Jeremy A. Feinberg; Catherine E. Newman; Gregory J. Watkins-Colwell; Matthew D. Schlesinger; Brian Zarate; Brian R. Curry; H. Bradley Shaffer; Joanna Burger

“ The long-term concealment and recent discovery of a novel anuran here is both surprising and biogeographically significant, and illustrates how new species can occur almost anywhere. It also raises potentially important conservation concerns: amphibians can be sensitive to disease, contaminants, and environmental perturbations, and their low vagility can be particularly problematic in fragmented and urban landscapes [9] . Also worrisome are enigmatic declines that have led to disappearances of leopard frogs from parts of the Northeast and mid-Atlantic US [10] – [11] ”

“ The overall conservation status of R. kauffeldi awaits further definition of distribution and habitat use and should be considered data deficient in the IUCN classification system. On-the-ground assessments, coupled with genetic and bioacoustic data, will be critical to this and allow for more complete mapping of boundaries and overlap with related taxa. If the distribution is indeed narrow and fragmented (as reported here) , it may pose some cause for concern as geographically restricted species are often at risk of extinction due to demographic stochasticity [73] . ”

“ Moving species to restore extirpated populations is a common conservation and management practice, but one that can have unintended risks and consequences. For example, had a leopard frog restoration been implemented on Long Island before the 2007 discovery of extant populations on nearby Staten Island (that were later found to be R. kauffeldi) , the incorrect species (R. sphenocephala) would have been moved from known populations farther to the south that harbor R. sphenocephala, not R. kauffeldi. ”

“ Few examples of undescribed vertebrate diversity exist in the recent literature from highly urbanized regions and areas with well-established taxonomic infrastructures.
The species we describe here was first identified by Newman et al. [3] via molecular data. It constitutes the newest member of the Rana pipiens complex and occupies parts of the lower Northeast and mid-Atlantic US within the densely populated and heavily industrialized Interstate-95 (I-95) corridor. This is one of the largest human population centers on earth [8] and a region where endemic vertebrate species are rare. ”

“ It also reinforces the critical role that basic natural history and alternative methods, such as bioacoustic techniques, can have in distinguishing potentially rare cryptic species. Lastly, it demonstrates that undocumented species can still reside in some of the most urbanized and densely inhabited parts of the world; these areas can harbor significant biodiversity and, with proper management, simultaneously protect that diversity and provide valuable educational opportunities to urban communities. ”

“ We collected morphometric measurement data from 283 specimens, including the holotype (YPM 13217) and 282 other museum specimens across four species (R. sp. nov., R. sphenocephala, R. pipiens, and R. palustris) , 30 US counties, seven eastern states, and Quebec, Canada (Table S1) . When genetic data were not available to confirm species identification, we used a combination of morphology and location to classify preserved specimens based on our knowledge of species habitat preferences and distributions (Fig. 1) . ”

“ The new species typically occurs in expansive open-canopied wetlands interspersed with upland patches, but centuries of loss and impact to these habitats give some cause for conservation concern. Other concerns include regional extirpations, fragmented extant populations, and a restricted overall geographic distribution. We assign a type locality within New York City and report a narrow and largely coastal lowland distribution from central Connecticut to northern New Jersey (based on genetic data) and south to North Carolina (based on call data) . ”

“ For one, this discovery clearly demonstrates that human knowledge of the natural world remains incomplete even in the best-known locales. Second, although new frog discoveries are generally uncommon north of Mexico, they do still occur periodically. Third, the two most recent examples (R. kauffeldi and P. fouquettei [6] ) are both cryptic species. Taken together, these points suggest that occasional future discoveries from well-cataloged areas may continue, but probably in the form of additional cryptic species rather than morphologically distinct taxa (which are likely already cataloged) . ”

“ Lastly, based on museum specimens from areas where leopard frogs are now extirpated, we note several isolated examples of possible R. sphenocephala from xeric eastern Long Island, NY, and R. pipiens from Queens and Bronx Counties, NY (Table S1) . Historical species composition in these areas remains unclear, however. These sparse samples may reflect natural historical populations (and potential areas of overlap with R. kauffeldi) or possible human introductions; isolated geographic records can suggest captive releases [3] , [22] , particularly in urban areas. ”

“ Potential sympatry is also possible in the yellow shaded areas, with R. sphenocephala (from Long Island southward) , or R. pipiens (north and west of Long Island) . The type locality for R. kauffeldi is indicated by an arrow. We looked for univariate differences in species morphology using boxplots and one-way ANOVAs followed by Tukey HSD post-hoc pairwise comparisons. We used discriminant function analysis (DFA) to examine variation in multivariate space and determine which variables best discriminated among species. ”

“ Findings such as this also provide invaluable opportunities to highlight and enhance access for increasingly urban societies to experience new species discoveries and taxa of high conservation concern firsthand. ”

“ Most offer large habitat areas, however, which may provide an important clue to survival. The surprising persistence of populations within these urban landscapes, while not completely understood, is encouraging and may have implications for management and restoration possibilities elsewhere, in the future. ”

“ Rana kauffeldi appears to occur parapatrically in this core area. Beyond the core area, we depict an extended area of potential occurrence (Fig. 1, yellow shading) based on habitat features and proximity to known bioacoustic confirmations in DE, MD, VA, and NC. Within the yellow shading we also note the potential for sympatry with R. sphenocephala (in the south) and R. pipiens (in the north) based on genetic, bioacoustic, and specimen sampling (see Discussion) . ”

“ We have not observed opportunistic mid-summer chorusing as we and others [19] , [63] have for R. sphenocephala, but we have observed occasional second breeding periods with the onset of cooler autumn temperatures and precipitation (late August through November) .
Individuals may exhibit a limited degree of color change around a general base color that can vary widely between frogs, from light green to dark brown. Holmes [64] noted that leopard frogs (sensu lato) tend towards darker nocturnal shading and brighter, more vivid diurnal colors (as a putative mode of camouflage) . ”

“ It may also provide further opportunity to investigate and verify species composition and boundaries throughout different parts of the range. This may be challenging, however, especially in states where leopard frogs (sensu lato) already receive legal protections and in areas where multiple species are found to co-occur. Thus, reliable, field-ready characters that distinguish similar taxa, and research on potential hybridization, are key priorities. ”

“ Table 2 "Mean primary (advertisement) call parameters for five species of Rana. (10.1371/journal.pone.0108213.t002) " Seven bioacoustic characters are listed as follows: call length (CL) , call rate (CR) , call rise time (CRT) , call duty cycle (CDC) , pulse number (PN) , pulse rate (PR) , and dominant frequency (DF) . Mean includes ± standard deviation (SD) . Note: the above values come from unadjusted (raw) data; in all other bioacoustic analyses CL and CR were corrected to a common temperature of 14°C, following Lemmon et al. [6] . ”

“ This species is usually abundant where it occurs, but populations in the NY/NJ-metro area tend to be disjunct and isolated from one another and often occur in highly fragmented landscapes with limited connectivity or dispersal opportunities. ”

“ Lower flank of holotype pale with light yellowish-green hues and smaller, lighter spots and mottles; these intrude onto ventral margins, throat, or body in some individuals. Tympanum finely granulated brown color with black flecks; central spot creamy and subtly defined in holotype; bright and well defined or entirely absent in some individuals. Reticulum and anterior ventral margin of thigh dark with distinct light flecks or mottles ”

“ Others, however, occurred enigmatically within less-developed coastal, suburban, and semi-rural areas (Fig. 1) ; this includes Long Island [3] , [11] , the largest island in the continental US and a former leopard frog stronghold [10] where potential causes of extirpations (e.g., disease, invasive species, and contaminants) are being assessed [75] (J. A. Feinberg and J. Burger, unpublished data) . Counterintuitively, R. kauffeldi persists in several locales within New York City (Staten Island) and the adjacent NJ Meadowlands. ”

“ Forearms relatively short and robust; unwebbed fingers; relative length III>I>II>IV. Fingers lack fringes; tips rounded without expansion; subarticular tubercles small, round, and moderately prominent. No palmer tubercles appear present. First finger slightly swollen at base with faint nuptial pad; all other fingers slender. Hindlimbs relatively long, moderately robust; thigh and shank length nearly equal. Relative toe lengths IV>V>III>II>I; toes have rounded tips without expansion; subarticular tubercles small, round, and prominent. ”

“ Species regulation in the Hyla versicolor complex. Texas J Sci 18: 361–364 ↑ Lande R (1988) Genetics and demography in biological conservation. Science 241: 1455–1460 doi:10.1126/science.3420403 pmid:3420403↑ Rorabaugh JC (2005) Rana pipiens , Schreber 1782 northern leopard frog. In: Lannoo MJ, editor. Amphibian declines: the conservation status of United States species. ”

“ A new North American chorus frog species (Amphibia: Hylidae: Pseudacris ) from the south-central United States. Zootaxa 1675: 1–30 1 2 3 4 5 6 7 8 9 10 Frost DR (2013) Amphibian species of the world: an online reference, v5.6 (9 January 2013) . New York, USA: American Museum of Natural History. Available: http://research.amnh.org/herpetology/amphibia/index.html Accessed 2013 November 19.1 2 Florida R, Gulden T, Mellander C (2008) The rise of the mega-region. Cambridge J Regions Econ Soc 1: 459–476 doi:10.1093/cjres/rsn0181 2 3 Wells KD (2007) The ecology and behavior of amphibians. ”

“ A new species of leopard frog (Anura: Ranidae) from the urban northeastern US. Mol Phylogenet Evol 63: 445–455 doi:10.1016/j.ympev.2012.01.021 pmid:223216891 2 3 Funk WC, Caminer M, Ron SR (2011) High levels of cryptic species diversity uncovered in Amazonian frogs. Proc R Soc B 279: 1806–1814 doi:10.1098/rspb.2011.1653↑ Köhler J, Vietes DR, Bonett RM, García FH, Glaw F, et al. . (2005) New amphibians and global conservation: a boost in species discoveries in a highly endangered vertebrate group. ”