Walsh et aL • HYBRIDIZATION IN NELSON'S AND SALTMARSH SPARROWS 
319 
TABLE 1. Mean and standard errors for four morphological features compared across three groups (Nelson s Sparrow. 
Sihmarsh Sparrow, and hybrids). Values with different letters are significantly different based on a Tukey's test. 
Measurements 
Nelson’s Sparrow (n = 34) Saltmarsh Sparrow <n = 29) 
Hybrids (n = 34) 
Culmen. mm 
8.42 ± 0.06 (B) 
9.22 ± 0.08 (A) 
9.26 ± 0.09 (A) 
Bill width, mm 
4.02 ± 0.04 (B) 
4.42 ± 0.05 (A) 
4.31 ± 0.05 (A) 
Bill depth, mm 
5.01 ± 0.03 (B) 
5.12 ± 0.04 (A) 
5.26 ± 0.04 (A) 
Body mass, g 
17.32 ± 0.25 (B) 
18.57 ± 0.28 (A) 
18.96 ± 0.27 (A) 
Hi nil specific restriction site and used it as the 
M>is for the development of a species-specific 
diagnostic assay. Amplified products were digest¬ 
ed ina 10.5 pi reaction (9 pi template DNA. 0.5 pi 
enryme Hi/j/7. and 1.0 pi of NEBuffer 4: New 
England BioLabs, Ipswich. MA. USA) and 
incubated overnight at 37 C. This restriction 
fragment length polymorphism (RFLP) analysis 
yielded two fragments (-100 and 550 base pairs 
In size) in Sallmarsli Sparrows, and three 
fragments(-100,150, and 400 base pairs in size) 
m Nelson's Sparrows when resolved on a 3% 
agarose gel. 
applied the diagnostic assay to the 426 
individuals identified morphologically (based on 
p'umage and morphometries) as Saltmarsh Spar¬ 
es from Maine to New York. Individuals with 
^* son s ituDNA were considered putative hybrids. 
" iese individuals were sequenced to confirm 
consistency among introgressed individuals. 
Morphological Comparisons. —We compared 
!n(, rphometric data of the putative hybrids (iden- 
"fad by our genetic assay as having Nelson's 
m^NA) to those of the allopatric Nelson's and 
saltmarsh sparrows sampled outside of the 
"'^ap zone (from the Penobscot River in Maine. 
Jn d Shirley and Oceanside. New York). Averages 
J| d standard errors were calculated for the four 
morphological features for the three groups 
%bnd./, = 33 . Nelson's, n = 34: and Saltmarsh. 
-9). ANOVA and a Tukey's test were used to 
“^differences in morphological characteristics 
an °ng the three groups and significance testing 
P e dormed using a Bonferroni correction in 
^ograrn JMP 8 (SAS Institute 2008). 
RESULTS 
^ consistently found the same species-specif- 
nucleotide variations at seven sites (1.2% 
^f s P ec ific variation) when comparing Nelson s 
Saltmarsh sparrow sequences at the COI 
reg,0n - Sequences showed no signs of double 
peaks, confirming that only mtDNA was ampli¬ 
fied. The absence of additional fragments or 
chimeric samples in the RFLP test also indicated 
nuclear copies of mtDNA were not amplified. 
There was no intraspecific variation within these 
seven sites with the exception of one Saltmarsh 
Sparrow at one nucleotide site (which was not 
within the restriction site), nor when comparing 
our Nelson's samples with those on GenBank. 
There also was no intraspecific variation when 
comparing our spccies-speeific samples collected 
within and outside of the overlap zone, confirming 
that our reference individuals tor each species 
were correctly identified in the field. We observed 
0.2% inlraspccific variation in Saltmarsh Spar¬ 
rows and no variation within Nelson’s Sparrows. 
Results from our genetic assay revealed that 34 
of 426 ( 8 %) putative Saltmarsh Sparrows had 
Nelson’s mtDNA (Fig. 1: pic charts). Over half 
(18) of the sparrows with Nelson’s mtDNA were 
captured in Parker River NWR, Newburyport, 
Massachusetts (Fig. I: pie charts). The most 
southern site at which we identified an individual 
with Nelson’s mtDNA was in John H. Chafee 
NWR in Narragansett, Rhode Island (1 of 54 
individuals). Sequences from the 34 individuals 
identified as putative hybrids were all consistent 
with sequences from Nelson’s Sparrows at the 
seven diagnostic nucleotide, sites. 
Results from the ANOVA indicated significant 
differences in mass (F , S8 - 10 . 8 , P < 0 . 0001 ) 
bill depth (F, , 75 = 10-5. P< 0.0001). bill w.dth 
... = ^0 9 P < 0.0001), and culmen length 
(F '' 75 = 40 . 1 , p < 0.0001). Tukey’s pairwise 
comparisons of the four morphological variables 
indicated bill measurements and body mass were 
significantly smaller in pure Nelson's Sparrow 
individuals than pure Saltmarsh Sparrow individ¬ 
uals. Hybrid measurements were statistically 
indistinguishable from those of Saltmarsh Spar¬ 
rows and larger then those of Nelson's Sparrows 
(Table 1). 
