases. The cumulative gene span covered 72 percent of the genome, with 27 percent occupied by 128,726 exons and 45 percent by 107,561 introns. The annotation achieved a complete BUSCO score of 95.1 percent, with 87.2 percent single-copy and 7.9 percent duplicated arthropod orthologs. The researchers consider this gene set a high-coverage first-pass annotation that likely includes a modest number of redundant or split models, particularly in repeat-rich regions, and expect future manual curation to refine total gene counts.

The ZZ and WW genomes show broadly similar gene numbers both genome-wide and on the proto-sex chromosomes themselves. Building on previous work demarcating the Z chromosome, the researchers estimate that the W and Z chromosomes contain 7,969 and 8,792 genes respectively, indicating that these proto-sex chromosomes have diverged in structure and repeat content but have not yet undergone extensive gene loss. This finding is consistent with the hypothesis that E. texana represents an early evolutionary stage of sex chromosome differentiation, where the Z and W chromosomes remain morphologically similar and retain comparable functional gene content. The researchers are currently undertaking follow-up analyses to identify genes specific to or enriched on the Z and W chromosomes and to investigate their potential roles in sex determination and sexual differentiation. Together with the WW hermaphrodite genome assembly, this ZZ genome provides a robust framework for mapping genomic and transcriptomic diversity between the Z and W sex chromosomes, advancing research on clam shrimp biology and sex chromosome evolution more generally.

Subject of Research: High-quality annotated ZZ male genome assembly of the clam shrimp Eulimnadia texana

Article Title: High‐Quality Annotated ZZ Male Genome Assembly of Eulimnadia texana

Article References: Ediriweera, C., & Weeks, S. C. (2026). High‐Quality Annotated ZZ Male Genome Assembly of Eulimnadia texana. Ecology and Evolution, 16(10), Article e74547. https://doi.org/10.1002/ece3.74547

Image Credits: AI Generated

DOI: 10.1002/ece3.74547

Keywords: Eulimnadia texana, clam shrimp, genome assembly, sex chromosomes, ZZ male, PacBio HiFi, androdioecy, BUSCO, genome annotation, transposable elements, proto-sex chromosomes, crustacean genomics