DOI: 10.5061/dryad.6q44t
title: Data from: Incorporating color into integrative taxonomy: analysis of the varied tit (Sittiparus varius) complex in East Asia

description: Species designations are critically important scientific hypotheses that
 serve as the foundational units in a wide range of biological
 subdisciplines. A growing realization that some classes of data fail to
 delimit species under certain conditions has led to increasingly more
 integrative taxonomies, whereby species discovery and hypothesis testing
 are based on multiple kinds of data (e.g. morphological, molecular,
 behavioral, ecological, etc.). However, although most taxonomic
 descriptions have been based on morphology, some key morphological
 features, such as color, are rarely quantified and incorporated into
 integrative taxonomic studies. In this paper, we applied a new method of
 ultraviolet digital photography to measure plumage variation in a
 color-variable avian species complex, the varied tit (Sittiparus varius).
 Plumage measurements corroborated species limits defined by morphometric,
 mitochondrial DNA, and nuclear DNA disjunctions and provided the only
 evidence for recognizing two recently evolved species. Importantly, color
 quantification also provided a justification for lumping putative taxa
 with no evidence of evolutionary independence. Our revised taxonomy thus
 refines conservation units for listing and management and clarifies the
 primary units for evolutionary studies. Species tree analyses, which
 applied the newly delimited species as operational taxonomic units,
 revealed a robust phylogenetic hypothesis for the group that establishes a
 foundation for future biogeographic analyses. Our study demonstrates how
 digital photography can be used to incorporate color character variation
 into integrative taxonomies, which should lead to more informed, more
 rigorous, and more accurate assessments of biodiversity.
description: ND2 locus
 alignmentND2 locus alignment in
 fasta formatVATI ND2 Align
 Final.fasta08352 locus
 alignment08352 locus phased
 alignment in fasta formatVATI CS08352 Align
 Final.fas09385 locus
 alignment09385 locus phased
 alignment in fasta formatVATI CS09385 Align
 Final.fas10179 locus
 alignment10179 locus phased
 alignment in fasta formatVATI CS10179 Align
 Final.fas11887 locus
 alignment11887 locus phased
 alignment in fasta formatVATI CS11887 Align
 Final.fas12884 locus
 alignment12884 locus phased
 alignment in fasta formatVATI CS12884B Align
 Final.fas15349 locus
 alignment15349 locus phased
 alignment in fasta formatVATI CS15349 Align
 Final.fasTable S1. Morphological
 sample listMuseum voucher
 information for all skin specimens of the varied tit (Sittiparus varius)
 complex examined in this study. Tissue voucher numbers are provided for
 the few cases in which an individual was sampled for both morphological
 and genetic data. Acronyms: National Museum of Nature and Science, Tokyo
 (NSMT); American Museum of Natural History (AMNH); Yamashina Institute of
 Ornithology (YIO); National Museum of Natural Science, Taiwan (NMNS),
 Taiwan Endemic Species Research Institute (TESRI).S1 Morphological sample
 list.pdfTable S2. Molecular
 sample listMuseum voucher
 information for all genetic samples of the varied tit (Sittiparus varius)
 complex used in this study. Stars indicate individuals selected for
 multilocus nuclear sequencing. Acronyms: National Museum of Nature and
 Science, Tokyo (NSMT); Taiwan Endemic Species Research Institute (TESRI);
 Liaoning University, China (LU); Cincinnati Museum Center (CMC); American
 Museum of Natural History (AMNH); University of Kansas University Natural
 History Museum (KUNMH).S2 Molecular sample
 list.pdfTable S3. Toepad primers
 & sequencing conditionsA list of primers used
 to amplify and sequence the mitochondrial NADH dehydrogenase subunit 2
 (ND2) gene from a toepad sample of Sittiparus varius orii. Detailed DNA
 extraction, PCR, and sequencing protocols for toepad samples are also
 given.S3
 Toepad primers & sequencing
 conditions.pdfFigure S4. Morphometric
 plotsBoxplots of morphometric
 size variation among subspecies of the varied tit (Sittiparus varius)
 complex: (a) bill length, (b) bill depth, (c) bill width, (d) tarsus
 length, (e) wing chord, and (f) tail length. The taxon castaneoventris
 showed non-overlapping size variation in bill depth, wing chord, and tail
 length. Heavy lines inside boxes represent the mediums (second quartiles).
 Each box is bounded by the first and third quartiles, and plot whiskers
 extend to 1.5 times the interquartile range or to the maximum and minimum
 value, as appropriate. Data points outside the whisker range are
 represented by open circles.S4 Morphometric
 plots.pdfFigure S5. Luminance
 plotsBoxplots of luminance
 measurements obtained from plumage patches with &lt;0.05 luminance
 reflectance: (a) mantle, (b) throat, and (c) black of head. Hue
 measurements were excluded from these patches because very dark colors
 yield random hues when converted into opponent colorspace. Each box is
 bounded by the first and third quartiles, and plot whiskers extend to 1.5
 times the interquartile range or to the maximum and minimum value, as
 appropriate. Data points outside the whisker range are represented by open
 circles.S5
 Luminance plots.pdfTable S6. Loci
 tableDescriptive statistics
 for the seven molecular loci used to construct phylogenetic relationships
 among S. varius, S. castaneoventris, S. owstoni, and S. olivaceus. The
 summary includes the length of the sequences (in base pairs), the number
 of alleles, the number of segregating sites (s), the number of
 parsimony-informative sites (PI sites), nucleotide diversity (π), and the
 nucleotide substitution model. Statistics are drawn from ingroup sequences
 only. Note that ND2 statistics are drawn from a reduced ND2 alignment,
 which comprised the 16 samples sequenced for nuclear
 loci.S6
 Loci table.pdfFigure S7. Nuclear
 intron networksStatistical parsimony
 networks of nuclear intron sequences: (a) 08352, (b) 09385, (c) 10179, (d)
 11887, (e) 12884, and (f) 15349. Each circle represents a haplotype; the
 size of the circle is proportional to a haplotype’s frequency. Small black
 circles represent inferred haplotypes. Colors distinguish the four species
 recognized in this study.S7 Nuclear intron
 networks.pdf