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.
ND2 locus alignment
ND2 locus alignment in fasta format
VATI ND2 Align Final.fasta
08352 locus alignment
08352 locus phased alignment in fasta format
VATI CS08352 Align Final.fas
09385 locus alignment
09385 locus phased alignment in fasta format
VATI CS09385 Align Final.fas
10179 locus alignment
10179 locus phased alignment in fasta format
VATI CS10179 Align Final.fas
11887 locus alignment
11887 locus phased alignment in fasta format
VATI CS11887 Align Final.fas
12884 locus alignment
12884 locus phased alignment in fasta format
VATI CS12884B Align Final.fas
15349 locus alignment
15349 locus phased alignment in fasta format
VATI CS15349 Align Final.fas
Table S1. Morphological sample list
Museum 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.pdf
Table S2. Molecular sample list
Museum 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.pdf
Table S3. Toepad primers & sequencing conditions
A 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.pdf
Figure S4. Morphometric plots
Boxplots 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.pdf
Figure S5. Luminance plots
Boxplots of luminance measurements obtained from plumage patches with <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.pdf
Table S6. Loci table
Descriptive 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.pdf
Figure S7. Nuclear intron networks
Statistical 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