DOI: 10.5061/dryad.b5mkkwhmw
title: Data from: Mating system variation and gene expression in the male reproductive tract of Peromyscus mice

description: Genes involved in reproduction often evolve rapidly at the sequence level
 due to postcopulatory sexual selection (PCSS) driven by male-male
 competition and male-female sexual conflict, but the impact of PCSS on
 gene expression has been under-explored. Further, though multiple tissues
 contribute to male reproductive success, most studies have focused on the
 testes. To explore the influence of mating system variation on
 reproductive tract gene expression in natural populations, we captured
 adult males from monogamous Peromyscus californicus and polygynandrous P.
 boylii and P. maniculatus. We generated RNAseq libraries, quantified gene
 expression in the testis, seminal vesicle, epididymis, and liver, and
 identified 3,627 mating system-associated differentially expressed genes
 (MS-DEGs), where expression shifted in the same direction in P.
 maniculatus and P. boylii relative to P. californicus. Gene expression
 variation was most strongly associated with mating behavior in the seminal
 vesicles, where 89% of differentially expressed genes were MS-DEGs,
 including key seminal fluid proteins Svs2 and Pate4. We also used
 published rodent genomes to test for positive and relaxed selection on
 Peromyscus-expressed genes. Though we did not observe more overlap than
 expected by chance between MS-DEGs and positively selected genes, 203
 MS-DEGs showed evidence of positive selection. Fourteen reproductive genes
 were under tree-wide positive selection but convergent relaxed selection
 in P. californicus and Microtus ochrogaster, a distantly related
 monogamous species. Changes in transcript abundance and gene sequence
 evolution in association with mating behavior suggest that male mice may
 respond to sexual selection intensity by altering aspects of sperm
 motility, sperm-egg binding, and copulatory plug formation.
description: This dataset includes three de novo assembled transcriptomes for
 Peromyscus maniculatus, Peromyscus boylii, and Peromyscus
 californicus. Gene expression data was generated from
 tissues sampled from wild-caught mice from Hastings Natural History
 Reserve (Carmel Valley, CA) and the Field Station for the Study of
 Behavior, Ecology, and Reproduction (Berkeley, CA). RNAseq data was
 generated from the testis, liver, epididymis, and seminal vesicle using
 the KAPA HyperPrep RNAseq library preparation kit and sequenced on an
 Illumina NovaSeq S4 at Vincent J. Coates Genome Sequencing Laboratory at
 UC Berkeley. Specimens were accessioned into the mammal collection at the
 Museum of Vertebrate Zoology and sample metadata is available on the
 Arctos museum database.  Transcriptomes were assembled
 starting from filtered, cleaned RNAseq data using the Trinity assembler.
 Transcripts with more than 95% similarity were collapsed and chimeric and
 mis-assembled sequences were removed before transcripts were annotated
 using Trinotate and the Peromyscus maniculatus reference genome (Lassance
 and Hoestra, NCBI GenBank: HU_Pman_2.1.3). Completeness was assessed with
 BUSCO (Euarchontoglires odb10 score: 87.8%). 
 Transcriptomes were used to quantify transcript abundance across
 tissues and species in downstream differential expression
 analyses. 
description: # Data from: Mating system variation and gene expression in the male
 reproductive tract of Peromyscus mice
 [https://doi.org/10.5061/dryad.b5mkkwhmw](https://doi.org/10.5061/dryad.b5mkkwhmw) ## Description of the data and file structure This dataset includes three de novo assembled transcriptomes for *Peromyscus maniculatus* (species abbreviation: PEMA) , *Peromyscus boylii* (PEBO), and *Peromyscus californicus* (PECA).  \**File names: **  *Peromyscus boylii*: P_boylii_transcriptome_Voss_Nachman_2024.fa *Peromyscus californicus*: P_californicus_transcriptome_Voss_Nachman_2024.fa *Peromyscus maniculatus*: P_maniculatus_transcriptome_Voss_Nachman_2024.fa Corrected_Supp_Table1_Voss_Nachman_2024.xlsx: list of samples included in study along with their Museum of Vertebrate Zoology (MVZ) catalog IDs, which can be searched for metadata and tissue loan and specimen availability on the [MVZ Arctos Museum Database](https://arctos.database.museum/search.cfm?guid_prefix=MVZ:Mamm).  **Experiment and file overview:**  This dataset contains transcriptomes that were individually assembled from RNAseq data for three species of *Peromyscus* mice. The goal of this study was to compare reproductive gene expression among male mice from species that vary in mating behavior and strength of postcopulatory sexual selection. We focused on four tissues: testis, seminal vesicle, epididymis, and liver. For each species, RNAseq gene expression data from one sample for each tissue was used to identify transcripts and assemble the resulting transcriptome. Each file is in fasta format and contains the sequences of all transcripts that were annotated to a known open reading frame from the *Peromyscus maniculatus* reference genome (Lassance and Hoekstra 2020). After transcriptome assembly, we used transcriptomes as a reference point from which to begin differential gene expression analyses. Raw read data for all samples and tissues where gene expression was quantified is available on NCBI under BioProject PRJNA1068126.  There is also an excel file that contains sampling localities, species identification, and museum catalog accession numbers. All specimens are being accessioned into the Museum of Vertebrate Zoology collection at the University of California, Berkeley.  *Peromyscus maniculatus*: polygynandrous (loosely affiliative multimale - multifemale) *Peromyscus boylii*: polygynandrous *Peromyscus californicus*: monogamous Study questions: Are different patterns of gene expression associated with different mating systems? Which male reproductive tissues show the greatest changes in gene expression in different mating systems? Which specific genes are expressed at very high levels in polygynandrous species and at low levels in monogamous species? **Specimen collection information**  Gene expression data was generated from tissues sampled from wild-caught mice from Hastings Natural History Reserve (Carmel Valley, CA) and the Field Station for the Study of Behavior, Ecology, and Reproduction (Berkeley, CA). RNAseq data was generated from the testis, liver, epididymis, and seminal vesicle using the KAPA HyperPrep RNAseq library preparation kit and sequenced on an Illumina NovaSeq S4 at Vincent J. Coates Genome Sequencing Laboratory at UC Berkeley. Specimens were accessioned into the mammal collection at the Museum of Vertebrate Zoology and sample metadata is available on the Arctos museum database.  **Transcriptome Assembly Methods** Transcriptomes were assembled starting from filtered, cleaned RNAseq data using the Trinity assembler (Haas et al., 2013). Transcripts with more than 95% similarity were collapsed (CD-Hit: Li and Godzik 2006; Fu et al., 2012) and chimeric and mis-assembled sequences were removed (RNAquast: Bushmanova et al., 2016) before transcripts were annotated using Trinotate (Bryant et al., 2017). Completeness was assessed with BUSCO (Euarchontoglires odb10 score: 87.8%; Simão et al., 2015). Transcriptomes were used to quantify transcript abundance across tissues and species in downstream differential expression analyses.  All software versions used in this pipeline are given below.  **Transcript Naming Conventions**  Transcripts are named according to a combination of annotated gene identifiers and Trinity-generated sequence IDs. The sequence ID begins with the Ensembl *P. maniculatus* gene code, followed by "_i#" to differentiate between unique isoforms of each annotated gene.  Trinity de novo assembler identifiers are preserved second in the sequence ID to retain the initial transcriptome assembly file structure, which includes more complete gene and isoform information for each species. Lastly, additional annotations from the *P. californicus* and *M. musculus* reference CDS are appended. These genomes were used to to provide support for and assess the accuracy level of *P. maniculatus* annotations. All reference genome identifiers are listed below.  **Differential Gene Expression Analyses** After transcriptomes were assembled, they were used as reference sequences to quantify gene expression from five individual per species for each of four tissues: testis, liver, epididymis, and seminal vesicle. We then used Salmon (v.1.10.0, Patro et al., 2017) to map cleaned fastq RNAseq reads to the reference transcriptome and DESeq2 (v.1.40.1, Love et al., 2014) to identify instances of differential expression between species for each tissue. Significantly differentially expressed genes that shifted in the same direction in both polygynandrous species relative to the monogamous species with reproductive gene ontology terms are provided in the supplementary materials for the manuscript, and complete differential expression results are available on request. All raw RNAseq data (n = 58 samples: 15 testis, 15 liver, 14 seminal vesicle, and 14 epididymis) generated for this study is available on the NCBI  Sequence Read Archive under BioProject PRJNA1068126. Metadata associated with each individual is available in the supplementary materials for the manuscript.  ## Sharing/Access information Data was derived from the following sources: * Raw RNAseq read data that was used to create transcriptomes is available at NCBI Sequence Read Archive BioProject PRJNA1068126. * Transcriptomes were annotated using: * *Peromyscus maniculatus* reference genome CDS available at NCBI GenBank: HU_Pman_2.1.3, GCA_003704035 (Lassance and Hoekstra 2020) * *Peromyscus californicus insignis* reference genome CDS available at NCBI GenBank: [ASM782708v3 ](https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_007827085.1/)GCA_007827085.3 (Trainor et al., 2022) * *Mus musculus* reference genome CDS available at NCBI GenBank: GRCm39 GCA_000001635.9 * Specimen metadata is available in the supplementary material for the associated manuscript (Voss and Nachman 2024, Molecular Ecology) and online in the Museum of Vertebrate Zoology (University of California, Berkeley) Mammal Collection catalog on the Arctos Database at [https://arctos.database.museum/home.cfm](https://arctos.database.museum/home.cfm).  ## Code/Software 1. Raw Illumina RNAseq reads were quality assessed with fastQC v.0.11.9 (Andrews 2010) and filtered and trimmed with fastp v.0.23.2 (Chen et al., 2018). 2. Transcriptomes were assembled using the de novo pipeline with Trinity v.2.15.1 (Haas et al., 2013). Then, we used CD-HIT v.4.8.1 (Li and Godzik 2006; Fu et al., 2012) to cluster sequences with greater than 95% sequence similarity and rnaquast v.2.2.2 (Bushmanova et al., 2016) to remove chimeric or misassembled sequences.  3. We assessed transcriptome completeness using Busco v.5.4.5 (Simão et al., 2015) with Euarchontoglires odb10 4. TransDecoder v.5.7.0 was used to predict open reading frames, followed by Trinotate v.3.2.2 (Bryant et al., 2017) with the Ensembl protein database for *P. maniculatus* (HU_Pman_2.1.3) to provide primary annotation.  5. All analyses were performed using bash scripting on the University of California, Berkeley HPC cluster (Savio). Scripts for transcriptome assembly and downstream differential gene expression analyses are available from the authors upon request. ## References Bryant, D. M., Johnson, K., DiTommaso, T., Tickle, T., Couger, M. B., Payzin-Dogru, D., Lee, T. J., Leigh, N. D., Kuo, T.-H., Davis, F. G., Bateman, J., Bryant, S., Guzikowski, A. R., Tsai, S. L., Coyne, S., Ye, W. W., Freeman, R. M., Jr, Peshkin, L., Tabin, C. J., … Whited, J. L. (2017). A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. *Cell Reports* ,*18* (3), 762–776. [https://doi.org/10.1016/j.celrep.2016.12.063](https://doi.org/10.1016/j.celrep.2016.12.063) Bushmanova, E., Antipov, D., Lapidus, A., Suvorov, V., & Prjibelski, A. D. (2016). rnaQUAST: a quality assessment tool for de novo transcriptome assemblies. *Bioinformatics* , *32* (14), 2210–2212. Chen, S., Zhou, Y., Chen, Y., & Gu, J. 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