Dramatic declines in neotropical salamanderpopulations are an important part of theglobal amphibian crisisSean M. Rovitoa, Gabriela Parra-Oleaa,b, Carlos R. Vasquez-Almazanc, Theodore J. Papenfussa, and David B. Wakea,1

aMuseum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720-3160; bInstituto de Biología,Universidad Nacional Autonoma de Mexico, AP 70-153, Ciudad Universitaria, CP 04510 Mexico DF, Mexico; and cMuseo de Historia Natural,Universidad de San Carlos, Calle Mariscal Cruz 1-56, Zona 10, Cuidad de Guatemala, Guatemala

Contributed by David B. Wake, December 20, 2008 (sent for review November 20, 2008)

We document major declines of many species of salamanders atseveral sites in Central America and Mexico, with emphasis on theSan Marcos region of Guatemala, one of the best studied and mostdiverse salamander communities in the Neotropics. Profound de-clines of several formerly abundant species, including 2 apparentextinctions, are revealed. Terrestrial microhabitat specialists atmid- to high elevations have declined more than microhabitatgeneralists. These terrestrial microhabitat specialists have largelydisappeared from multiple sites in western Guatemala, including inwell-protected areas, suggesting that the phenomenon cannot beexplained solely by localized habitat destruction. Major declines insouthern Mexican plethodontid salamanders occurred in the late1970s to early 1980s, concurrent with or preceding many reportedfrog declines. The species in decline comprise several major evo-lutionary lineages of tropical salamanders, underscoring that sig-nificant portions of the phylogenetic diversity of Neotropicalsalamanders are at risk. Our results highlight the urgent need todocument and understand Neotropical salamander declines as partof the larger effort to conserve global amphibian diversity.

climate change � elevational transect � Guatemala � Mexico �Plethodontidae

I t is estimated that one-third of all amphibian species worldwideare endangered or threatened with extinction (1). Efforts to

understand the causes of this alarming decline, known as theglobal amphibian crisis, have focused primarily on frogs; com-paratively little attention has been paid to salamanders (2–5).Reasons for this bias include the fact that most salamanders aresecretive in nature, so population trends may not be apparent asin frogs. At well-studied sites in Central America wheresalamanders are present, they are often relatively uncommoncompared with frogs (6). Nonetheless, given the precipitousdeclines and recently documented extinctions (2, 6, 7) in an arrayof tropical frog species, tropical salamanders demand attentionas well.

The lungless salamanders (family Plethodontidae) include theonly tropical salamanders, which have radiated dramatically inthe Neotropics and now account for 40% of all salamanders(www.amphibiaweb.org). Nuclear Central America (between theIsthmus of Tehuantepec and the Nicaraguan lowlands) andsouthern Mexico contain a high diversity of plethodontid species(8). The salamander fauna has long been of interest to herpe-tologists; past studies and associated museum collections providea rich historical database of abundance and distribution. One ofthe richest datasets available for tropical salamanders comesfrom the studies of Wake and colleagues (9, 10), who sampledalong an elevation gradient on the southern slopes of VolcanTajumulco in the Department of San Marcos, Guatemala. Thisdataset provides the best opportunity to examine changes insalamander populations over time. Here, we assess salamanderpopulation status by using historical (1970s) and recent (2005–2007) survey data from sites in Guatemala and Mexico across

multiple elevations. Our sampling focuses on high elevation andcloud forest communities, which harbor the highest diversity ofsalamander species (9, 10). Our data include a broad phyloge-netic sample of the Middle American salamanders across a widegeographic area (Fig. 1). According to this dataset, salamanderspecies exhibit differences in elevational distribution, habitat,and microhabitat preference, allowing us to identify factorsassociated with declines or changes in population status anddifferentiate among hypotheses to explain these changes.

ResultsDramatic declines in abundance were seen for most species fromthe upper cloud forest and high elevation salamander assem-blages on the San Marcos transect (Table 1). Two of the mostcommon salamander species in the 1970s, Pseudoeurycea brun-nata and Pseudoeurycea goebeli (10), were not found on any ofour recent visits (see Methods) at either of the 2 sites where theypreviously occurred in great abundance (Fig. 2). The third,undescribed species of Pseudoeurycea (P. sp. nov.‘‘San Marcos’’)was also not found on any of our recent surveys, despite beingfound on 15 of 25 visits to the El Rincon site in the 1970s. At ElRincon, Bolitoglossa rostrata was found on 22 of 25 visits in thepast, generally in high abundance (mean � 64.4 salamanders pervisit, n � 22 visits), but not a single specimen was seen on ourrecent surveys. This site represents the lower elevational limit ofB. rostrata, and its absence provides evidence of an elevationalrange contraction on the transect. The species found at thehighest elevations on the transect, Pseudoeurycea rex, was onlyfound on 2 of our 5 visits, at a significantly lower encounter rate[0.58 salamanders found per person-hour vs. 8.92 in the 1970s(Student’s t test: t � �8.338, P � 0.023)] and lower abundance(mean � 1.8 individuals per visit vs. 77.7 in the past). The specieswas absent on our 2 surveys at a site in the high elevation zonewhere 174 specimens were found on a single visit in 1972. Threeof the 4 species of Pseudoeurycea found on the upper part of theSan Marcos transect (P. rex, P. brunnata, and P. goebeli) were alsoknown from nearby Volcan Chicabal. Despite 2 intensivesearches in 2006 and 2007, we found only a single specimen ofP. goebeli and no P. rex or P. brunnata. This finding represents aprecipitous decline in encounter rate for all 3 of these species(Table 1 and Fig. 2), and raises the possibility that P. brunnatamay be extinct.

Author contributions: S.M.R., G.P.-O., T.J.P., and D.B.W. designed research; S.M.R., G.P.-O.,C.R.V.-A., T.J.P., and D.B.W. performed research; S.M.R., G.P.-O., T.J.P., and D.B.W. analyzeddata; and S.M.R. and D.B.W. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

1To whom correspondence should be addressed. E-mail: wakelab@berkeley.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0813051106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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Surveys of other sites in the region reveal that the decline insalamander abundance seen for upland species is not unique toSan Marcos. Either no P. rex or only a single individual was foundat other sites in western Guatemala and Chiapas (Table S1).Populations of this species were denser (usually 10 to �100salamanders per visit) than any other salamander in Guatemalain the past (11), and large areas of suitable habitat still exist.Although B. rostrata remains fairly common in some areas, wedid not find it at several sites (Table S1) where it was common

in the 1970s (210 collected in 6 visits at the high point on thePanamerican Highway, contrasted with none on 3 recent visits).

Two species from the San Marcos upper cloud forest assem-blage displayed no evidence of population declines. Bolitoglossamorio was found on each of our 3 recent visits to El Rincon vs.about half of the visits in the 1970s and was also foundsignificantly more often than in the past at Buena Vista (Wil-coxon test: Z � 3.0228, P � 0.0025). Bolitoglossa lincolni was alsofound on all recent visits to El Rincon vs. approximately half ofthe visits in the 1970s, and the species displayed similar abun-dance at Buena Vista compared with past surveys (Wilcoxontest: Z � �0.8645, P � 0.387) (Table 1). In addition to beingmore abundant than in the past at San Marcos, B. morio wascollected above its previous elevational limit at the high point onthe Panamerican Highway, where only B. rostrata and P. rexoccurred previously. Although Dendrotriton bromeliacius was notfound at Finca Insula in 2005–2007, it was found at both ElRincon and Buena Vista. All 4 species occupying the lower cloudforest at San Marcos were found less often on recent visits andin much lower abundance than in the 1970s (Table 1), althoughthe limited amount of search time on recent surveys makes itdifficult to draw strong conclusions about population status. Atthe lowest elevation site, Finca Santa Julia, Bolitoglossa occiden-talis was found on 1 of 2 visits with relatively little search effort.

The 4 sites in Mexico north of the Isthmus of Tehuantepecdemonstrate similar patterns to the declines in Guatemala,despite having no species in common. Both high-elevationspecies of Pseudoeurycea from Cerro San Felipe, Oaxaca, showsteep declines in density beginning in the early 1980s, and neitherhas been seen at the site in 10 years despite recent search efforts(Fig. 3). The same trend is evident at El Chico National Park,where a sharp decline in encounter rate of 2 species of Chirop-terotriton, C. multidentatus and C. dimidiatus, occurred between1974 and 1976 (Fig. 3). On Cerro Chicahuaxtla, surveys in the1980s found decreasing numbers of salamanders, and nosalamanders at all were encountered in 8 person-days of field-work in 1999. The number of Thorius found at Puerto del Airedeclined dramatically between 1977 and 1982, and few or nosalamanders were found on subsequent visits through 2001, apattern consistent with the other sites (Table S2). These resultssuggest a decline in abundance of salamanders at this site in thelate 1970s to early 1980s.

DiscussionThe results of our surveys indicate a collapse of the upper cloudforest and high elevation salamander assemblages at most sitesand demonstrate changes in abundance at most sites surveyed.At our focal survey sites in San Marcos, the complete disap-pearance of 3 species of Pseudoeurycea from the upper cloudforest is especially striking, given the remarkable abundance ofboth P. brunnata and P. goebeli in the past. P. goebeli, P. brunnata,P. rex, and B. rostrata are terrestrial microhabitat specialists (10)that make little or no use of arboreal bromeliads. B. morio and

Fig. 1. Survey sites in Guatemala and Mexico. San Marcos elevationalgradient: A) Upper slopes of Volcan Tajumulco, B) El Rincon, C) Buena Vista, D)Finca Insula and E) Finca Santa Julia, F) Volcan Chicabal. Guatemalan sites: 1)High point on Panamerican Highway; 2) Cerro Tecpan; 3) Rancho de Tejo; 4)Sierra de los Cuchumatanes. Mexican sites: 5) Cerro Mozotal; 6) San Cristobalde las Casas area; 7) Cerro San Felipe; 8) Puerto del Aire; 9) Cerro Chicahuaxtla;10) Parque Nacional El Chico. Volcan Tajumulco is visible near the center of theLandsat image (Inset); the Guatemalan Plateau is at the upper right, and thePacific coastal plains are in the lower left.

Table 1. Encounter rate (salamanders per person per hour) for selected species from San Marcos upper cloud forest sites

SiteTime

periodNo. ofvisits P. rex

P.brunnata

P.goebeli

P. sp. novSan Marcos

B.rostrata

B.lincolni

B.morio

D.bromeliacius

El Rincon 1970s 25 ** 0.18 (13/25) 0.43 (13/25) 0.17 (15/25) 4.87 (22/25) 0.20 (12/25) 0.12 (13/25) 0.23 (5/25)(2,700 m) 2005–2007 3 ** 0 (0/3) 0 (0/3) 0 (0/3) 0 (0/3) 0.28 (3/3) 0.47 (3/3) 0.52 (2/3)

Buena Vista 1970s 36 (27) ** 1.77 (23/27) 1.51 (21/27) 0.02 (15) ** 0.45 (17/36) 0.07 (12/36) 1.30 (26/36)(2,400 m) 2005–2007 8 (5) ** 0 (0/5) 0 (0/5) 0 (0/5) ** 0.0625 (3/8) 0.92 (6/8) 0.007 (1/8)

Volcán Chicabal(2,700 m)

1970s 4 1.15 (3/4) 1.69 (3/4) 3.07 (4/4) ** ** ** ** **2005–2007 2 0 (0/2) 0 (0/2) 0.04 (1/2) ** ** ** ** **

** indicates that a species did not occur at a site or was not considered at that site for analysis. Number of visits encountered per total visits is shown inparentheses.

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B. lincolni, which were encountered with equal or greaterfrequency than in the past, are microhabitat generalists, foundnot only in terrestrial habitats but also in bromeliads, logs, andunder bark (10). D. bromeliacius, an arboreal bromeliad special-

ist, persists Buena Vista and El Rincon, where appropriate foresthabitat still exists. Thus, there appears to be a strong correspon-dence between terrestrial microhabitat preference and declinesin encounter rate. This pattern holds when considering the

Fig. 2. Mean number of salamanders per visit in the 1970s and from 2005 to 2007 at upper cloud forest and high-elevation sites on the San Marcos transectand microhabitat associations of species. (A) Slopes of Volcan Tajumulco. (B) El Rincon. (C) Buena Vista. Asterisk indicates that species was not detected in timeperiod. Arrow in C indicates bar for D. bromeliacius (mean � 0.017 salamanders per visit). Microhabitat classifications of upper cloud forest and high-elevationspecies from ref. 10 are shown at bottom right. (D) Location of 3 sites on San Marcos transect, with elevational extirpations shown in pink. Upper cloud forestand high-elevation sites are outlined in black, with arrows indicating elevation of sites A–C. Transect diagram has been adapted from ref. 9.

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Mexican salamander communities; the mid- to high elevationspecies of Pseudoeurycea, Chiropterotriton, and Thorius showingmajor declines are all terrestrial microhabitat specialists. Thecontinued presence of 3 species at Finca Insula and the abun-dance of B. occidentalis at lower elevations suggest that there isan elevational pattern to the declines as well, with the mostseverely affected species inhabiting the highest 2 elevationalzones. The lower-elevation species of Bolitoglossa also make useof arboreal microhabitats (e.g., bromeliads and leaf axils), muchlike B. morio and B. lincolni (10).

The decline and disappearance of Pseudoeurycea in the uppercloud forest of San Marcos cannot be attributed to habitat lossalone. Both P. goebeli and P. brunnata were found in disturbedareas near forest in the past, and some cloud forest remains onthe San Marcos transect. Similarly, Volcan Chicabal is within aprotected reserve and contains primary cloud forest where wesurveyed. The marked decrease in encounter rate of P. rex andB. rostrata at many sites throughout the western highlands ofGuatemala and adjacent areas of Chiapas suggests that thefactors causing these changes in populations are regional inscope. At least some suitable habitat remains at all of the sites,and at the sites in southern Mexico, but salamanders are absentor occur at dramatically lower densities than in the past.

In addition to an association between high elevation, terres-trial microhabitat preference and population declines, thereappears to be a phylogenetic pattern. Of the salamanders on theSan Marcos transect, all of the Bolitoglossa involved in ouranalysis except B. occidentalis are part of the subgenus Magna-digita (12); we found conclusive evidence for the decline of onlyone of these species (B. rostrata). All of the Pseudoeurycea fromSan Marcos are close relatives (13, 14). At the broader regionalscale, several major clades (Pseudoeurycea, Thorius, Chiroptero-triton) seem to be most severely affected (Fig. S1). This strongphylogenetic clustering of decline indicates that large portions ofthe evolutionary history of the tropical salamanders are at riskof being lost. Furthermore, the recently released results of the2008 Global Amphibian Assessment (15) show that, in additionto the declines that we report here, a high percentage of speciesof tropical salamanders is threatened with extinction (Fig. S1);indeed, more than half of the species in all but 1 genus (Oedipina)are in the 3 highest International Union for Conservation ofNature threat categories.

We cannot determine the exact timing of salamander popu-lation declines in San Marcos from our dataset, given thesampling gap between 1979 and 2005 caused by political condi-tions in Guatemala. The Mexican sites, however, have morecontinuous sampling through the early 1980s and provide abetter estimate of the time of decline. Salamander declines atthese 4 sites seem to have occurred between the mid-1970s andearly 1980s (Fig. 3). At Puerto del Aire, Thorius were consis-tently found in moderate to high numbers until 1977 (Table S2).These declines appear to have taken place before or at the sametime as many of the reported frog population declines in CentralAmerica (2, 6, 7)

The fungal disease chytridiomycosis, caused by the pathogenBatrachochytrium dendrobatidis (Bd) (16), has been implicated inthe decline of multiple species of Middle American amphibians(4, 17). Analysis of skin swabs from adult salamanders collectedin 2006 and 2007 found only 7 of 62 individual salamanders tobe positive for Bd (Table S3). Four of the 7 positive salamandersfrom the San Marcos transect were from the abundant low-elevation species B. occidentalis; the other 3 were from upper-cloud forest species that show no declines in abundance. Thenegative samples include some of the upper-cloud forest andhigh-elevation species that have undergone major declines (P. rex,B. rostrata). No direct evidence currently links Guatemalansalamander declines to Bd, although its importance in otherNeotropical amphibian declines suggests that it may potentiallyhave played a role.

The most drastic declines in encounter rate of salamanders onthe San Marcos transect occurred in terrestrial species, which donot use arboreal bromeliads as refuges. These species would beexpected to respond more directly to changes in climate, par-ticularly changes in humidity and precipitation, because they lackthe moist, buffered microenvironment that bromeliads provide.Cloud water provides an important source of moisture in tropicalmontane cloud forests during the dry season (18, 19), whensalamanders would be expected to be under the highest physi-ological stress; most cloud forest habitats contain little or nostanding water, and plethodontid salamanders depend on ade-quate moisture for surface activity because of their permeableskins (20). Lawton et al. (21) and Nair et al. (22) showed thatdeforestation in lowland and premontane regions leads to anelevational increase of the cloud base, and Ray et al. (23) useda modeling approach to demonstrate that deforestation in thelowlands of Costa Rica has led to an increase in mist-freeconditions in the cloud forest at Monteverde. The Pacificlowlands and premontane areas of Guatemala have been exten-sively deforested; although the Pacific plain deforestation oc-curred before the salamander sampling in the 1970s, much of thepremontane forest disappeared during and after this time (24).

Fig. 3. Salamander encounter rate of selected species at 3 sites in Mexiconorth of the Isthmus of Tehuantepec. (A) Cerro San Felipe, Oaxaca. (B) ParqueNacional El Chico, Hidalgo. (C) Cerro Chicahuaxtla, Veracruz. All sites show amajor decline in salamander abundance between the late 1970s and mid-1980s, with very few (if any) salamanders found in recent years. Arrowsindicate visits with nonzero encounter rates. Asterisks indicate visits when nosalamander of any species was found.

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The results of the studies cited above indicate that lowland andpremontane deforestation could have had a substantial impacton moisture conditions in the areas where the greatest declinesoccurred. Pounds et al. (25) used modeling to show that large-scale warming led to a greater decrease in relative humidity atMonteverde compared to that caused by deforestation. Speciesof cloud forest salamanders that can still be found rely at leastin part on bromeliads. Bromeliads depend on cloud waterdeposition and are predicted to be particularly vulnerable toclimate change (26, 27). Therefore, if climate change is in partresponsible for the declines we observed, arboreal salamanderspecies that are presently not in decline may soon suffer the samefate as the fully terrestrial species.

The results of this study point to widespread and severedeclines of upland salamanders at multiple sites in Guatemalaand Mexico, including the most intensively-studied salamandertransect in the neotropics. Although the causes of these declinesare not yet well understood, the drastic reductions in salamandernumbers and changes in community composition in this regionindicate that the salamander populations of many upland speciesare in need of protection. Until the forces causing these declinesare identified, however, an effective conservation strategy can-not be devised. Protecting habitat, although important, is insuf-ficient to conserve populations of many of these species. Fur-thermore, other recent studies have also provided evidence ofdeclining salamander populations in the neotropics (4, 5). Theglobal amphibian crisis, usually discussed in terms of frogs,clearly involves Middle American salamanders as well.

MethodsMost salamanders analyzed in this study were collected as voucher specimensand deposited in the Museum of Vertebrate Zoology (MVZ). Detailed fieldnotes and specimen catalogues were recorded and are stored at the MVZ,including data on numbers of salamanders seen but not collected. We made3 visits to the San Marcos transect in November 2005, September 2006, andAugust 2007 during the wet season when salamanders, especially terrestrialspecies, were found in large numbers in the past. We revisited 5 of the samesites along the San Marcos transect that were worked by D. Wake andcolleagues in the 1970s, summarized in ref. 10 (Fig. 1 and Table S4). In additionto the San Marcos sites, we surveyed a nearby Volcano with primary uppercloud forest in a protected reserve, Volcan Chicabal, in an attempt to distin-guish between habitat alteration and other potential reasons for decline.Several of the same collectors who worked in the 1970s (D.B.W., T.J.P., locals)were involved in the more recent surveys, allowing us to visit the exact sitesthat were surveyed in the past and to use similar methods.

We collected all salamanders seen and quantified collecting effort byrecording the number of collectors and search time. Each of the 5 sites wasvisited at least twice, and efforts were made to search at each site underfavorable weather conditions to maximize the chance of finding salamanders.We attempted to survey the best habitat available, which included areas ofprimary forest, well-established secondary forest with extensive epiphytegrowth, and more disturbed areas such as road banks and cleared areas.

We used the field notes of the collectors who worked most extensively onthe San Marcos transect in the 1970s (D.B.W., J. Lynch, T.J.P., and L. Houck) andseveral other MVZ collectors to quantify the encounter rate of salamanders atthe 5 focal sites on the transect (see SI Text for a detailed description ofmethods for field note data). Catalogues and field journal entries were used

to record the number of salamanders of each species collected or observedduring each collecting event. The search time and number of collectors werealso recorded in the journal entries in most cases. Collecting events wereassigned to the 5 focal sites (listed in Table S4), and only species known to bepresent at a site based on the detailed surveys of the 1970s were consideredat each site (Table S4). For each collecting event, the number of individualsfound was divided by the number of people searching and the number ofhours spent searching to give a measure of salamanders encountered perperson per hour for each species. The mean encounter rate and proportion ofvisits on which a species was found were calculated for each one of the sites.The same procedure was repeated for the 2005–2007 visits.

Although our systematic survey efforts concentrated on the San Marcosarea, records were also kept of species detection in other areas of westernGuatemala and Chiapas, Mexico that share species in common with SanMarcos (Fig. 1 and Table S1). P. rex, B. rostrata, and P. goebeli were found atthese sites in the 1970s. Adult salamanders collected in 2006 and 2007 wereswabbed and tested for B. dendrobatidis by using the real-time PCR assaydescribed by Boyle et al. (28).

To compare population trends in a broader geographic and phylogeneticscope, data from 4 sites in Mexico north of the Isthmus of Tehuantepec wereanalyzed: Cerro San Felipe in Oaxaca, Parque Nacional El Chico in Hidalgo,Cerro Chicahuaxtla in Veracruz, and Puerto del Aire on the Puebla/Veracruzborder (Fig. 1). These sites, similar in elevation, habitat type, species diversity,and patterns of elevational zonation (10) to the Guatemalan sites, provideindependent communities in which to analyze salamander population trends.In addition to Pseudoeurycea, they contain salamanders of the genera Thoriusand Chiropterotriton not found in Guatemala. The 4 sites were visited onmultiple occasions by researchers (D.B.W., T.J.P., J. Lynch, and G.P.-O.) whokept detailed field notes. For the first 3 sites, the number of salamanders ofeach species found was recorded and totaled by year, and the number ofcollectors and field days were also recorded. Encounter rate (salamanders perperson per day) for each species was estimated by dividing the number ofsalamanders found each year by the number of people searching and thenumber of days spent searching. For the Puerto del Aire site, the number ofThorius found per visit was obtained from 5 museums (American Museum ofNatural History, Natural History Museum of Los Angeles County, MVZ, Na-tional Museum of Natural History, University of Michigan Museum of Zoology)dating back to 1970, with no quantification of collection effort. Although 3species are present at that site (Thorius dubitus, Thorius magnipes, Thoriustroglodytes), identification to species level was not always possible, so only thetotal number was used. All 3 of these species use terrestrial microhabitats, and2 (T. dubitus and T. troglodytes) are exclusively terrestrial (29).

ACKNOWLEDGMENTS. We thank C. Moritz, M. H. Wake, J. Patton, V. Vreden-burg, T. Devitt, M. Fujita, G. Goldsmith, S. Schoville, S. Singhal, and C. Moritz’sand D.B.W.’s laboratory groups for comments and discussion; A. MunozAlonso and E. Recuero for valuable assistance in the field; El Consejo Nacionalde Areas Protegidas for providing Guatemalan research and collecting per-mits; R. Perez for logistical assistance and permission to do fieldwork at FincaInsula; L. D. Houck and J. F. Lynch, whose work and detailed field notes fromSan Marcos made this study possible; and L. D. Houck and E. D. Brodie, Jr. forreviewing the manuscript. S.M.R. was supported by a National Science Foun-dation Graduate Research Fellowship and a Berkeley graduate fellowship, andG.P.O. was supported by a University of California Institute for Mexico and theUnited States visiting scholar fellowship. Secretaria de Educacion Publica/Consejo Nacional de Ciencia y Tecnologia No. 50563 and Programa de Apoyoa Proyectos de Investigacion e Innovacion Tecnologica/Universidad NacionalAutonoma de Mexico IN211808. Fieldwork was supported by University ofCalifornia Institute for Mexico and the United States Research Grant 022043,the AmphibiaTree Project (National Science Foundation Grant EF-0334939)and the Museum of Vertebrate Zoology. Funding for analysis of swabs forchytrid fungus was provided by George Rabb and National Science Founda-tion Grant DEB-0139273.

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22. Nair US, Lawton RO, Welch RM, Pielke RA (2003) Impact of land use on Costa Ricantropical montane cloud forests: Sensitivity of cumulus cloud field characteristics tolowland deforestation. J Geophys Res D 10.1029/2001JD001135.

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28. Boyle DG, Boyle DB, Olsen V, Morgan JA, Hyatt AD (2004) Rapid quantitative detectionof chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay. Dis Aquat Organ 60:141–148.

29. Hanken J, Wake DB (1998) Biology of tiny animals: Systematics of the minutesalamanders (Thorius: Plethodontidae) from Veracruz and Puebla, Mexico, with de-scriptions of five new species. Copeia 1998:312–345.

3236 � www.pnas.org�cgi�doi�10.1073�pnas.0813051106 Rovito et al.

Supporting InformationRovito et al. 10.1073/pnas.0813051106SI TextRT-PCR Batrachochytrium dendrobatidis Assay. This assay uses spe-cies-specific primers ITS1–3 Chytr and 5.8S Chytr and the probeChytrMGB2 to amplify ITS-1 and 5.8S region (1). DNA wasextracted by using the PrepMan Ultra protocol for DNA extrac-tion (Applied Biosystems). DNA standards (provided by A. D.Hyatt, Commonwealth Scientific and Industrial Research Or-ganisation, Geelong, Australia) were diluted to give 100, 10, and1 genome equivalents for use in Taqman assay (1). Results fromthe assay are presented quantitatively as the number of genomicequivalents or zoospore equivalents recovered from tissue fromeach specimen.

Analysis of Historical Field Note Data. The quantity and identifica-tion of the specimens were confirmed by checking the MVZonline database (mvzarctos.berkeley.edu) and, when necessary,by examining specimens in the collection. In cases where only aminimum number of salamanders found was given, this numberwas used to provide a conservative estimate of encounter rate.When only approximate search times were given, the followingclassification was used to quantify search time: brief stop � 1 h,a couple of hours � 2 h, a few hours � 3 h, morning orafternoon � 3 h, most of a day � 4 h, full day � 6 h. In caseswhere the exact number of collectors was not recorded, thefollowing numbers were assigned: several � 5 collectors, many �10 collectors. These approximations were necessary because theoriginal data were not collected for analysis in this framework.In most cases, only a portion of this time was actually spent

actively searching, and fewer collectors were probably involved.The high estimates of search time and number of collectors werechosen to provide an extremely conservative estimate ofsalamander encounter rate, to avoid reporting an artifact as adecline in encounter rate. Mean encounter rate per species in the1970s and from 2005 to 2007 was compared by using a t test (incases of similar sample size) or a Wilcoxon test (in cases ofunequal sample sizes) for the highest site on the gradient and atBuena Vista, where sample sizes from 2005 to 2007 weresufficiently large for statistical analyses.

Collections from the Buena Vista site were made primarily bylocal collectors in the past who may have brought salamandersfrom both the upper and lower cloud forest zones. Thesecollectors consistently worked in the same areas, however,justifying the inclusion of these collecting events in a single site.At this site, P. brunnata, P. goebeli, and P. sp. nov. San Marcoswere only ever found in a very limited elevational range by MVZresearchers. For this reason, only collecting events between2,350- and 2,400-m elevation were counted when calculatingencounter rates for the three species of Pseudoeurycea. AlthoughBolitoglossa rostrata occurs at high elevations on the upper slopesof Volcan Tajumulco, many of the sites visited on this part of thetransect in the past were above its upper elevational limit. Forthis reason, B. rostrata was analyzed only for the El Rincon site.Two seldom-encountered species from Finca Santa Julia, Oe-dipina stenopodia and Bolitolgossa salvinii, were excluded fromthe analysis because of a lack of data from the 1970s (19 O.stenopodia and 0 B. salvinii collected from 1970–1979) and a lackof recent search effort.

1. Boyle DG, Boyle DB, Olsen V, Morgan JA, Hyatt AD (2004) Rapid quantitative detectionof chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples usingreal-time Taqman PCR assay. Dis Aquat Organ 60:141–148.

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Fig. S1. Phylogeny of Neotropical plethodontid genera (redrawn from ref. 1], with branch lengths proportional to time. Genera containing species withreported declines in the literature (2 ,3) are shown in bold; numbers on branches indicate number of species shown to be declining in this study. At right, numberof threatened species based on 2008 global amphibian assessment (IUCN categories CR, EN, VU)/total number of species in genus. Pseudoeurycea, shown to beparaphyletic in Wiens et al. (1), is shown as a single lineage (including Lineatriton) for simplicity.

1. Wiens JJ, Parra-Olea G, García-París M, Wake DB (2007) Phylogenetic history underlies elevational diversity patterns in tropical salamanders. Proc R Soc London Ser B 274:919–928.2. Whitfield SM, et al. (2007) Amphibian and reptile declines over 35 years at La Selva, Costa Rica. Proc Natl Acad Sci USA 104:8352–8356.3. Lips KR, et al. (2006) Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proc Natl Acad Sci USA 103:3165–3170.

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Table S1. Total collection numbers for B. rostrata, B. morio, P. rex at localities in western Guatemala and Chiapas, 2005–2007

Site Department/State Visits B. rostrata B. morio P. rexSearch time inperson � hours

Sierra de los Cuchumatanes Huehuetenango 2 (Oct. 2005, Aug. 2007) 56, �20 ** 1, 0 19.35, 23Rancho de Tejo Totonicapan 1 (Nov. 2005) 39 ** ** 3.75High point on Panamerican Highway Totonicapan/Solola 2 (Nov. 2005, Aug. 2007) 0, 0 0, 5 0, 1 3.75, 10Cerro Tecpan Chimaltenango 2 (Oct. 2005, Aug. 2006) 0, 0 1, 0 0, 0 10, 5.4Cerro Tzontehuitz Chiapas 1 (Aug. 2006) 2 ** ** 4Cerro Mozotal Chiapas 1 (Aug. 2006) ** ** 0 8

Double asterisk indicates that site is outside the range of a species. Sierra de los Cuchumatanes totals do not include sites below lower elevational limit of P. rex.

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Table S2. Collection totals for Thorius at Puerto del Aire, Veracruz/Puebla, Mexico

Month Total Thorius collected Museum

April, 1970 2 MVZSeptember, 1971 10 LACMSeptember, 1972 82 LACMJanuary, 1973 9 MVZJanuary, 1974 55 MVZJanuary, 1974 14 MVZJanuary, 1974 6 MVZJanuary, 1974 74 MVZApril, 1974 148 LACMJuly, 1974 286 LACMJuly, 1974 49 LACMSeptember, 1975 60 NMNHFebruary, 1976 1 MVZFebruary, 1976 8 MVZFebruary, 1976 29 MVZJuly, 1976 12 NMNHJuly, 1976 2 MVZJuly, 1976 17 MVZJuly, 1976 12 MVZJuly, 1977 27 MVZJuly, 1977 3 MVZJuly, 1977 61 MVZJanuary, 1982 1 MVZOctober, 1997 4 MVZApril, 1999 2 MVZAugust, 1999 0 MVZJanuary, 2000 1 MVZSeptember, 2000 1 MVZJanuary, 2001 3 MVZ

MVZ, Museum of Vertebrate Zoology; LACM, Natural History Museum of Los Angeles County; NMNH,Smithsonian Institution National Museum of Natural History.

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Table S3. Results of RT-PCR assay for Batrachochytrium dendrobatidis from salamanders on the San Marcos transect and other sitesin western Guatemala and Chiapas

Site No. tested Positives Species testing positive Zoospore equivalent

San Marcos: Tajumulco 6 0San Marcos: El Rincon 35 1 D. bromeliacius 20.5San Marcos: Buena Vista 5 1 B. lincolni 475.2San Marcos: �South transect� 8 1 B. franklini � lincolni hybrid 9894.8Lowland sites (�1,500 m elevation) 8 4 B. occidentalis 0.74, 2.1, 83.2, 356.8San Marcos Total 62 7Sierra de los Cuchumatanes 17 0High point on Panamerican Highway 6 0San Cristobal de las Casas area 2 0Volcan Chicabal 2 0Western Guatemala and Chiapas Total 27 0

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Table S4. Species present within each elevational zone on the San Marcos transect, elevation ranges of these zones, and survey sites

Elevational zone Species present Elevational range, m Survey sites

High-elevation grassland andwoodland

Pseudoeurycea rex, Bolitoglosssa rostrata 2,800- 4,200 Upper slopes of Volcán Tajumulco(�3,000 m)

Upper cloud forest Pseudoeurycea goebeli, Pseudoeurycea brunnata,Pseudoeurycea sp. nov. �San Marcos�,Bolitoglossa rostrata, Bolitoglossa lincolni,Bolitoglossa morio, Dendrotriton bromeliacius

2,400–2,800 El Rincon (2,700 m), Buena Vista(2,400 m)

Lower cloud forest Bolitoglossa engelhardti, Bolitoglossa franklini,Bolitoglossa flavimembris, Dendrotritonbromeliacius

1,600–2,400 Finca Insula (2,200 m)

Lower wet forest Bolitoglossa occidentalis, Bolitoglossa flaviventris,Bolitoglossa salvinii, Oedipina stenopodia

0–1,600 Finca Santa Julia(1,100 m)

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