DOI: 10.5061/dryad.q573n5th0
title: The interplay of color and bioacoustic traits in the differentiation of a Southeast Asian songbird complex

description: Morphological traits have served generations of biologists as a taxonomic
 indicator, and have been the main basis for defining and classifying
 species diversity for centuries. A quantitative integration of behavioural
 characters, such as vocalizations, in studies on biotic differentiation
 has arisen more recently, and the relative importance of these different
 traits in the diversification process remains poorly understood. To
 provide a framework within which to interpret the evolutionary interplay
 between morphological and behavioral traits, we generated a draft genome
 of a cryptic Southeast Asian songbird, the Limestone
 Wren-babbler Napothera crispifrons. We re-sequenced
 whole genomes of multiple individuals of all three traditional subspecies
 and of a distinct leucistic population. We demonstrate strong genomic and
 mitochondrial divergence among all three taxa, pointing to the existence
 of three species-level lineages. Despite its great phenotypic
 distinctness, the leucistic population was characterized by shallow
 genomic differentiation from its neighbor, with only a few localized
 regions emerging as highly-diverged. Quantitative bioacoustic analysis
 across multiple traits revealed deep differences especially between the
 two taxa characterized by limited plumage differentiation. Our study
 demonstrates that differentiation in these furtive songbirds has resulted
 in a complex mosaic of color-based and bioacoustic differences among
 populations. Extreme color differences can be anchored in few genomic loci
 and may therefore arise and subside rapidly.
description: <i>Bioacoustic analysis: </i>A total of 25 sound
 recordings were measured using Raven Pro 1.5 and analysed using PCA and
 the Isler criterion. <i>Whole genome resequencing
 library preparation: </i>15 blood, tissue or feather samples were
 extracted using DNeasy Blood & Tissue Kit (Qiagen, Germany). The
 extracted DNA were sheared with a Bioruptor. Libraries were prepared using
 NEBNext Ultra II DNA Library PrepKit (New England Biolabs, UK). Each
 sample was prepared with a unique dual index barcode using NEBNext
 Multiplex Oligos for Illumina (New England Biolabs, UK). The libraries
 were sequenced using the Illumina Hiseq 4000 platform with 150 bp
 paired-end runs. <i>Draft genome assembly:
 </i>KK05 (~38.8X coverage)<i> </i>was assembled
 with<i> </i>MaSuRCA 3.3.0. Repeat regions were filtered using
 repeat regions with RepeatMasker 4.0.9. Completeness were assessed using
 BUSCO 3.1. Chromosomal position of the assembled draft genome was inferred
 with Satsuma 3.1.0 using <i>Taeniopygia guttata </i>as a
 reference. <i>Raw data processing and SNP
 calling: </i>Cutadapt 1.18 was applied to remove adapter
 contamination and reads with a quality score lower than 20. Each sample
 was aligned to the <i>Napothera </i>draft genome using BWA-MEM
 0.7.15 (Li, 2013). Picard 1.8 MarkDuplicates was conducted to filter out
 PCR duplicates and GATK 3.8.1.0 IndelRealigner was applied to perform
 local realignment around insertions and deletions to minimise mismatches.
 SNP calling was conducted using ANGSD 0.923 and SNPs were pruned using
 PLINK 1.90. PCA and STRUCTURE were plotted. The Python script
 popgenWindows.py (https://github.com/simonhmartin/genomics_general) was
 used to compute d<sub>XY </sub>for each population pair. The
 peak regions found in the net d<sub>XY </sub>comparisons were
 identified and associated genes were inferred using the Ensembl database
 (Yates et al., 2019). Trees were constructed using SNP, genomic sequence
 and mitochondrial data with RAxML 8.2.12.
description: Raw fastq files of all 15 individuals can be downloaded
 from NCBI PRJNA669821 <em>Uploaded
 here:</em> Vocal measurements, ND2 alignment, draft genome of
 <em>Napothera crispifrons</em>, VCF, bed, bim and fam files of
 the final set of 104,092 SNPs, maximum likelihood trees constructed with
 for each chromosome, and supporting information.