The Wilson Journal of Ornithology 123(2):316—322, 2011 
GENETIC BARCODE RFLP ANALYSIS OF THE NELSON’S AND 
SALTMARSH SPARROW HYBRID ZONE 
JENNIFER WALSH. 1 ADRIENNE I. KOVACH. 1 4 OKSANA P. LANE.-’ 
KATHLEEN M. O'BRIEN, 1 AND KIMBERLY J. BABBITT 1 
ABSTRACT.—Hybridization between Saltmarsh Sparrow (Ammodramus caudacutus ) and Nelson's Sparrow (A 
nelsom) has been documented in areas where the two species occur sympatrically. increasing the difficulty of accurate 
species identification. We developed a DNA barcoding restriction fragment iength polymorphism (RFLP) test to 
discriminate between Nelson’s Sparrows and Saltmarsh Sparrows and applied it to 426 putative Saltmarsh Spaiir"> 
sampled from Maine to New York. USA. All individuals were identified in the field as Saltmarsh Sparrows based on 
morphology, but 34 (Wo hud Nelson's specific mitochondrial DNA. indicating they were of hybrid origin, This 
discrepancy m morphological and genetic data highlights the difficulties associated with accurate field identification and 
may hinder conservation efforts by confounding attempts to identify and monitor "pure" populations. Mitochondrial DNA 
of Nelson s Sparrow was prevalent at the most southern point of the previously documented overlap zone and was also 
found in one individual 150 km south of the overlap zone. Our findings offer new insights into the extent of hybridization 
between the two species and underscore the need for further investigation into the consequences of hvbridi/aucn mi 
conservation ol Saltmarsh Sparrows. Received 23 August 2010. Accepted I January 2011. 
The distribution and taxonomic classification of 
Nelson s Sparrow ( Ammodramus nelsoni) and 
Saltmarsh Sparrow ( A. caudacutus) have been a 
topic of ornithological debate for over a century 
(Greenlaw 1993, Rising and Avise 1993, Shriver 
et al. 2005). In 1995. the five previously 
recognized subspecies of A. caudacutus (Ameri¬ 
can Ornithologists’ Union 1957) were split into 
the two species recognized today. The elevation to 
species status was based on observed variation in 
morphological features, mitochondrial DNA 
(mtDNA), and behavior in Nelson's and Salt- 
marsh sparrows (Greenlaw 1993, Rising and 
Avise 1993) and the assumption of limited 
interbreeding (American Ornithologists' Union 
1995), despite recommendations for retaining the 
subspecies classification (Rising and Avise 1993). 
Both species breed in coastal marshes along the 
northeastern United States. A subspecies of 
Nelson’s Sparrow (A. n. subvirgatus) breeds in 
marshes from coastal Quebec to northeastern 
Massachusetts, and the two subspecies of Salt¬ 
marsh Sparrow (A. c. caudacutus and A. c. 
diversus) breed from Maine to New Jersey and 
New Jersey to Virginia, respectively (Greenlaw 
'.Department of Natural Resources and the Environment, 
University of New Hampshire. James Hall, 56 College 
Road, Durham, NH 03824. USA, 
2 Biodiversity Research Institute. 19 Flaggy Meadow 
Road. Gorham, Ml! 04038, USA. 
3 U.S. Fish and Wildlife Service, Rachel Carson National 
Wildlife Refuge, 321 Port Road, Wells, ME 04090, USA. 
Corresponding author; e-mail: 
Adrienne.Rovach @ unh.edu 
and Woolfenden 2007). The two species occur 
sympatrically in tidal marshes along the Atlantic 
Coast, bul Nelson’s Sparrows also inhabit less 
tidal, brackish marshes as well as upland habitats, 
including grasslands and hayfields (Nocera et al. 
2007); Saltmarsh Sparrows are restricted to 
expansive, intertidal areas that are influenced 
strongly by tidal flow (Greenlaw 1993. Shriver et 
al. 2005). 
Hybridization between Nelson's and Saltmarsh 
sparrows, despite interspecific variation (morpho¬ 
logical, behavioral and genetic), has been previ¬ 
ously documented within an overlap zone from 
the Weskeag River estuary in South Thomaston. 
Knox County. Maine (44 04.60' N. 69 08.66' 
W) to Parker River National Wildlife Refuge 
(NWR) in Newburyport. Essex County. Massa¬ 
chusetts (42’ 77.42' N, 70' 80.86' W) (Rising and 
Avise 1993, Hodgman et al. 2002, Shriver el al- 
2005). Shriver et al. (2005) found a concordance 
between genotypic and phenotypic variation in 
hybrid sparrows from three marshes in southern 
Maine. The authors suggested hybrids occur 
wherever the two species are sympatric, which 
would manifest in a I90-km reduction in the range 
of "pure" Saltmarsh Sparrow populations. The 
implications of this range reduction suggest the 
need for additional research to monitor the rate ol 
occurrence of hybrids in marshes throughout the 
overlap zone. 
Hybridization is common in nature and espc 
daily in birds, for which it has been recorded in 
almost one of 10 species (Grant and Grant 199-) 
It typically occurs in young pairs of sibling 
316 
