318 
THE WILSON JOURNAL OF ORNITHOLOGY • Vol. 123. No. 2. June 2011 
FIG. I. Saltmarsh and Nelson’s sparrow sampling locations in the northeastern United States. Boxes represent marshes 
where pu re Nelson’s (PON) and Saltmursh (WNWR and MNC) sparrow samples were collected (from Penobscolt. Maine 
and W ertheim NWR and Marine Nature Center in Shirley and Oceanside. New York, respectively). Circles indicate when; 
pmanv-e Sal"marsh Sparrows were sampled and pie charts represent the proportion of sampled individuals identified as 
hybrid. Matsh names are followed by individual marsh codes that correspond to the map and numbers of hybrids identified 
are in parentheses: Scarborough State Wildlife Management Area. Scarborough. Maine. SCAR (4); Rachel Carson NYVR 
Furbish marsh Wells. Maine. R( F (2): Chapman’s Landing. Stratham. New Hampshire. CL (5); Fairhill marsh. Rye, New 
Hampshire. FH (0). Hampton Beach. New Hampshire, HB (4); Parker River NWR. Massachusetts. PR HS>: and John H. 
Chafee NWR, Narragansett. Rhode Island. JHC (1). Crosshatched area represents the currently recoeni/cd Nd«nV 
Saltmarsh sparrow overlap zone. 
York (Marine Nature Center: 40 37.27' N 71 
37.38' W). 
Genetic Identification.—We used a DNA bar¬ 
coding approach (Hebert et al. 2003) to develop 
our assay for species identification. DNA was 
extracted from known Nelson’s (n = 11) and 
Saltmarsh sparrow (;j = 12) samples using a 
DNeasy Blood Kit (Qiagen, Valencia. CA. USA). 
Universal avian primers (BirdFl and BirdR2) 
were used to amplify a 648 base pair region of the 
cytochrome c oxidase I (COI) gene in a 12.5 pi 
polymerase chain reaction following Hebert et al. 
(2004). Samples were sequenced by Geneway 
Research LLC (Hayward. CA. USA) or by the 
Hubbard Center for Genome Studies at the 
University of New Hampshire. 
Nelson’s and Saltmarsh sparrow sequences 
(Genbank accession numbers HM230725- 
HM230747) were edited and aligned in Geneious 
Pro 4.7.6 (Biomatters Ltd. Auckland. NZ> t° 
identity variation within and between species. We 
included three Nelson’s Sparrow sequences front 
GenBank (accession numbers DQ433298. 
DQ432709, and DQ432708) in our alignntenL 
These specimens originated from Minnesota and 
Illinois (inidwestem U.S.) and were identical t" 
the sequences from our Nelson’s reference 
individuals despite being representative ot a 
different subspecies (A. n. nelsoni). 
We found seven diagnostic nucleotide differ 
ences between Nelson's and Saltmarsh sparrow 
We identified a single nucleotide difference in a 
