• Stewart, Kathryn A.
  • Austin, James
  • Zamudio, Kelly R.
  • Lougheed, Stephen C.


Characterizing the genetic and behavioural consequences of contact between previously geographically isolated lineages provides insights into the mechanisms underlying diversification and ultimately speciation. The spring peeper (Pseudacris crucifer) is a widespread Nearctic chorus frog with six divergent mitochondrial DNA (mtDNA) lineages, many of which came into secondary contact during the Holocene. We examined genetics, morphology, advertisement calls and female preference for two lineages that began diverging in allopatry in the Pliocene and now overlap in southwestern Ontario, Canada. We found non-coincident clines in mtDNA and nuclear DNA, mirroring directionality of premating isolation barriers. We also found divergence in a range of traits between these two lineages, displacement in male call attributes and female preference for calls of their natal lineage in sympatry. Hybrids were morphologically distinct from both parental lineages, but hybrid male calls were acoustically intermediate. Female hybrids showed asymmetrical preference for Eastern male calls. These results considered together provide evidence of either unidirectional hybridization or selection against hybrids, potentially implying reproductive character displacement. Our work demonstrates the utility of integrated, multi-character approaches to understanding the processes of divergence and the nature of speciation.


Study species

Spring peepers are small (20 to 30 mm snout–vent length) chorus frogs with females slightly larger than males. Mate selection by females is mediated primarily by male vocalizations (Forester and Czarnowsky, 1985). In North America, spring peepers are among the first frogs to breed in spring, and their breeding season is prolonged. In southern Ontario, Canada, male spring peepers typically call from early April until early June, with peak breeding in May.

Sampling design

We examined geographic variation in female preference, neutral DNA markers and acoustic and morphological traits of spring peepers (P. crucifer) in a zone of secondary contact in southwestern Ontario (Figure 1), consisting of admixture of Interior and Eastern mtDNA lineages. In total, 32 populations were sampled from 2003 to 2011 to obtain genetic, morphological, acoustic and female preference data (Figure 1 and Table 1).

We sampled 579 reproductive adults between April and June (2100 to 0200 h local time) collected across a 580-km straight-line sampling distance. Of these, 185 males from a total of 25 populations were used for call analysis: 10 mtDNA contact zone populations (breeding aggregations consisting of both Eastern and Interior lineages), 6 pure Interior populations (that is, no Eastern mtDNA) and 9 pure Eastern populations (that is, no Interior mtDNA). Full genetic and morphological analysis used data from 429 individuals (including 86 females) sampled from 4 previously delineated mtDNA contact zone populations, 5 pure Interior and 5 pure Eastern populations (Table 1). Of the 429 individuals, we were unable to amplify 6 spring peeper (4 male, 2 female) mtDNA sequences sampled within the contact zone and thus were left with 423 individuals.