Soley et al. • HATCH ORDER OF EASTERN BLUEBIRDS 
773 
the day the female lays the penultimate egg (M. 
Liu. unpubl. data). Incubation lasts —15 days, and 
all nestlings in the brood hatch over a period of 
between 6 and 36 hrs (Gowaty and Plissner 1998). 
The rectrices and rentiges of nestling bluebirds 
begin to emerge within 11 days after hatching and 
exhibit UV-blue ornamental coloration. The color 
of remiges can be quantified by 14 days after 
hatching (Siefferman and Hill 2007). Young 
bluebirds retain these juvenile wing and tail 
feathers as pan of their first nuptial plumage 
iGowat) and Plissner 1998). Experimental ma¬ 
nipulations of food availability to nestlings 
demonstrate that male nestling bluebirds reared 
in poor natal conditions grow more slowly and 
display duller wing color compared to those 
reared in better natal environments (Siefferman 
and Hill 2007). Parents exhibit preferences for the 
more-colorful fledglings (Ligon and Hill 2010). 
Our objectives were to: (1) test the assumption 
that laying order reflects hatching order. (2) 
investigate whether sex ratio varies between 
early- and late-hatched nestlings. (3) ascertain 
whether late-hatched chicks remain smaller than 
early-hatched siblings throughout the nestling 
period. (4) test whether late-hatched nestlings 
have higher circulating corticosterone and beg 
more vigorously than their early-hatched siblings, 
and (5) investigate whether late-hatched nestlings 
have duller plumage coloration than their early- 
hatched siblings. 
METHODS 
Study Area and Field Proceedures, —We stud¬ 
ied a population of Eastern Bluebirds breeding in 
nes < boxes in Lee County. Alabama. USA 
f 32 35' 52" N. 85 28' 51" W; elevation 21b m) 
in 2007. The study site includes pasture and edge 
habitat. We monitored first nests of Eastern 
Bluebirds every other day during the nest building 
Mage. We visited nests each day during the laying 
l^hod and marked each new egg with a Sharpie® 
barter to establish laying order. We identified 
,yvo groups of eggs in each brood in relation to 
la ying order, defined as early- and late-laid eggs. 
E ?gs laid in the first half of the clutch were early- 
kid in clutches with even number of eggs and 
'hose laid in the last half were late-laid eggs. The 
middle egg was considered a late-laid egg in nests 
w * t h an odd number of eggs. 
w e ascertained which chick hatched from each 
e 8g during the hatching period by visiting each 
nest every 3 hrs (0600-1900 hrs) during daylight 
until all eggs hatched. We identified individual 
nestlings by marking their tarsi with a unique 
color of Sharpie® marker. Nestlings were assigned 
the same hatching order if more than one egg 
hatched in the same 3-hr interval but were given 
different markings. 
Nestlings that hatched in the first 3-hr interval 
during which hatching occurred were ‘early- 
hatched nestlings'. These were nestlings with 
early spots in the hierarchy of hatching positions. 
Similarly, nestlings that hatched after the first 3-hr 
hatching interval were ‘late-hatched nestlings’. 
These were nestlings with late spots in the 
hierarchy of hatching positions. We monitored 
>200 clutches but were only able to assign egg 
laying and nestling hatch order to 31 broods 
because most eggs hatched during the night. 
We defined the age of the brood by the hatching 
date of the first-hatched nestling (day ! = hatch 
day). W'e measured mass of nestlings to the 
nearest 0.1 g on day 2, 5. 8, 11. and 14 post hatch. 
We banded nestlings at 8 days of age and 
collected a 150-ul blood sample within 3 min of 
first handling each nestling. We spun the blood 
sample in a centrifuge, separated the sera and 
plasma, and froze the samples. We measured the 
right tarsi and wing to the nearest 0.1 mm at day 
14 post hatch. Nestlings increase rapidly in mass 
from hatching until they are about 11 days of age, 
but by 13 days of age. the mass of nestlings begins 
to reach the asymptote (Pinkowski 1975). Thus, 
mass at 14 days is an accurate estimate of fledging 
muss. Nestlings generally fledge between 15 and 
18 days post hatch. 
Nestlings at 8 days of age have feather sheaths. 
Feathers begin to emerge from the feather sheaths 
at 11 days of age and 2 cm of the feathers have 
emerged from the sheaths at 14 days of age. We 
cut the distal 2 cm of both primary 5 feathers of 
nestlings for spectrophotomctrie plumage analysis 
on day 14 post hatch. We stored the feathers in 
envelopes in a climate-controlled environment 
until spectrophometric analyses were conducted, 
juvenile Eastern Bluebirds are sexually dichro¬ 
matic. We classified male and female nestlings 
using sexually dichromatic plumage coloration. 
Previous experience with plumage coloration and 
molecular classification showed that 95% of 
young could be properly classified using plumage 
coloration (L. Siefferman, pers. obs.). 
Nestling Begging Behavior .—We stimulated 
nestlings to beg and video recorded nestling 
begging behavior in the morning when the oldest 
