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Supporting Information Supporting Information Rovito et al. 10.1073/pnas.0813051106 SI Text actively searching, and fewer collectors were probably involved. RT-PCR Batrachochytrium dendrobatidis Assay. This assay uses spe- The high estimates of search time and number of collectors were cies-specific primers ITS1–3 Chytr and 5.8S Chytr and the probe chosen to provide an extremely conservative estimate of ChytrMGB2 to amplify ITS-1 and 5.8S region (1). DNA was salamander encounter rate, to avoid reporting an artifact as a extracted by using the PrepMan Ultra protocol for DNA extrac- decline in encounter rate. Mean encounter rate per species in the tion (Applied Biosystems). DNA standards (provided by A. D. 1970s and from 2005 to 2007 was compared by using a t test (in Hyatt, Commonwealth Scientific and Industrial Research Or- cases of similar sample size) or a Wilcoxon test (in cases of ganisation, Geelong, Australia) were diluted to give 100, 10, and unequal sample sizes) for the highest site on the gradient and at Buena Vista, where sample sizes from 2005 to 2007 were 1 genome equivalents for use in Taqman assay (1). Results from sufficiently large for statistical analyses. the assay are presented quantitatively as the number of genomic Collections from the Buena Vista site were made primarily by equivalents or zoospore equivalents recovered from tissue from local collectors in the past who may have brought salamanders each specimen. from both the upper and lower cloud forest zones. These collectors consistently worked in the same areas, however, Analysis of Historical Field Note Data. The quantity and identifica- justifying the inclusion of these collecting events in a single site. tion of the specimens were confirmed by checking the MVZ At this site, P. brunnata, P. goebeli, and P. sp. nov. San Marcos online database (mvzarctos.berkeley.edu) and, when necessary, were only ever found in a very limited elevational range by MVZ by examining specimens in the collection. In cases where only a researchers. For this reason, only collecting events between minimum number of salamanders found was given, this number 2,350- and 2,400-m elevation were counted when calculating was used to provide a conservative estimate of encounter rate. encounter rates for the three species of Pseudoeurycea. Although When only approximate search times were given, the following Bolitoglossa rostrata occurs at high elevations on the upper slopes classification was used to quantify search time: brief stop ϭ 1h, of Volcan Tajumulco, many of the sites visited on this part of the a couple of hours ϭ 2 h, a few hours ϭ 3 h, morning or transect in the past were above its upper elevational limit. For afternoon ϭ 3 h, most of a day ϭ 4 h, full day ϭ 6 h. In cases this reason, B. rostrata was analyzed only for the El Rincon site. where the exact number of collectors was not recorded, the Two seldom-encountered species from Finca Santa Julia, Oe- following numbers were assigned: several ϭ 5 collectors, many ϭ dipina stenopodia and Bolitolgossa salvinii, were excluded from 10 collectors. These approximations were necessary because the the analysis because of a lack of data from the 1970s (19 O. original data were not collected for analysis in this framework. stenopodia and 0 B. salvinii collected from 1970–1979) and a lack In most cases, only a portion of this time was actually spent of recent search effort. 1. Boyle DG, Boyle DB, Olsen V, Morgan JA, Hyatt AD (2004) Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Organ 60:141–148. Rovito et al. www.pnas.org/cgi/content/short/0813051106 1of6 Fig. S1. Phylogeny of Neotropical plethodontid genera (redrawn from ref. 1], with branch lengths proportional to time. Genera containing species with reported 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, number of threatened species based on 2008 global amphibian assessment (IUCN categories CR, EN, VU)/total number of species in genus. Pseudoeurycea, shown to be paraphyletic 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. Rovito et al. www.pnas.org/cgi/content/short/0813051106 2of6 Table S1. Total collection numbers for B. rostrata, B. morio, P. rex at localities in western Guatemala and Chiapas, 2005–2007 Search time in Site Department/State Visits B. rostrata B. morio P. rex person ϫ hours Sierra de los Cuchumatanes Huehuetenango 2 (Oct. 2005, Aug. 2007) 56, Ͼ20 ** 1, 0 19.35, 23 Rancho de Tejo Totonicapan 1 (Nov. 2005) 39 ** ** 3.75 High point on Panamerican Highway Totonicapan/Solola 2 (Nov. 2005, Aug. 2007) 0, 0 0, 5 0, 1 3.75, 10 Cerro Tecpan Chimaltenango 2 (Oct. 2005, Aug. 2006) 0, 0 1, 0 0, 0 10, 5.4 Cerro Tzontehuitz Chiapas 1 (Aug. 2006) 2 ** ** 4 Cerro 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. Rovito et al. www.pnas.org/cgi/content/short/0813051106 3of6 Table S2. Collection totals for Thorius at Puerto del Aire, Veracruz/Puebla, Mexico Month Total Thorius collected Museum April, 1970 2 MVZ September, 1971 10 LACM September, 1972 82 LACM January, 1973 9 MVZ January, 1974 55 MVZ January, 1974 14 MVZ January, 1974 6 MVZ January, 1974 74 MVZ April, 1974 148 LACM July, 1974 286 LACM July, 1974 49 LACM September, 1975 60 NMNH February, 1976 1 MVZ February, 1976 8 MVZ February, 1976 29 MVZ July, 1976 12 NMNH July, 1976 2 MVZ July, 1976 17 MVZ July, 1976 12 MVZ July, 1977 27 MVZ July, 1977 3 MVZ July, 1977 61 MVZ January, 1982 1 MVZ October, 1997 4 MVZ April, 1999 2 MVZ August, 1999 0 MVZ January, 2000 1 MVZ September, 2000 1 MVZ January, 2001 3 MVZ MVZ, Museum of Vertebrate Zoology; LACM, Natural History Museum of Los Angeles County; NMNH, Smithsonian Institution National Museum of Natural History. Rovito et al. www.pnas.org/cgi/content/short/0813051106 4of6 Table S3. Results of RT-PCR assay for Batrachochytrium dendrobatidis from salamanders on the San Marcos transect and other sites in western Guatemala and Chiapas Site No. tested Positives Species testing positive Zoospore equivalent San Marcos: Tajumulco 6 0 San Marcos: El Rincon 35 1 D. bromeliacius 20.5 San Marcos: Buena Vista 5 1 B. lincolni 475.2 San Marcos: ЉSouth transectЉ 81B. franklini ϫ lincolni hybrid 9894.8 Lowland sites (Ͻ1,500 m elevation) 8 4 B. occidentalis 0.74, 2.1, 83.2, 356.8 San Marcos Total 62 7 Sierra de los Cuchumatanes 17 0 High point on Panamerican Highway 6 0 San Cristobal de las Casas area 2 0 Volcan Chicabal 2 0 Western Guatemala and Chiapas Total 27 0 Rovito et al. www.pnas.org/cgi/content/short/0813051106 5of6 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 and Pseudoeurycea rex, Bolitoglosssa rostrata 2,800- 4,200 Upper slopes of Volcán Tajumulco woodland (Ͼ3,000 m) Upper cloud forest Pseudoeurycea goebeli, Pseudoeurycea brunnata, 2,400–2,800 El Rincon (2,700 m), Buena Vista Pseudoeurycea sp. nov. ЉSan MarcosЉ, (2,400 m) Bolitoglossa rostrata, Bolitoglossa lincolni, Bolitoglossa morio, Dendrotriton bromeliacius Lower cloud forest Bolitoglossa engelhardti, Bolitoglossa franklini, 1,600–2,400 Finca Insula (2,200 m) Bolitoglossa flavimembris, Dendrotriton bromeliacius Lower wet forest Bolitoglossa occidentalis, Bolitoglossa flaviventris, 0–1,600 Finca Santa Julia Bolitoglossa salvinii, Oedipina stenopodia (1,100 m) Rovito et al. www.pnas.org/cgi/content/short/0813051106 6of6.
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