Focal taxa

Although the existence of three distinct northeastern leopard frogs has been well demonstrated through recent molecular and bioacoustical research [21,23], the natural history of these species has remained uncertain since the discovery of R. kauffeldi. This recent uncertainty is pre-dated by a long history of earlier confusion: despite the fact that some herpetologists had suggested that there might be undocumented leopard frog species in the northeastern U.S. [24–26], all of the region’s leopard frogs were considered R. pipiens for a substantial part of the previous century. Eventually that single-species framework gave way to a two-species taxonomy in the eastern US (adding R. utricularia, later renamed R. sphenocephala) following Pace [27]. Decades later, Newman et al. [23] documented a distinct genetic lineage in leopard frogs from northern New Jersey, southern New York, and central Connecticut, and Feinberg et al. [21] formally described this new species as R. kauffeldi, reporting a broader range (Connecticut to North Carolina) documenting for the first time differences in the primary mating call, morphology, and color pattern compared to either R. pipiens, mainly to the north, and R. sphenocephala, whose more southerly range appeared to overlap partially with that of R. kauffeldi.

The preliminary range map in Feinberg et al. [21] was based on several lines of evidence: genetics from the New York City metropolitan area, museum specimens whose locality and physical appearance could be reliably associated with each species, and bioacoustical sampling from as far south as North Carolina. Those authors used data from three sites with co-located bioacoustics and genetic information to support their conclusion that the distinct mating call they documented was from a genetically distinct species. In addition, Feinberg et al. [21] built on previous observations of leopard frog patterning and morphology [24,25,27,28] to propose a set of characteristics for reliable distinction among the three species. Feinberg et al. [21] concluded that additional research was needed to ensure that the patterns reported based on their few samples were reliable range-wide, and so could be used to resolve areas of distributional uncertainty across the entire putative range of R. kauffeldi and to confirm characters that could be used to reliably distinguish animals in the field without genetics or bioacoustics.

In this paper, we provide a particularly complete example of the follow-up research that is necessary when the taxonomy is sorted out for a cryptic species complex, using the leopard frogs of the eastern U.S. as a case study. We refined the range of R. kauffeldi relative to the two other recognized leopard frog species in the region so that managers and researchers in the ten states represented by our authorship team were better informed about which species were present in their state, and mapped new, ecologically informed ranges for all three taxa. Updated knowledge of the three species’ distributions then informed our updated assessments of conservation status, which were especially important in areas where leopard frogs (sensu lato) are already of concern. Finally, we used confirmatory genetic data to improve methods for separating the three species using acoustical and morphological field characters that should facilitate future inventory, monitoring, status assessments, and conservation actions.

Call and visual surveys

To locate populations of leopard frogs and identify sites for subsequent sampling, we conducted call surveys in a variety of wetland habitats throughout the study area. Dates of surveys ranged from late February to late March in southern latitudes and from late April to early June in northern latitudes in 2014 and 2015. Because many survey partners had existing frog monitoring programs, and volunteers comprised a portion of the workforce, we allowed for considerable flexibility in survey methodology. At a minimum, observers recorded the GPS coordinates of sampling locations, time spent listening, and species detected. Sampling locations were selected by observers based on habitat suitability, access, and safety considerations. Large wetlands could have multiple sampling locations. Observers were asked to spend a minimum of 3 minutes (though some spent fewer) at each sampling location and to record the survey duration. If survey duration was not recorded, we assumed that it was 3 minutes. Surveys began no earlier than one half-hour after sunset and ended by 1:00 a.m. Surveys were not conducted when temperatures were below 4 degrees Celsius or in heavy rain or high wind. Observers made audio recordings of calls of suspected leopard frogs and the acoustically similar (to R. kauffeldi) Wood Frog (R. sylvatica) for subsequent confirmation from members of the team most familiar with the species.

From spring through fall in 2014 and 2015, we visited sites where we had confirmed the presence of leopard frogs bioacoustically to capture frogs for photographic and genetic analyses. We also visited sites of unknown occupancy and suspected historical occupancy for visual surveys to determine presence. If we encountered leopard frogs, we captured and photographed them following standardized digital imaging protocols (below), clipped the last digit of one toe as a tissue samples, stored the sample in 95% ethanol and shipped them to the University of California, Los Angeles (UCLA) for genetic analysis. We followed the State University of New York College of Environmental Science and Forestry’s Institutional Animal Care and Use Committee protocol #140102, developed specifically for this project, for the temporary care and handling of captured frogs.

Photography

To aid in the identification of potentially reliable field characters, we photographed captured frogs from several specific angles (Fig 1) to clearly show the dorsal and ventral surfaces, snout profile, femoral reticulum, tympanum (right and left), and hind foot toe webbing, and matched each image with its corresponding tissue sample. This allowed us to explore many possible diagnostic features, including some of those referenced by Feinberg et al. [21] as well as earlier studies by Porter [29] and Moore [28].

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Fig 1.

Example of photographs of a captured leopard frog taken from prescribed angles (clockwise from lower left): right side, left side, dorsal, ventral, femoral reticulum, hind foot; and photo measurements (lower right) of head width (A), head length (B), snout width (C), and snout length (D).

https://doi.org/10.1371/journal.pone.0205805.g001

We used our photographic data set to test the validity of these characters across the suspected range of R. kauffeldi. A single observer (ELW) with no explicit knowledge of character states suspected to differentiate these species evaluated photos of each genotyped frog to assess 1) number of dorsal spots from snout to vent occurring between the dorsolateral folds (conjoined spots were counted as two spots); 2) snout spot (large, small, or absent); 3) snout shape (blunt, pointed, or intermediate); 4) reticulum coloration (predominantly dark, predominantly light, or intermediate); 5) reticulum pattern (mostly large, connected blotches; or mostly small, unconnected dots); 6) left and right tympanum spot pattern (sharp dot, sharp blotch, or present but indistinct); 7) left and right tympanum spot color (white/cream, green, or brown/bronze); and 8) webbing on the first toe of the left and right hind food (curves all the way to the tip or stops about halfway to the tip). For all characters evaluated qualitatively, the observer had exemplar photographs to guide in interpretation (S1 Appendix).

We also used our dorsal photographs of genetically confirmed R. kauffeldi and R. sphenocephala to quantify snout shape and reticulum coloration, since these characters had been suggested previously to help distinguish these two species [21,29]. For snout shape, we imported each photo into ArcGIS 10.3 [30] and drew lines in four locations (Fig 1): A) along the posterior edge of the eyes, perpendicular to the spine of the frog, connecting the visible edges of the head (“head width”); B) perpendicular from the tip of the snout to line (A) (“head length”); C) along the anterior edge of the eyes but otherwise as in (A) (“snout width”); D) from the tip of the snout to line (C) (“snout length”). We recorded the length of each line in arbitrary map units, and calculated three ratios: head length to head width (B/A), snout length to snout width (D/C), and snout width to head length (C/B). We characterized a subset of reticula using image processing software (S2 Appendix).

Genetic analysis

Genomic DNA extraction and PCR amplification were performed at UCLA following the methods in Newman et al. [23]. We amplified the nuclear genes neurotrophin-3 (NTF3, 599 bp), tyrosinase (Tyr 557–585 bp), Rag-1 (647–683 bp), seven-in-absentia (SIA, 362–393 bp), and chemokine receptor 4 (CXCR4, 550 bp). PCR products were bidirectionally sequenced at Beckman Coulter Genomics (Danvers, MA, USA). Contigs were assembled and trimmed in Geneious version 6.1.6 [31]. Consensus sequences for each locus were aligned with sequences from Newman et al. [23] using ClustalW in Geneious and manually adjusted.

To determine how all of the samples grouped into genetic population clusters, we used Structure version 2.3.2 [32,33] with an allelic data set (12–15% missing data) derived from our sequence data. We inferred haplotypes for each locus with a Bayesian algorithm using Phase version 2.1.1 [34,35]. Each allele was represented by a single haplotype. Phase input files were formatted from NEXUS files using a Perl script (RC Thomson, unpublished).

Following the same parameters as Newman et al. [23], we used Structure to determine the number of genetically distinct clusters (K) in our complete data set. We used the admixture model [32] and assumed correlation of allele frequencies among clusters and no other a priori population information. We ran 20 iterations of K values from 1 to 10, each with 100,000 generations plus a burn-in of 100,000 generations. We chose the appropriate K value by obtaining likelihood scores using the Evanno method [36]. We then assigned species identifications based on their genetic makeup: frogs were designated as “pure” if they had a 90% or greater match with a single species, while those with a species match between 10% and 90% were designated as “admixed.” We confirmed the robustness of genetic assignment of individuals to PHASE parameter choice and that these results were further validated with likelihood-based phylogenetic methods (S3 Appendix).

Observation data and museum records

To supplement our field work and inform our reconstructions of the historical and current ranges of each species, we gathered observational and survey data from a variety of sources, including those outside the project team in cases where call recordings were available to confirm species identification. To determine the historical ranges of the three leopard frogs, we examined 2,522 museum specimens from the following institutions: American Museum of Natural History, Carnegie-Mellon Museum of Natural History, Cornell University Museum of Vertebrates, Harvard Museum of Comparative Zoology, National Museum of Canada, North Carolina Museum of Natural Sciences, Southern Connecticut State University, Smithsonian National Museum of Natural History, Texas Cooperative Museum, University of Connecticut, and Yale Peabody Museum of Natural History. Based on identification characters from Feinberg et al. [21] and our own findings, we determined probable species identifications that we used to inform maps of historical ranges. In a small percentage of cases (1–2% of specimens examined) where color patterns had faded or the typical characters used to determine species were unclear, we either made our best informed identification or did not assign a species; these few specimens did not substantially affect our historical distribution assessments. Most or all of these historical specimens had been preserved in formalin, making molecular examination challenging [37].

Data analysis

Determining conservation status

To help prioritize conservation and management actions, we suggested conservation status ranks for R. kauffeldi for its entire range and for each state in which it was documented. We used the NatureServe and Natural Heritage Program methodology [45,46], to determine conservation status by evaluating a suite of factors representing rarity, threats, and trends to arrive at a G-rank (“global” rank, for the entire range; Table 1) or S-rank (for a state or other subnational jurisdiction). We focused on three rarity factors that we felt our data could best inform—range extent (area encompassed by the outer boundary of presence points), area of occupancy (number of 4-km² grid cells occupied), and number of occurrences (estimated by counting a detection as a separate occurrence if separated by 5 km of suitable habitat or 1 km of unsuitable habitat; [47]). For each of these categories, the methodology uses wide value ranges and provides the opportunity to select multiple scores to encapsulate uncertainty.

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Table 1. Conservation status ranks used in the NatureServe methodology [45,46].

From http://www.natureserve.org/conservation-tools/conservation-status-assessment. At the subnational level, S-ranks are used.

https://doi.org/10.1371/journal.pone.0205805.t001

Our data were less well suited to addressing threat and trend factors, largely due to the lack of species-specific information on threats. Thus, we used the optional factor intrinsic vulnerability, a surrogate for threats representing susceptibility to impacts from human activities based on life history. Amphibians are highly sensitive to disease [48] and aquatic pollutants [49,50], and R. kauffeldi’s proximity to the coast makes it vulnerable to habitat degradation and loss from coastal storms and rising sea levels [21,51]. Rana kauffeldi and R. sphenocephala were both found to be susceptible to several amphibian diseases (including chytridiomycosis, ranavirus, and a perkinsus-like organism) when reared in outdoor pens on Long Island, in wetlands where leopard frog complex members are now extinct (JAF, unpublished data). On the other hand, R. kauffeldi is known from wetlands near heavy industry [21], suggesting it may be relatively insensitive to environmental toxins if suitable freshwater habitat exists. Some of these industrial wetlands in southern NY and northern NJ are threatened by development. We characterized intrinsic vulnerability as the combination rating “High or Moderate” for all jurisdictions to reflect the balance between these susceptibilities and apparent tolerances.

Information on trends was mostly lacking given the recent discovery of this species and the difficulty of identifying older museum specimens with certainty. We calculated a G-rank for the overall range and S-ranks for each state in two ways: using NatureServe’s element rank calculator [52] and based on our interpretation of the data.

Call surveys

We conducted call surveys at 1,004 point locations throughout the northeastern U.S. (Fig 2). Most points were surveyed once, with some surveyed multiple times, for a total of 2,159 surveys. Survey durations ranged from 1 to 110 minutes (mean = 5.76 minutes) and totaled approximately 207 hours. Surveys were conducted between 21 February 2014 and 15 June 2015. Additional call survey data dating back to 2006 were included as available.

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Fig 2. Leopard frog presence and non-detections from call surveys and genetic analysis in the northeastern U.S. Question marks are directly below presence points where identifications from genetic analysis were questionable.

Shaded relief map from https://nationalmap.gov/.

https://doi.org/10.1371/journal.pone.0205805.g002

Calls from R. kauffeldi were documented as early as 10 March 2014 in MD and 14 March 2015 in DE. Outside the formal window of our field work, calls were documented on 3 February 2016 in NC, 21 February 2017 in VA, and 8 March 2016 in NY. The last call dates for R. kauffeldi in the two project years were 23 April 2014 in PA and 15 April 2015 in NJ. Calls have been heard sporadically in late April and May in NY (JAF, unpublished data), and 12 June 2006 (with the identification confirmed later) in NJ’s Meadowlands [53].

Genetics

We collected tissue samples from 254 individual frogs for genetic analysis from throughout the putative range of R. kauffeldi and beyond to include samples of all three species across the region. Of these samples, 251 were successfully extracted, amplified, and sequenced. Three samples had DNA concentrations that were too low for successful PCR amplification and were removed from the analysis. Aligned sequence lengths for nuclear loci were similar to those for a broader set of leopard frog samples for the same loci [23]: 527 bp (CXCR4), 530 bp (NTF3), 648 bp (Rag-1), 388 bp (SIA), and 549 bp (TYR). There were between 31 and 49 variable sites per locus. Individuals that received greater than three null haplotypes were removed from the analysis. In total, 243 of the original 251samples plus 50 individuals determined to be pure R. kauffeldi, R. pipiens, or R. sphenocephala by Newman et al. [23] went into the final analysis, for a total of 293 frogs. The results of Structure analyses from both the 80% and 90% probability Phase data sets were quantitatively similar (S3 Appendix); given that the 80% Phase probability set was slightly larger, we use it in the remaining analyses.

Bayesian cluster analysis in Structure resolved three clusters (lnL = 4314.60, DK = 130.93). Individuals with a cluster probability greater than 90% were assigned to that cluster, and individuals with a cluster assignment between 10% and 90% were designated as admixed. We assigned species identification to each cluster using the individuals from Newman et al. [23] as reference samples. A total of 262 individuals fell into one of the three clusters unambiguously, including 111 R. kauffeldi, 79 R. sphenocephala, and 72 R. pipiens (Fig 2). The remaining 32 individuals were considered potential hybrids that we identified by the dominant species based on admixture proportions.

The concatenated maximum likelihood phylogeny identified three major, but poorly supported, clades representing R. kauffeldi, R. pipiens, and R. sphenocephala. There were also a few individuals that could not be attributed to any of these three clades and were generally identified as admixed in the Structure analysis.

We identified and omitted from further analysis four samples that appeared, upon genotyping, to have identification or locality errors. Two samples from one site in central CT appeared to have their labels switched: one was visually R. pipiens but was genotyped as R. kauffeldi, while the other was visually R. kauffeldi but was genotyped as R. pipiens. Both species are known to occur at this site based on other individuals, and together with another site in central CT are the only known sympatric sites for these two species. Two additional samples are identified with question marks in Fig 2. One frog from the southern tip of the Delmarva Peninsula (Northampton Co., VA) appeared visually to be R. sphenocephala but was genotyped as R. kauffeldi. Only calls of R. sphenocephala have been documented in the area and appropriate habitat for R. kauffeldi does not appear to exist at that locality. An additional frog in western NJ near the Delaware River (Gloucester Co.) was genotyped as R. sphenocephala, but no other R. sphenocephala are known from that mesic area of the state. No photographs were associated with this sample, but another frog from the site appears visually to be R. kauffeldi. Leopard frogs from along the Delaware River (Delaware Co., PA) show field characters of both species, so we cannot rule out either species just across the river in NJ. All the above cases of confusion may be highly admixed frogs, and further investigation of these potential hybrid zones is certainly needed.

Species distributions

Rana kauffeldi.

We confirmed Rana kauffeldi in eight eastern U.S. states: CT, NY, NJ, PA, DE, MD, VA, and NC. We did not detect R. kauffeldi in MA or RI, where only R. pipiens was detected. The two locations farthest from one another in CT and NC are 746 km apart, close to the 780 km estimated by Feinberg et al. [21]. The range of R. kauffeldi that we estimate (Fig 3) covers just over 46,500 km² from 208 m elevation in northern NJ to sea level in multiple locations. No R. kauffeldi were detected south of the 17°C isocline (i.e., annual average temperature greater than 17°C). Confirmed extremes of the range of R. kauffeldi were as follows (in WGS84): northernmost—Middlesex County, CT (lat 41.63, long -72.62); southernmost—Washington County, NC (lat 35.79, long -76.41); easternmost—Middlesex County, CT (lat 41.60, long -72.61); and westernmost—Sussex County, VA (lat 36.98, long -77.27). A frog collected in 1989 farther south in Hyde, NC (USNM 292558) appears to be R. kauffeldi, but we lack bioacoustic and genetic confirmation at that location.

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Fig 3. Range of Rana kauffeldi, with presence points confirmed by bioacoustics or genetics, compared to ranges of R. pipiens and R. sphenocephala.

Historical ranges for R. kauffeldi and R. pipiens are based on examination of museum specimens and recent detections. Question marks denote locations where museum specimens appear to be R. kauffeldi but no bioacoustic or genetic evidence is available, or where genetic determinations were questionable.

https://doi.org/10.1371/journal.pone.0205805.g003

The species does not occur far from coastally influenced habitats. The maximum distance the species was documented from the ocean, bays, and estuaries was 40 km near the border of NJ and NY. Eighty-nine percent of R. kauffeldi locations were within 20 km, 77% were within 10 km, and just under 50% were within 1 km of coastal waters.

In portions of its range, R. kauffeldi overlaps with its close congeners. Rana pipiens and R. kauffeldi were documented to be syntopic at the two aforementioned sites in CT, and the ranges of R. sphenocephala and R. kauffeldi overlap broadly from central NJ south to NC, including several instances of syntopy. In central NJ, DE, and MD, they appear to be less frequently sympatric than in VA and NC.

Our survey data also support the view promoted by Feinberg et al. [21] that R. kauffeldi has disappeared from a large part of its historical range in southern NY and CT (Fig 3), including much of the Hudson Valley and all of Long Island. We could not verify recent reports of leopard frogs from these areas despite considerable survey effort.

Match of calls to genetics

We found a near-perfect match of population-level calling with genetics of individual frogs. At 16 sites for R. kauffeldi, 18 sites for R. sphenocephala, and 3 sites for R. pipiens, genetically pure frogs of each species were confirmed where calling by that species was documented within 100 m; the only mismatch was one location (Sussex Co., DE) where R. kauffeldi was documented calling but the genetic identification of an individual from that location was R. sphenocephala. The same concordance between calls and genetics held for situations where calling frogs were documented within 300 m of genetic samples (23 sites for R. kauffeldi, 21 for R. sphenocephala, and 11 for R. pipiens); the only potential exceptions were three genetically R. kauffeldi sites that had both R. kauffeldi and R. sphenocephala calling, and the above-noted genetically R. sphenocephala site with R. kauffeldi calls.

Morphological and color characters

We examined 912 photographs of 220 leopard frogs with genetic identities as follows: 80 pure R. kauffeldi, 16 admixed R. kauffeldi, 45 pure R. pipiens, 5 admixed R. pipiens, 64 pure R. sphenocephala, and 10 admixed R. sphenocephala. Not all 220 frogs had suitable photographs of all characters, so sample sizes for each character varied slightly. The comparisons below were based on examination of pure individuals only.

Rana kauffeldi had fewer dorsal spots on average than both R. pipiens and R. sphenocephala in a one-way ANOVA (F_174,2 = 15.08, P < 0.0001, with Tukey HSD post-hoc tests), although there was considerable overlap among species (S4 Appendix; Fig 4). Rana sphenocephala and R. pipiens had similar numbers of spots.

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Fig 4. Boxplot of number of dorsal spots for three species of leopard frogs in the northeastern US.

Open circles are statistical outliers.

https://doi.org/10.1371/journal.pone.0205805.g004

Rana kauffeldi was readily distinguished from R. pipiens by smaller body spots (85% of R. kauffeldi had spots smaller than the eye vs. 36% for R. pipiens), the frequent absence of a snout spot (85% absent vs. 29%), reticula characterized as predominantly dark (97% vs. 0%), and overall duller coloration (S4 Appendix; Fig 5).

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Fig 5.

Typical patterns in femoral reticula (top row) and dorsal spotting (bottom row) in three species of leopard frogs in the northeastern U.S.

https://doi.org/10.1371/journal.pone.0205805.g005

Rana kauffeldi and R. sphenocephala were more challenging to differentiate, as no single character reliably distinguished the two species. Nearly all R. kauffeldi reticula appeared predominantly dark with small, unconnected dots of light pigment, while most R. sphenocephala reticula appeared predominantly light with large, connected blotches of dark pigment (S4 Appendix; Fig 5). The overall coloration of R. kauffeldi was typically duller than that of R. sphenocephala. Rana kauffeldi had sharp tympanum spots less frequently (58% of frogs) than R. sphenocephala (91%), and the snout of R. kauffeldi was more frequently characterized as “blunt” (62% of frogs) than that of R. sphenocephala (13%). The three head measurement ratios were not significantly different between the two species in a one-way ANOVA, although the ratio of snout width to head length was nearly so (F_121,1 = 3.899, 0.05 < P < 0.10). The extent of toe webbing and the color of reticulum spots and blotches did not consistently differentiate the three species.

Characters used in combination provided accurate identification in most cases. Random forest analysis using 144 individuals with missing values imputed correctly classified R. kauffeldi and R. sphenocephala in over 90% of cases (out-of-bag estimate of error rate = 9.72%). The most important field characters in distinguishing between species were reticulum color, reticulum pattern, and snout shape. When the analysis was run with cases with missing values omitted (leaving n = 33 individuals), the error rate was similar (9.09%), although the order of important variables shifted somewhat, with ratio of snout width to head length, reticulum color, reticulum pattern, and ratio of head length to head width being most important.

Sample sizes of admixed individuals were insufficient for statistical analyses, but examination of the raw numbers (S4 Appendix) did not show the intermediacy of characteristics that would be expected to result from hybridization.

Habitat association and distribution model

Our surveys found that the basic habitat description in Feinberg et al. [21] holds. South of the glacial maximum, R. kauffeldi is a habitat specialist restricted to large coastal and riparian wetlands. In the southern portion of its range, it occurs primarily in riparian cypress-gum swamps. Along the Delaware River and Bay, it occupies large freshwater impoundments, tidal guts, and tidal freshwater marshes often dominated by introduced Common Reed (Phragmites australis) marshes that may be subject to salinity intrusions. In the northern, recently glaciated portion of its range, R. kauffeldi typically occupies large freshwater wetlands with open canopies that otherwise are indistinguishable from similar wetlands where it was not detected. Where R. sphenocephala is sympatric with R. kauffeldi, R. sphenocephala is a generalist, being found in nearly any semi-permanent (isolated) or permanent wetlands, created or natural, including tire ruts, fish hatchery ponds, waterfowl impoundments, and cypress-gum swamps. It also appears to be less restricted to xeric habitats in the southern portion of its range than in more northerly locations (e.g., New Jersey).

Our distribution model shows that suitable habitat for R. kauffeldi is concentrated along the coast and in riparian corridors (Fig 6). The model overall was very accurate, with an out-of-bag error estimate of 1.11%, although absences were predicted with greater accuracy (99%) than presences (34%). The factors most closely associated with the presence of R. kauffeldi were low elevation, high impervious surface and more wetlands in the surrounding landscape, and short distances to calcareous bedrock, saltwater or freshwater emergent wetlands, freshwater forested wetlands, and lakes and rivers.

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Fig 6. Distribution model for the full range of R. kauffeldi along the coastal northeastern U.S.

Inset: Delaware Bay and surrounding states. Values reflecting habitat suitability were broken into 10 quantiles, with the top five displayed.

https://doi.org/10.1371/journal.pone.0205805.g006

Conservation status of Rana kauffeldi

Because we recognized that a two-year survey, even when supplemented with pre-survey data, could not reveal all locations of R. kauffeldi, we embraced the uncertainty allowed by the NatureServe approach. For example, when our counts of number of grid cells or number of occurrences fell near the boundaries of ranking categories, we selected both categories. We identified a considerable decline in NY and CT based on our surveys and Feinberg et al. [21], but the evidence for statewide decline in other states within the range was weaker, so we did not use trends factors for those states. We estimated a slight range-wide decline based on our surveys (Table 2).

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Table 2. Conservation status ranking of Rana kauffeldi using the NatureServe methodology [45,46].

Range extent is defined by the smallest polygon that encapsulates known occurrences. Area of occupancy is the area within the range in which the species actually occurs. Number of occurrences is intended to reflect number of populations, based on taxon-specific distances within which animals are assumed to be interacting. Long-term trend was estimated based on historical literature or museum specimens. Values given for these rank factors are the ranges encompassed by NatureServe’s categories. Calculated rank was generated by NatureServe’s element rank calculator [46], while Expert rank was assigned by biologists familiar with the species in that state.

https://doi.org/10.1371/journal.pone.0205805.t002

The ranks generated by the rank calculator tended toward greater concern than those based on our expert opinion (compare the last two columns in Table 2), but we report both to be transparent about our process. In the core of R. kauffeldi’s range (NJ, DE, VA, and perhaps MD), we believe it to be secure, with many apparently large populations in protected wetlands. At the northern edge of its range (including eastern PA, southern CT, southern NY, and extreme northeastern NJ), R. kauffeldi is exceedingly rare and appears to have declined substantially. In NY, for example, the species once occurred across 11 counties and likely more than 100 populations; today it is known from only three counties and fewer than 10 populations. Declines in NY’s leopard frogs have been suspected for decades [56]. While the species is common along the Delaware River, only a sliver of its range falls within PA, hence its suggested S1 status in that state. At the southern edge of our survey limits—and possibly the actual southern range limit for R. kauffeldi—in NC, it may be rare too, but this area needs additional field surveys to confirm the species’ status and actual southern limit. Throughout its range a rank of G3G4 (Vulnerable to Apparently Secure) seems appropriate.

Decrypting Rana kauffeldi

Our multi-year, 10-state investigation demonstrated conclusively that R. kauffeldi is a habitat specialist with a small range centered in the most densely populated region of the United States, and one of the most heavily populated mega-regions on earth [57]. In several northern states, it is extremely rare, while in the southern portion of its range it is still broadly distributed and can be common. We have a much better idea of its distribution than we did just a few years ago, but some unexplained gaps remain. Fortunately, for those interested in surveying for this frog, methods for its identification in the field are also now better understood. The unique breeding call identified by Feinberg et al. [21] was reliably associated in our study with frogs genetically determined to be R. kauffeldi. Separation in the hand from R. pipiens based on morphology and color patterns is very reliable, while separation from R. sphenocephala is correct as much as 90% of the time.

Distribution and biogeography.

In the core of its mid-Atlantic range south of the Pleistocene glacial maximum, R. kauffeldi is a species exclusively of the Coastal Plain (Fig 7), and the degree to which its apparent western range margin matches that of the Coastal Plain is striking. The Coastal Plain physiographic region of the U.S. covers the Gulf and Atlantic coasts from southern Texas east to Florida and north to Long Island, NY [58]. The region is characterized by low elevation, minimal topography, and unconsolidated sediments [59] and has recently been recognized as a global biodiversity hotspot [60]. Within this physiographic region, several ecoregions—areas with similar geology, soils, climate, and vegetation [61]—have been identified, of which the Mid-Atlantic Coastal Plain (MACP), Chesapeake Bay Lowlands (CBY), and North Atlantic Coast (NAC) are occupied in part by R. kauffeldi. The MACP has been described as “a factory for the generation of new and novel species” because of its dynamism and juxtaposition of natural communities [62].

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Fig 7. Physiographic regions of coastal northeastern U.S. ([58]; https://water.usgs.gov/lookup/getspatial?physio) with presence points for Rana kauffeldi confirmed by bioacoustics and/or genetics.

Last glacial maximum is from Ray and Adams [63].

https://doi.org/10.1371/journal.pone.0205805.g007

But since the last glacial maximum occurred only 20,000 years ago, R. kauffeldi has clearly moved out of its core region post-glacial retreat, (re)colonizing some previously glaciated regions along major river valleys to the north and east, and now occurs in the Piedmont, New England, and Valley and Ridge physiographic provinces. These ecoregions are characterized by greater topographic variation and a greater diversity of habitat types and soils than the core of the species range, perhaps reflecting the competitive or predator release that can characterize the opening of newly available habitat.

Apparent gaps in the range in areas with at least some suitable habitat—the northwestern shore of the Delmarva Peninsula and the western shore of the Chesapeake Bay from Baltimore, MD to mid-coastal VA—may be clarified with further sampling and continued examination of museum specimens and historical recordings. Examination of USNM museum specimens from the western shore of the Chesapeake Bay yielded little evidence of R. kauffeldi across that region apart from two specimens from Calvert County, MD that more closely resemble R. kauffeldi than they do R. sphenocephala. We note, however, that the distribution model identified little suitable habitat in the MD-to-VA gap. Our range map is based on confirmed observations, with the expectation that new observations may add to the known range. Our distribution model represents suitable habitat at a finer scale, with coastal impoundments and river valleys standing out and a clear signature of having sampled in part along roads in both the map and the variable importance rankings. The low success rate of the model in predicting presence may reflect local extirpations from still-suitable habitat. Future distribution modeling could use likely absence points instead of random background points, which may allow for more accurate predictions of presence points. The combined use of modeling, interpretation of aerial imagery, and field survey may also help fill these gaps over time.

Ecological research agenda for cryptic species and conclusion

Cryptic species remain a fascinating, and rising challenge to systematics, ecology, and conservation, but one that is increasingly met with new research technologies. Much has been written about the problem of identifying cryptic species [8,10,18] and the implications for assessments of species diversity within taxa and globally [104–107]. Less attention has been paid to the follow-up studies necessary to answer basic questions about ecological, behavioral, and morphological differences among species in a cryptic complex. Here we present a framework for answering these important questions in phylogenetics, biogeography, ecology and behavior, morphology, conservation, and implications for scientific literature that should be a standard part of our ecological research agenda following cryptic species discovery (Table 3). This framework differs from one that might reasonably be applied to any newly discovered species in that it focuses on teasing out differences between two or more cryptic species within a complex.

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Table 3. Research agenda for follow-up studies on a cryptic species discovery, specifically for cases in which one species is split off from another, presumably through genetic analysis.

https://doi.org/10.1371/journal.pone.0205805.t003

As with any newly described species, there is still much to learn about R. kauffeldi’s ecology and natural history. Descriptions of R. kauffeldi egg masses and tadpoles are lacking [108], and the ability to distinguish species based especially on tadpoles would be a boon to detailed natural history, ecological, and demographic studies. For example, adaptations of the various life stages to particular environmental conditions and stressors may help explain the geographic and ecological distributions of the species. Given the highly urban setting of many populations, the susceptibility to environmental contaminants of the various life stages [109], and potential impacts of noise on anuran communication [110], are of great interest. Research into possible competitive interactions may shed light on any niche separation given the overlap with R. sphenocephala and R. pipiens and the suggestion of hybridization in both the genetic and morphological data, and help explain observed declines. Finally, additional taxonomic research using a combination of genetics, morphology, and bioacoustics may yet reveal more cryptic diversity within the North American leopard frog complex.