DOI: 10.5061/dryad.612jm64c9
title: Chromosome assembly and preliminary gene and repeat annotations for Myzomela tristrami reference genome

description: Secondary contact between closely related taxa represents a “moment of
 truth” for speciation — an opportunity to test the efficacy of
 reproductive isolation that evolved in allopatry and to identify the
 genetic, behavioral, and/or ecological barriers that separate species in
 sympatry. Sex chromosomes are known to rapidly accumulate differences
 between species, an effect that may be exacerbated for neo-sex chromosomes
 that are transitioning from autosomal to sex-specific inheritance. Here we
 report that, in the Solomon Islands, two closely related bird species in
 the honeyeater family — Myzomela cardinalis and Myzomela tristrami — carry
 neo-sex chromosomes and have come into recent secondary contact after ~1.1
 my of geographic isolation. Hybrids of the two species were first observed
 in sympatry ~100 years ago. To determine the genetic consequences of
 hybridization, we use population genomic analyses of individuals sampled
 in allopatry and in sympatry to characterize gene flow in the contact
 zone. Using genome-wide estimates of diversity, differentiation, and
 divergence, we find that the degree and direction of introgression varies
 dramatically across the genome. For sympatric birds, autosomal
 introgression is bidirectional, with phenotypic hybrids and phenotypic
 parentals of both species showing admixed ancestry. In other regions of
 the genome, however, the story is different. While introgression on the
 Z/neo-Z-linked sequence is limited, introgression of W/neo-W regions and
 mitochondrial sequence (mtDNA) is highly asymmetric, moving only from the
 invading M. cardinalis to the resident M. tristrami. The recent
 hybridization between these species has thus enabled gene flow in some
 genomic regions but the interaction of admixture, asymmetric mate choice,
 and/or natural selection has led to the variation in the amount and
 direction of gene flow at sex-linked regions of the genome.
description: This data repository contains <em>Myzomela
 tristrami</em> reference genome files. The sequences associated with
 this assembly are available on NCBI sequence read archive at https://www.ncbi.nlm.nih.gov/sra/?term=SRA%20SRR29254783. We sequenced a <em>M. tristrami </em>female at the University of Delaware DNA sequencing & Genotyping Cener. HiFi libraries were prepared with SMRTbell prep kit, followed by Blue Pippin size selection (15-20Kbp) before sequencing on a PacBio Sequel IIe. We generated a <em>de novo</em> assembly using hifiasm v0.13-r308 with default parameters using the resulting long reads (Cheng et al. 2021, 2022). We used GeMoMa (v1.8) and the annotation from zebra finch genome bTaeGut1.4.pri to infer a rough annotation of genes in the <em>Myzomela </em>genome. We then used these rough annotations, comparing contigs against both zebra finch and the chicken genome bGalGal1.mat.broiler.GRCg7b to infer synteny relationships, remove duplicate haplotigs, and, finally, scaffold contigs into chromosomes in <em>Myzomela.</em> The resulting assembly uses the zebra finch numbering system for chromosomes 1-29; chromosome 30-40 were named in descending order of size. Final chromosomes and contigs were aligned with those of related species— helmeted honeyeater (<em>Lichenostomus melanops cassidix</em>), and blue-faced honeyeater (<em>Entomyzon cyanotis)</em>— using Mauve (version 2015-02-25), and visualized using FastANI (v1.33) (Darling et al. 2004, Jain et al. 2018, Robledo-Ruiz et al. 2022, Burley et al. 2023). We generated repetitive DNA libraries using the RepeatModeler v2 pipeline (Flynn et al. 2020). RepeatModeler employs a combination of <em>de novo</em> and homology-based characterization of different classes of repeats. The repeat library was annotated and combined with Repbase, and manually curated repeat libraries from other studies (Suh et al. 2018, Boman et al. 2019, Weissensteiner et al. 2020, Peona et al. 2021). We then used RepeatMasker ( v4.1.0) to identify and mask repetitive regions of the genome (Smit et al. 2013). Boman, J., C. Frankl-Vilches, M. D. S. dos Santos, E. H. C. de Oliveira, M. Gahr, and A. Suh. 2019. The genome of Blue-capped Cordon-bleu uncovers hidden diversity of LTR retrotransposons in Zebra Finch. Genes 10. Burley, J. T., S. C. M. Orzechowski, S. Y. W. Sin, and S. V. Edwards. 2023. Whole-genome phylogeography of the blue-faced honeyeater (<em>Entomyzon cyanotis</em>) and discovery and characterization of a neo-Z chromosome. Molecular Ecology 32:1248–1270. Cheng, H., G. T. Concepcion, X. Feng, H. Zhang, and H. Li. 2021. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nature Methods 18:170–175. Cheng, H., E. D. Jarvis, O. Fedrigo, K. P. Koepfli, L. Urban, N. J. Gemmell, and H. Li. 2022. Haplotype-resolved assembly of diploid genomes without parental data. Nature Biotechnology 40:1332–1335. Darling, A. C. E., B. Mau, F. R. Blattner, and N. T. Perna. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Research 14:1394–1403. Flynn, J. M., R. Hubley, C. Goubert, J. Rosen, A. G. Clark, C. Feschotte, and A. F. Smit. 2020. RepeatModeler2 for automated genomic discovery of transposable element families. Proceedings of the National Academy of Sciences of the United States of America 117:9451–9457. Jain, C., L. M. Rodriguez-R, A. M. Phillippy, K. T. Konstantinidis, and S. Aluru. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications 9:5114. Peona, V., O. M. Palacios-Gimenez, J. Blommaert, J. Liu, T. Haryoko, K. A. Jønsson, M. Irestedt, Q. Zhou, P. Jern, and A. Suh. 2021. The avian W chromosome is a refugium for endogenous retroviruses with likely effects on female-biased mutational load and genetic incompatibilities. Philosophical Transactions of the Royal Society B: Biological Sciences 376. Robledo-Ruiz, D. A., H. M. Gan, P. Kaur, O. Dudchenko, D. Weisz, R. Khan, E. Lieberman Aiden, E. Osipova, M. Hiller, H. E. Morales, M. J. L. Magrath, R. H. Clarke, P. Sunnucks, and A. Pavlova. 2022. Chromosome-length genome assembly and linkage map of a critically endangered Australian bird: the helmeted honeyeater. GigaScience 11:giac025. Smit, A., R. Hubley, and P. Green. 2013, 2015. RepeatMasker Open-4.0. Suh, A., L. Smeds, and H. Ellegren. 2018. Abundant recent activity of retrovirus-like retrotransposons within and among flycatcher species implies a rich source of structural variation in songbird genomes. Molecular Ecology 27:99–111. Weissensteiner, M. H., I. Bunikis, A. Catalán, K. J. Francoijs, U. Knief, W. Heim, V. Peona, S. D. Pophaly, F. J. Sedlazeck, A. Suh, V. M. Warmuth, and J. B. W. Wolf. 2020. Discovery and population genomics of structural variation in a songbird genus. Nature Communications 11:1–11.
description: # Chromosome assembly and preliminary gene and repeat annotations for
 Myzomela tristrami reference genome I. Files (GENOME) Mt_v1.0_MAIN.fa.gz
 Primary genome, (largely) scaffolded to chromosome-level, plus other
 primary assembled contigs Mt_v1.0_MAIN.gff.gz Simple gene annotations for
 primary genome, annotated using GeMoMa v1.8 and a zebra finch
 (bTaeGut1.4.pri) annotation reference Mt_v1.0_extra.fa.gz Additional
 contigs, not for use in most analyses but some may be of interest This set
 is a combination of hand-identified haplotigs of the main genome, and
 assembler-identified "alternate" (haplotig) contigs
 (ORIGINAL_ASSEMBLY_CONTIGS) Mt_hifi.asm.p.fa.gz "primary"
 assembly contigs, output from hifiasm (v0.13-r308) Mt_hifi.asm.a.fa.gz
 "alternate" assembly contigs, output from hifiasm (v0.13-r308)
 (REPEAT_MASKING) TElib_Myzo_preliminary.fa.gz Preliminary Myzomela-tuned
 TE/repeat library, generated using RepeatModeler (v.2)
 Mt_v1.0_MAIN_RM_sites_to_filter.txt List of sites masked by RepeatMasker
 (v4.1.0), "MAIN" genome file, using Myzomela-specific library
 Mt_v1.0_extra_RM_sites_to_filter.txt Same for the "extra"
 contigs (SCAFFOLDING_ANNOTATIONS) Mt_hifi_zf_reference_gene_table.tabular
 Tabular list of genes annotated in the M. tristrami hifiasm output, using
 zebra finch (bTaeGut1.4.pri) annotations and GeMoMa (v1.8) software
 Mt_hifi_ch_reference_gene_table.tabular Tabular list of genes annotated in
 the M. tristrami hifiasm output, using chicken
 (bGalGal1.mat.broiler.GRCg7b) annotations and GeMoMa (v1.8) software
 zf_zf_reference_gene_table.tabular Tabular list of genes from GeMoMa
 (v1.8) reannotation of zebra finch (bTaeGut1.4.pri), using its own
 annotations as reference ch_ch_reference_gene_table.tabular Tabular list
 of genes from GeMoMa (v1.8) reannotation of chicken
 (bGalGal1.mat.broiler.GRCg7b, using its own annotations as reference
 Mt_v1.0_MAIN_key.txt Key relating contigs to their Mt_v1.0_MAIN.fa
 scaffold/chromosome Brief notes about the scaffolded genome
 (Mt_v1.0_MAIN.fa): \-- While the bulk of the primary genome has been
 scaffolded into chromosomes, the sex chromosomes have only been partially
 scaffolded. In particular, the four large contigs that comprise W-linked
 sequence have been left un-joined. These W-linked contigs are nevertheless
 thought to be physically contiguous, as are the "Z1" and
 "Z2" portions of chrZ (along with their copy of the diploid
 "neoPAR" sequence that, in this assembly, is linked to a W
 contig). \-- We identified and removed haplotigs from the primary genome
 using hand-curation rather than automated pipelines. This means that this
 genome will retain *genuinely* repetitive regions that might be of
 interest (satellite arrays, etc.), but we also expect that there will be
 at least some erroneous duplications and misassemblies. \-- chr30-40
 should be regarded as highly tentative. Standards for naming/inclusion in
 this genome version: 1\. greater than 1Mb in length 2\. at least 10
 potential gene annotations 3\. clear haplotig sequence, roughly syntenic
 barring a handful of rearrangements (ie, reasonably well-behaved diploid)
 4\. sorted/named by length. ``` We were conservative about joining contigs
 (doing so only when supported by an alternate haplotig) to form these
 chromosomes, and it is likely that at least some of them are actually
 fragments of larger chromosomes. We included them as named chromosomes
 here but stress that the arrangements and the specific names used here are
 highly likely to change in future genome versions as more data become
 available. ``` \-- A large number of un-scaffolded contigs from the
 hifiasm primary assembly are retained in the "MAIN" genome. Many
 of these sequences seem to derive from non-specific repetitive sequence
 (telomeres and telomere-adjacent sequence, for example), although there
 are some with gene annotations. Where possible, haplotigs of these contigs
 have been moved to the "extra" file, but there are undoubtedly
 many remaining duplications in the retained contigs.