1 2 DR. RUDOLF VON MAY (Orcid ID : 0000-0003-3981-2615) 3 4 5 Received Date : 29-Jul-2016 6 Revised Date : 06-Jan-2017 7 Accepted Date : 28-Jan-2017 8 Article type : Original Research 9 10 11 Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical 12 elevational gradient 13 14 Rudolf von May,1,2,3 Alessandro Catenazzi,4 Ammon Corl,2 Roy Santa-Cruz,5 Ana Carolina 15 Carnaval,6 Craig Moritz2,7 16 17 1 Department of Ecology and Evolutionary Biology, Museum of Zoology, University of Michigan, 18 2051 Ruthven Museums Building, 1109 Geddes Ave., Ann Arbor, MI 48109, USA 19 2 Museum of Vertebrate Zoology, University of California, Berkeley, 3101 Valley Life Sciences 20 Bldg., Berkeley, CA 94720, USA 21 3 Email: [email protected] 22 4 Department of Zoology, Southern Illinois University Carbondale, Carbondale, Life Science II 23 Bldg. 326, IL 62901, USA 24 5 Área de Herpetología,Author Manuscript Museo de Historia Natural de la Universidad Nacional de San Agustín 25 (MUSA), Av. Alcides Carrión s/n, Arequipa, Perú This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ece3.2929 This article is protected by copyright. All rights reserved 26 6 Department of Biology, The City University of New York, Marshak Science Building J-526, 160 27 Convent Avenue, NY 10031, USA 28 7 Centre for Biodiversity Analysis and Research School of Biology, The Australian National 29 University, Canberra, Australia 30 31 Running head: Divergence of critical thermal limits 32 Key Words: Amazon, Andes, critical thermal limits, CTmax, CTmin, physiological divergence. 33 Data Archival location: GenBank (molecular sequence data), Dryad (phylogenetic trees). 34 Abstract 35 36 Critical thermal limits are thought to be correlated with the elevational distribution of species 37 living in tropical montane regions, but with upper limits being relatively invariant compared to 38 lower limits. To test this hypothesis, we examined the variation of thermal physiological traits in 39 a group of terrestrial breeding frogs (Craugastoridae) distributed along a tropical elevational 40 gradient. We measured the critical thermal maximum (CTmax; n = 22 species) and critical 41 thermal minimum (CTmin; n = 14 species) of frogs captured between the Amazon floodplain (250 42 m asl) and the high Andes (3800 m asl). After inferring a multi-locus species tree, we conducted 43 a phylogenetically informed test of whether body size, body mass, and elevation contributed to 44 the observed variation in CTmax and CTmin along the gradient. We also tested whether CTmax and 45 CTmin exhibit different rates of change given that critical thermal limits (and their plasticity) may 46 have evolved differently in response to different temperature constraints along the gradient. 47 Variation of critical thermal traits was significantly correlated with species’ elevational midpoint, 48 their maximum and minimum elevations, as well as the maximum air temperature and the 49 maximum operative temperature as measured across this gradient. Both thermal limits showed 50 substantial variation, but CTmin exhibited relatively faster rates of change than CTmax, as 51 observed in other taxa. Nonetheless, our findings call for caution in assuming inflexibility of 52 upper thermal limits, and underscore the value of collecting additional empirical data on species’ 53 thermal physiology across elevational gradients. Author Manuscript 54 55 56 This article is protected by copyright. All rights reserved 57 58 Introduction 59 60 In a rapidly changing world, many species are faced with shrinking habitat and novel climatic 61 conditions. As a result, there has been widespread interest in understanding species responses to 62 past and present climatic variation in order to predict how best to conserve species in future 63 climatic conditions (e.g., Sinervo et al. 2010, Moritz & Agudo 2013). While much attention has 64 been given to modeling and predicting elevational range shifts in montane organisms, especially 65 in the context of climate change, most predictions about future geographic ranges are based on 66 correlative models that ignore species’ evolutionary history and eco-physiology (Colwell et al. 67 2008, Laurance et al. 2011, VanDerWal et al. 2012). Tropical montane regions are of special 68 concern because they are centers of biodiversity and endemism (Graham et al. 2014). Mountain 69 uplift, climatic fluctuations, and the emergence of new ecological conditions have been 70 hypothesized to promote the diversification of organisms at high elevations (Moritz et al. 2000, 71 Hoorn et al. 2010). As a result, species living at high elevation often exhibit narrowly 72 overlapping (i.e., parapatric) distributions, many of which are assumed to have greater tolerance 73 to cold (Janzen 1967, Navas 2005, Ghalambor et al. 2006). However, empirical data on critical 74 thermal limits of most tropical montane taxa remain unknown. Furthermore, tropical lowland 75 taxa, especially ectotherms, are thought to live near their thermal optimum, so increased 76 temperatures due to changing climates would lead to decreased fitness (Colwell et al. 2008, Huey 77 et al. 2009, Sunday et al. 2014). As with species living at higher elevations, empirical data on 78 species’ critical thermal limits are not available for most tropical lowland taxa. 79 80 Several hypotheses have been proposed to explain the potential causes of diversity patterns along 81 elevational gradients (Terborgh 1970, MacArthur 1972, Hofer et al. 1999, Lomolino 2001, 82 McCain & Grytnes 2010, McCain & Colwell 2011, Graham et al. 2014, Peters et al. 2016). One 83 of these hypotheses proposes that climatic conditions along the gradient restrict species’ 84 distributions (von Humboldt 1849, Janzen 1967). Air temperature is the main environmental Author Manuscript 85 factor that predictably decreases with increasing elevation as a result of adiabatic cooling (on 86 average 5.2−6.5 °C decrease per 1000 m elevation; Colwell et al. 2008). Critical thermal 87 maximum (CTmax) and critical thermal minimum (CTmin) are two measures that have been used This article is protected by copyright. All rights reserved 88 to infer species’ critical thermal limits. Numerous studies have shown that ectotherms exhibit a 89 general trend of decreasing critical thermal limits with elevation (Heatwole et al. 1965, Christian 90 et al. 1988, Gaston & Chown 1999, Navas 2003, Catenazzi et al. 2014). Moreover, it is likely 91 that critical thermal limits change at different rates in response to different temperature 92 constraints along elevational gradients (McCain & Grytnes 2010). Specifically, CTmax is thought 93 to be relatively inflexible across elevation (e.g., Hoffman et al. 2013, Muñoz et al. 2014, 2016), 94 with a narrow upper limit and low plasticity (Sunday et al. 2011; Gunderson & Stillman 2015). 95 96 Although many researchers have examined the relationship between critical thermal limits and 97 the elevational distribution of species living in montane gradients, only a few have combined 98 empirical (CTmax and CTmin) data and accounted for the effect of phylogenetic relatedness 99 among species (Muñoz et al. 2014, 2016, Sheldon et al. 2015). Phylogenetic comparative 100 methods are particularly useful for this purpose because they allow researchers to examine 101 evolutionary transitions in physiological traits and account for statistical non-independence of 102 interspecific data when studying life history evolution among closely related species (Harvey & 103 Pagel 1991, Garland et al. 1992, Revell 2008). 104 105 We investigated the role of physiological divergence among closely related species distributed 106 along an elevational gradient of > 3500 m in southern Peru. Although 80% of Peruvian Andean 107 frogs (ca. 250 species) occur within relatively narrow elevational ranges (Aguilar et al. 2010), 108 little is known about the relationship between their critical thermal limits and their elevational 109 distributions. We focused on 22 species of terrestrial-breeding frogs, Craugastoridae, the most 110 diverse amphibian family in the Tropical Andes (Hedges et al. 2008, Duellman & Lehr 2009, 111 Padial et al. 2014). These direct-developing frogs (Figure 1) are ideal model organisms in which 112 to test hypotheses about divergence across environmental gradients because they have low 113 vagility (resulting in local genetic structure), small body size (a trait that makes them amenable 114 for physiological experiments), and limited geographic and elevational ranges (suggesting strong 115 potential for local adaptation). Author Manuscript 116 117 Our goal was to examine how CTmax and CTmin vary in relation to the elevational distribution of 118 species and to test whether life history traits such as body size and body mass, and elevational This article is protected by copyright. All rights reserved 119 range midpoint explain differences in CTmax and CTmin among species. Altogether, we used four 120 metrics relating to elevation (elevational minimum, maximum, midpoint, and range) and two 121 metrics relating to temperature (maximum air temperature and maximum operative temperature) 122 as proxy for thermal environments. We reconstructed a phylogeny to determine the evolutionary 123 relatedness among species and