Aves

Single linkage clustering is performed on the aligned sequence data. This approach ordinarily requires the generation of a distance matrix for all pairs of sequences followed by a clustering step where sequences are grouped based on a pre-selected distance threshold [45] (link), [46] . RESL performs distance calculations and clustering concurrently, employing the transitive property to avoid distance determinations for sequences that are certain to possess a divergence above the threshold. This strategy is implemented by flushing all clusters to disk, and retaining one or more representative sequences, depending on the diameter of the cluster, for each cluster and inter-cluster distance statistics in active memory, excepting those clusters whose members show high variability (max intra-cluster distance >2.2%, see below). The sequence divergence between each new sequence and the representative(s) of all existing clusters is then calculated. If its distance to any existing cluster is more than twice the threshold [>4.4%], it is recognized as the founder of a new cluster. If, on the other hand, it shows lower divergence, all members of the closest cluster(s) are retrieved from disk to enable more detailed analysis of sequence variation. This approach considerably reduces computational requirements without compromising accuracy, and analysis is further expedited by moving clusters to disk when they have seen no activity ( =  gained new members) for a number of cycles.
The implementation of single linkage clustering requires the selection of a threshold parameter, t, which represents the level of sequence divergence for the designation of OTUs. Early work [13] suggested that a threshold value of 2% was effective because most specimens showing more than this level of divergence represented different species, while those with less divergence were usually conspecific. However, this issue was examined in more detail by inspecting the patterning of OTU recovery with variance in the distance threshold for eight datasets (Table 1). Sixty single linkage cluster analyses were generated for each dataset by stepping the distance threshold parameter by an increment of 0.1% across the range from 0.1%–6.0%. The OTUs recovered at each threshold were subsequently evaluated for their concordance with recognized species boundaries (Figure 2). These analyses revealed that maximal concordance was achieved by thresholds that varied from a low of t = 0.7% (in North American birds) to a high of t = 1.8% (in Bavarian moths). It also showed that performance, as measured by the number of correctly recognized species, dropped steeply when the threshold deviated on either side of optimality. Thresholds higher than optimal inflated the number of cases where members of different species were merged in a single OTU, while thresholds lower than the optimal value increased the cases where members of what are thought by current taxonomy to be a single species were split into two or more OTUs. Based on these analyses, a threshold (t) of 2.2% was adopted as it represents the upper 99% confidence limit for the optimal thresholds in the eight test datasets SD  = 0.40). Its adoption will lead to the merger of some distinct clusters, but such cases are addressed in the third step of the analysis.

Five bird species with profoundly black plumage and two species with normal black plumage were identified by visual observation of museum study skins from the Yale Peabody Museum (YPM), Harvard Museum of Comparative Zoology (MCZ), American Museum of Natural History (AMNH), and the University of Kansas Biodiversity Institute (KU). Details of the specimens and plumage patches studied are summarized in Supplementary Table 1. To the human observer, super black plumage had a strongly matte appearance with so little specular reflectance that it was difficult to focus on the surface of the plumage and distinguish individual feathers. The species with normal black plumage lacked any conspicuous glossy specular highlights. Individual contour feathers were sampled from museum skins for scanning electron microscopy (SEM) and synchrotron-radiation X-ray microtomograhy (nano-CT). We could not obtain SEM of Lophorina superba back feathers or CT scans for Lophorina superba back and display cape feathers due to availability of material. Visual inspection of the Lophorina back plumage using a light microscope confirmed that the barbules have normal morphology, without the modified barbule arrays present in super black feathers.

According to the previous studies on avian species and a chick brain atlas (Kuenzel and Masson, 1988 ; Kang et al., 2009 (link); Kang et al., 2020 (link)), two major 5-HTergic regions in the brainstem, DRN and CRN, and VTA regions were dissected in a cryostat microtome. The dimensions of the dissected section are as follows: 2.5–3 mm (W) x 1–1.5 mm (H) x 2.5–3.0 mm (L) for DRN; 2–2.5 mm (W) x 1–1.2 mm (H) x 2.5–3.0 mm (L) for CRN; and 3–3.5 mm (W) x 2–3 mm (H) x 1–1.2 mm (L) for VTA. The thickness (W, H, and L) of the dissected brain tissue block was proportionally increased from young birds to older birds based on the brain size and structure. Inside the cryostat, the brain areas shown as rectangles were dissected from each flattened brain section using a scalpel handle and blade (#11) and were quickly transferred to TRIzol reagent and then stored at -80°C until total RNA extraction.

One-day-old broilers (Cobb 700, mixed sex, and 19,200 birds/house) were housed in four commercial broiler houses (Tyson Foods Broiler Welfare Research Farm (BWRF)). Four replicate trials were performed, and each house was composed of four quadrant sections (compartment). Each quadrant of the house was placed with 4,800 chicks with all source flocks equally represented in each quadrant. Birds were raised for 56, 51, 49, and 55 days in trails 1, 2, 3, and 4, respectively. Each house was equipped with standard feeders, waterers, and brooders (12.8 m × 122 m, wood shavings). Two of the houses have a 60-cm-wide strip of clear plastic that runs the length of the houses from 120 to 180 cm high on the sidewall and allows for natural light to enter. The natural light window can be sealed to convert that house to internal illumination. In each trial, four different light intensity lighting programs were installed, and the light intensity (LED) was measured at nine different areas of the house. Averages of light intensity in 5-lx, 20-lx, natural-light (NL), and VL houses were 6.16 ± 0.16 lx, 26.16 ± 0.70 lx, 483.76 ± 42.02 lx, and 2.07/40.4 ± 0.04 lx, respectively. A diet was formulated to meet minimum industry standards (Council, 1994 ). Light was switched on at 6 a.m. on day 1–3 (23L1D_40 lx), and then on day 4–7, the photoperiod schedule was changed to 20L4D_20 lx. The NL house received supplemented light for maintaining the same photoperiod. On day 7, lighting programs were started for 5-lx, 20-lx, NL, and VL houses (16L8D; light switched on at 6 a.m.). The VL house received about 40 lx light intensity over the feed lines and dimmer light intensity at the sidewalls (2–5 lx). Data on dustbathing holes and other natural behaviors were obtained weekly without interruption of the time schedules. In trials 1 and 3, the brains of the birds in each section were sampled on days 14, 28, and 42. Birds in each section (n = 12/treatment; male) were randomly selected, weighed, and transported to the brain sampling room. Daily body weight gain and feed conversion ratio were obtained from the processing plant at the end of the trial for each house. The guidelines for care and experimental use of animals were followed, and all birds were maintained in accordance with the protocol of Tyson Foods BWRF.