DOI: 10.5061/dryad.zgmsbccc8
title: Describing biodiversity in the genomics era: Description of a new species of Nearctic Cynipidae gall wasp and its genome

description: Gall wasps (Hymenoptera: Cynipidae) specializing on live oaks in the genus
 Quercus (subsection Virentes) are a relatively diverse and well-studied
 community with 14 species described to date, albeit with incomplete
 information on their biology, life history, and genetic structure.
 Incorporating an integrative taxonomic approach, we combine morphology,
 phenology, behavior, genetics, and genomics to describe a new species,
 Neuroterus valhalla sp. nov. The alternating generations of this species
 induce galls on the catkins and stem nodes of Quercus virginiana and Q.
 geminata in the southern United States. We describe both generations in
 the species’ life cycle, and primarily use samples from a population in
 the center of Houston, Texas, thus serving as an example of the
 undescribed biodiversity still present in well-traveled urban centers. In
 parallel, we present a draft assembly of the N. valhalla genome providing
 a direct link between the type specimen and reference genome. The genome
 of N. valhalla is the smallest reported to date within the tribe Cynipini,
 providing an important comparative constrast to the otherwise large genome
 size of Cynpids. While relatively small, the genome was found to be
 composed of &gt;64% repetitive elements, including 43% unclassified
 repeats and 11% retrotransposons. A preliminary ab-initio and
 homology-based annotation revealed 32,005 genes, and a subsequent
 orthogroup analysis grouped 18,044 of these to 8,186 orthogroups, with
 some evidence for high levels of gene duplications within Cynipidae. A
 mitochondrial barcode phylogeny linked each generation of the new species
 and a phylogenomic ultraconserved element (UCEs) phylogeny indicates that
 the new species groups with other Nearctic Neuroterus. However, both
 phylogenies present the genus Neuroterus in North America as polyphyletic.
description: <strong>GENOME SEQUENCING, ASSEMBLY AND
 ANNOTATION</strong><br> <br> To sequence N. valhalla’s
 genome, we extracted whole genomic DNA from a single female from the
 catkin generation using the DNeasy Blood and Tissue Kit (Qiagen, Germany)
 as described above. A paired-end sequencing library was then constructed
 with the Illumina TruSeq kit (Illumina, USA) using the standard adapters,
 and sequencing was performed at Genewiz (New Jersey, USA) on an Illumina
 X-Ten sequencing platform. Raw data was submitted to Genbank Short Read
 Archive (Accession number SRX7007139). Then, fastq files were filtered and
 trimmed by Trimmomatic v.0.39 (Bolger et al., 2014), first removing
 sliding windows of 5 nucleotides with quality average below 20, followed
 by a hard-trailing trim of nucleotides with quality below 15. Trimmed
 reads were then assembled de novo by SPAdes v3.14.0 (Bankevich et al.,
 2012) using kmer-sizes of 27,37,47,57,67,77,87,97,107,117 and 127.
 Assembly quality and completeness were accessed by quast v5.0.2 (Gurevich
 et al., 2013) and BUSCO v4.0.6 (Simão et al., 2015).<br> <br>
 For annotation, repetitive sequences were inferred using RECON (Bao et
 al., 2002), RepeatScout (Price et al., 2005) and LTR_retriver (Ou
 & Jiang, 2018), as applied by RepeatModeler (Smit &
 Hubley, 2015). Then, these sequences were classified with RepeatClassifier
 according to RepBase v25.04 (Bao et al., 2015), and representation of
 these sequences in the genome were accessed by RepeatMasker v4.0 (Smit et
 al., 2015), which also masked the assembly for subsequent gene annotation.
 Augustus (Stanke et al., 2006) was applied for Ab-initio gene prediction,
 using Nasonia vitripennis (Walker) (Werren et al., 2010) training
 parameters, the closest organism with a training set available. Then, for
 protein-based annotation, we used a database containing all known genes
 from the most closely related organism with an annotated genome,
 Belonocnema kinseyi Weld (GenBank 17056478), and used Exonerate v.2.2
 (Slater & Birney, 2005). Both annotations were then combined using
 EvidenceModeler v1.1.1 (Haas et al., 2008), with equal weight to either
 approach. The final .gff file was then filtered and analyzed with gFACs
 v1.0 (Caballero & Wegrzyn, 2019) for gene statistics. <br>
 Gene ortholog analysis was performed using Orthofinder (Emms &
 Kelly, 2019). The analysis encompassed nine annotated genomes, including
 Belonocnema kinseyi; every proteome available in UniProt database
 belonging to the Infraorder Parasitoida (three total, including above
 mentioned N. vitripennis); Apis mellifera L. and Drosophila melanogaster
 Meigen for their status as most well characterized insect genomes; and
 Arabidopsis thaliana (L.) Heynh. and Homo sapiens L. as
 outgroups. <strong>ULTRACONSERVED ELEMENTS
 PHYLOGENY</strong><br> <br> To build the UCE phylogeny,
 we downloaded the UCE assemblies from all Cynipini and selected outgroups
 from other tribes from Blaimer et al. (2020) and assembled the contigs
 using SPAdes. Additionally, UCE loci were extracted from all available
 Cynipini genomes on NCBI not already present in Blaimer et al. (2020)
 dataset, including N. valhalla, following tutorial III of the PHYLUCE
 pipeline, using the Hym-V2P probe set developed by Branstetter et al.
 (2017). All assemblies were aligned using MAFFT and trimmed using Gblocks
 v0.91b-2 (Castresana, 2000) using the following settings: b1=0.5, b2=0.5,
 b3=12, b4=7. Then, we used Spruceup 0.95 lognormal distribution or manual
 cutoff of select samples to remove any potentially misaligned regions as
 they can produce exaggerated branch lengths (Borowiec, 2019). We selected
 the 50% complete matrix as the final dataset and inferred the maximum
 likelihood phylogeny using IQ-TREE using best models for each locus
 selected by ModelFinder. To assess nodal support, we performed 1000
 ultrafast bootstrap replicates (UFBoot2, Hoang et al., 2017), along with
 “-bnni” to reduce risk of overestimating branch supports; and a
 Shimodaira-Hasegawa approximate likelihood-rate test (SH-aLRT, Guindon et
 al., 2010) with 1000 replicates. Only nodes with support values of UFBoot2
 ≥ 95 and SH-aLRT ≥80 were considered robust.  
description: # Supplementary data on the "Describing biodiversity in the genomics
 era: Description of a new species of Nearctic Cynipidae gall wasp and its
 genome" https://doi.org/10.5061/dryad.zgmsbccc8 Files in this folder
 include: **c\_valhalla\_assembly\_v1.fasta** Fasta file of the Neuroterus
 valhalla assembly version used in the manuscript.
 **EVM\_cvalhalla.all.gff3** A gff filing containing the gene annotation in
 the N.valhalla genome. See methods for further details.
 **Repeats\_annotation.gff** A gff file containing the annotation of the
 repeat sequences in the genome. See methods for further details.
 **cvalhalla-families.fa** A fasta file containing the repeat sequences
 families from the N. valhalla genome. See methods for further details.
 **Neuroterus\_COI.treefile** Tree file for constructing figure 2 (the
 genetic barcoding COI phylogeny). See methods and supplementary materials
 for further details on treebuilding methods and sequences used.
 **Neuroterus\_COI** A Fasta file containing the alignment of all sequences
 used in figure 2 (the genetic barcoding COI phylogeny)
 **Neuroterus\_UCE.treefile** Tree file for constructing figure 6 (the UCE
 barcoding phylogeny). See methods for further details.
 **Cyn\_50p\_0.95trim.phylip** UCE phylip file that was trimmed at 95%
 lognormal cutoff from spruceup. It contains sequences used to build the
 UCE phylogeny (figure 6) For further questions or if additional files are
 required to replicate the results presented, feel free to contact the
 authors directly. Enjoy!; Pedro