Evolution, 59(9), 2005, pp. 2000±2016

PHYLOGENETIC ANALYSIS OF ECOMORPHOLOGICAL DIVERGENCE, COMMUNITY STRUCTURE, AND DIVERSIFICATION RATES IN DUSKY SALAMANDERS (PLETHODONTIDAE: DESMOGNATHUS)

KENNETH H. KOZAK,1,2 ALLAN LARSON,1 RONALD M. BONETT,3 AND LUKE J. HARMON1 1Department of Biology, Box 1137, Washington University, Saint Louis, Missouri 63130 3Department of Integrative Biology and Museum of Vertebrate Zoology, 3101 Valley Life Sciences Building, University of California, Berkeley, California 94720-3160

Abstract. An important dimension of adaptive radiation is the degree to which diversi®cation rates ¯uctuate or remain constant through time. Focusing on plethodontid salamanders of the genus Desmognathus, we present a novel synthetic analysis of phylogeographic history, rates of ecomorphological evolution and species accumulation, and community assembly in an adaptive radiation. Dusky salamanders are highly variable in life history, body size, and ecology, with many endemic lineages in the southern Appalachian Highlands of eastern North America. Our results show that life- history evolution had important consequences for the buildup of plethodontid-salamander species richness and phe- notypic disparity in eastern North America, a global hot spot of salamander biodiversity. The origin of Desmognathus species with aquatic larvae was followed by a high rate of lineage accumulation, which then gradually decreased toward the present time. The peak period of lineage accumulation in the group coincides with evolutionary partitioning of lineages with aquatic larvae into seepage, stream-edge, and stream microhabitats. Phylogenetic simulations dem- onstrate a strong correlation between morphology and microhabitat ecology independent of phylogenetic effects and suggest that ecomorphological changes are concentrated early in the radiation of Desmognathus. Deep phylogeographic fragmentation within many codistributed ecomorph clades suggests long-term persistence of ecomorphological features and stability of endemic lineages and communities through multiple climatic cycles. Phylogenetic analyses of com- munity structure show that ecomorphological divergence promotes the coexistence of lineages and that repeated, independent evolution of microhabitat-associated ecomorphs has a limited role in the evolutionary assembly of Des- mognathus communities. Comparing and contrasting our results to other adaptive radiations having different biogeo- graphic histories, our results suggest that rates of diversi®cation during adaptive radiation are intimately linked to the degree to which community structure persists over evolutionary time.

Key words. Adaptive radiation, Appalachian Highlands, community ecology, comparative method, North America, phylogeography, speciation.

Received January 12, 2005. Accepted July 3, 2005.

Adaptive radiations, clades that exhibit rapid divergence restrict opportunities for diversi®cation once available geo- into an exceptional variety of adaptively disparate forms graphical and niche spaces are occupied (Stanley 1973; (Schluter 2000; Losos and Miles 2002), have served for over Schluter 2000; Harmon et al. 2003). A thorough understand- a century as model systems for investigating the processes ing of the processes that in¯uence diversi®cation rates there- that contribute to the origin and maintenance of biodiversity fore requires simultaneous consideration of morphological (Osborne 1902; Simpson 1953; Schluter 2000). Classic mod- evolution, species accumulation, phylogeographic history, els of adaptive radiation link the buildup and maintenance and community structure. Recent studies have explored one of species richness with the evolution of phenotypic disparity. or a few of these aspects of adaptive radiation; however, For example, evolution of key traits and/or colonization of simultaneous analysis of the relationship between diversi®- competitor-free environments appear to promote diversi®- cation rates, phylogeographic history, and community assem- cation by giving lineages access to new adaptive zones bly is a new approach. Here we examine the temporal and (Simpson 1953; Schluter 2000). Similarly, ecomorphological geographical accumulation of species richness and ecomor- divergence may in¯uence diversi®cation, long-term persis- phological disparity in a widespread and ecologically diverse tence, and coexistence of lineages by reducing competitive clade of eastern North American plethodontid salamanders, interactions (Barraclough et al. 1999). genus Desmognathus. An important, but less-studied dimension of adaptive ra- In the southern Appalachian Highlands and adjacent phys- diation is to what degree diversi®cation rates ¯uctuate or iographic provinces of eastern North America, plethodontid remain constant through time. Both paleontological and salamanders have undergone extensive evolutionary diver- neontological studies of adaptive radiation suggest that the si®cation. The southern Appalachian plethodontid fauna accumulation of lineages and ecomorphological disparity combines high species richness with great disparity in habitat may be intimately linked to the persistence of species as- use and life history. Species occupy fully aquatic to terrestrial sociations over evolutionary time. For example, rates of di- habitats and exhibit developmental strategies ranging from versi®cation may decline in taxa whose lineages show ex- long aquatic larval periods to direct development (Petranka tensive long-term coexistence because ecological interactions 1998). The highlands of Central America represent another global hot spot of salamander species richness; however, di- 2 Present address: Department of Ecology and Evolution, Stony versity patterns in this region differ in two important respects. Brook University, Stony Brook, New York 11794-5245; E-mail: First, although regional species richness is greater in Central [email protected]. America (168 vs. 57 species; Campbell 1999; Duellman and 2000 ᭧ 2005 The Society for the Study of Evolution. All rights reserved. DIVERSIFICATION OF DUSKY SALAMANDERS 2001

TABLE 1. Microhabitat, length of aquatic larval period, and geographic distribution of the 18 formally recognized species of Desmognathus included this study. Microhabitat categories follow Titus and Larson (1996). Larval periods are from Bernardo (2000), Camp et al. (2000), and Tilley and Bernardo (1993). Geographic distribution follows Duellman and Sweet (1999).

Species Microhabitat Larval period (months) Geographic distribution D. aeneus terrestrial direct development Southern Appalachians, Piedmont D. apalachicolae seepage 9 Gulf Coastal Plain D. auriculatus stream edge 12 Southern Atlantic Coastal Plain, Gulf Coastal Plain, Missis- sippi Embayment Pine, Woodlands D. brimleyorum stream edge 12 Interior Highlands D. carolinensis seepage 9±10 Southern Appalachians D. conanti stream edge 12 Interior Lowlands, Alleghany Plateau, Southern Appala- chians, Piedmont D. folkertsi stream 34 Southern Appalachians D. fuscus stream edge 8±12 Interior Lowlands, Alleghany Plateau, Northern Appala- chians, Southern Appalachians, Northern Atlantic Coastal Plain D. imitator seepage 10 Southern Appalachians D. marmoratus stream 35 Southern Appalachians D. monticola stream edge 9.5±12.5 Alleghany Plateau, Southern Appalachians, Piedmont, Gulf Coastal Plain D. ochrophaeus seepage 8 Alleghany Plateau, Northern Appalachians, Piedmont D. ocoee seepage 9±10 Southern Appalachians D. orestes seepage 8±9 Southern Appalachians D. quadramaculatus stream 35±48 Southern Appalachians D. santeetlah stream edge 12 Southern Appalachians D. welteri stream edge 24 Alleghany Plateau, Southern Appalachians D. wrighti terrestrial direct development Southern Appalachians

Sweet 1999), fewer species coexist locally than in the south- al. 2000). If so, ecomorphs may have evolved repeatedly as ern Appalachians (maximum of 11 species vs. 18 species; lineages diversi®ed to form local communities in which eco- Duellman and Sweet 1999; GarcõÂa-ParõÂs et al. 2000). Second, logical interactions among species generated selective re- tropical plethodontids lack aquatic larval periods and sub- gimes promoting subdivision of the stream-forest ecotone divide arboreal and terrestrial adaptive zones (Wake and (Tilley and Bernardo 1993). Alternatively, some evidence Lynch 1976; GarcõÂa-ParõÂs et al. 2000), except for a single suggests that cladogenesis and ecomorphological divergence stream-associated species now considered extinct (Wake and are concentrated early in the history of the Desmognathus Campbell 2001). radiation and that communities have formed by spatial over- Desmognathus is a distinctive element of the eastern North lapping of stable, ecomorphologically disparate lineages (Ti- American plethodontid fauna. In contrast to most plethodon- tus and Larson 1996). These alternative hypotheses of evo- tid genera (Larson 1984), this clade of 19 recognized species lutionary radiation have important implications for under- exhibits a wide range of variation in body size, life history, standing the role of phylogenetic history in Desmognathus and microhabitat use. Up to seven species of Desmognathus community assembly. This ®rst hypothesis predicts recurring occur in local sympatry in the southern Appalachians (Bruce in situ evolution of ecomorphological divergence between 1991), where species partition microhabitats (Hairston 1949; codistributed lineages, whereas the second hypothesis pre- Organ 1961; Tilley 1968) along an aquatic-to-terrestrial mi- dicts a strong phylogenetic inertia for ecomorphological char- crohabitat gradient (Table 1). The largest species have long acteristics and community structure (Webb et al. 2002). aquatic larval periods (two to four years) and occupy the Distinguishing these hypotheses of evolutionary diversi- channels of streams with nearly permanent ¯ow regimes, ®cation and community assembly requires a phylogenetic whereas the smallest species have direct development and analysis that includes comprehensive geographic sampling of occupy terrestrial habitats. Species with intermediate body genetic and morphological variation across the range of the sizes and larval periods (Յ1 year) inhabit stream edges and group. We present the ®rst combined analysis of phylogeo- interstream seepage areas. The ecological basis of local com- graphic history, morphometric evolution, and community as- munity structure has been studied extensively; both predation sembly in an adaptive radiation. This integrative approach and competition among congeners maintain an association permits us to examine (1) constancy versus ¯uctuation in accumulation of lineages and ecomorphological disparity between body size and the spatial position that a species throughout the phylogenetic history of dusky salamanders, occupies along the stream-forest ecotone (Krzysik 1979; and (2) the relationship between phylogeny, ecomorphol- Keen 1982; Keen and Sharp 1984; Hairston 1986, 1987; ogical divergence, and coexistence of lineages. Southerland 1986a,b; Roudebush and Taylor 1987). The relationship between cladogenesis, ecomorphological MATERIALS AND METHODS divergence, and dusky-salamander community assembly is poorly understood. Evolutionary transitions in ecomorphol- Phylogenetic Analyses ogy might be accomplished easily by changes in length of We sampled 113 specimens representing 18 formally rec- the larval period and age at maturity (Bruce 1996; Camp et ognized species of Desmognathus and its sister taxon, 2002 KENNETH H. KOZAK ET AL.

Phaeognathus (Figure 1; Appendix 1, available online only verged if their ln-likelihood scores approached similar mean at http://dx.doi.org/10.1554/05-024.1.s1). Aneides ¯avipunc- values (Huelsenbeck and Bollback 2001). tatus and Eurycea wilderae were used as outgroups. We imported the post-burn-in sample of topologies from Genomic DNA was extracted from liver using the Qiagen our Bayesian analysis into PAUP* and used the tree-®lter (Valencia, CA) QIAamp tissue kit. Ampli®cation of genomic option to count the number of topologies consistent with the DNA was conducted using primers L4437 (5Ј-AAGCTTTC monophyly of stream, stream-edge, seepage, or terrestrial GGGCCCATACC-3Ј) and H6159 (5Ј-GCTATGTCTGGGG haplotypes. Each microhabitat group was tested separately. CTCCAATTA-3Ј) (Macey et al. 1997; Weisrock et al. 2001) The proportion of trees in which haplotypes from a given with denaturation at 94ЊC for 35 sec, annealing at 50ЊC for microhabitat category are monophyletic corresponds to the 35 sec, and extension at 70ЊC for 150 sec with 4 sec added Bayesian posterior probability of that hypothesis. A hypoth- to the extension time per cycle, for 30 cycles. Negative con- esis is rejected if its posterior probability is Յ0.05. trols were run for all ampli®cations. Ampli®ed products were To test whether lineages accumulated nucleotide substi- puri®ed with Qiagen gel-extraction kits. Primers L4882b (5Ј- tutions at signi®cantly different rates we conducted relative- TGACAAAAAATTGCNCC-3Ј) and H5617a (5Ј-AAAATR rate tests as implemented in the program RRTree (Robinson- TCTGRGTTGCATTCAG-3Ј) were used as internal sequenc- Rechavi and Huchon 2000). ing primers (Macey et al. 1997, 2000; Weisrock et al. 2001). Sequencing reactions were run using Big-Dye Terminator Delimitation of Independent Evolutionary Lineages Ready-Reaction Kits (Perkin Elmer, Wellesley, MA) with an initial denaturation at 96ЊC for 2 min, then denaturation at Our sampling of Desmognathus includes all recognized 96ЊC for 15 sec, annealing at 50ЊC for 1 sec, and extension species except a single geographically restricted species de- at 60ЊC for 4 min for 40 cycles. All sequences were then scribed too recently to be included (Anderson and Tilley visualized on an ABITM 373A automated sequencer (PE Ap- 2003); thus, our taxon sampling is nearly comprehensive at plied Biosystems, Foster City, CA). the level of described species. However, phylogeographic Sequence data collected in this study included the genes structure within recognized species suggests that phyloge- encoding ND2 (NADH dehydrogenase subunit 2), tRNATrp, netically and geographically distinct lineages that would tRNAAla, tRNAAsn, tRNACys, and tRNATyr, the origin for qualify as species under the phylogenetic or evolutionary species concepts (Mayden 1997; de Queiroz 1998) are un- light-strand replication (OL), and 154 base pairs of the CO1 (cytochrome oxidase subunit 1) gene. Sequences were derestimated by current taxonomy. aligned manually. Sequences encoding ND2 and CO1 were To delineate boundaries of putatively independent evolu- translated to amino acids analytically using MacClade (Mad- tionary lineages, we employed the tree-based method of dison and Maddison 1992) to verify alignments and to check Wiens and Penkrot (2002), which uses geographic patterns for premature stop codons. The ®ve tRNA sequences were of coalescence among DNA haplotypes to test for gene ex- aligned manually based on models of secondary structure change among populations of a focal species and one or more (Macey and Verma 1997). closely related species. Two sampling-design components are Phylogenetic trees were estimated using parsimony and required: inclusion of as many species as possible to test for Bayesian methods. PAUP* 4.0b10 (Swofford 2002) was used exclusivity (monophyletic grouping of haplotypes within a to reconstruct phylogenetic relationships among haplotypes population lineage) of the focal-species haplotypes, and in- using maximum parsimony and 100 heuristic searches with clusion of at least two individuals from as many localities random addition of sequences and tree bisection-reconnection as possible to evaluate gene ¯ow among populations. Dis- branch swapping. The nonparametric bootstrap was con- cordance between the phylogenetic relationships among hap- ducted using 500 bootstrap pseudoreplicates with 10 random lotypes and the geographic areas from which they are sampled taxon-addition-sequence replicates per bootstrap pseudore- is interpreted as evidence for gene ¯ow among populations plicate. Decay indices (Bremer 1994) were calculated for all of the focal species (Slatkin and Maddison 1989). Well-sup- nodes using Treerot version 2a (Sorenson 1999). Following ported groupings of haplotype clades that are concordant with selection of the GTR ϩ I ϩ⌫model using MrModelTest 1.1b geography delimit independent populational lineages con- (http://www.ebc.uu.se/systzoo/staff/nylander.html), Bayesian cealed by previous taxonomy. Inclusion of as many species phylogenetic analyses were implemented in MrBayes 3.01 as possible that are closely related to the focal species is (Huelsenbeck and Ronquist 2001). Flat priors were used for crucial, because a focal species may be nonexclusive for hap- all parameter estimates, and random trees were used to begin lotypic variation and may comprise multiple cryptic lineages each Markov chain. Four incrementally heated Markov chains that are not each other's closest relatives (e.g., Sinclair et al. were run for 2 ϫ 106 generations, sampling every 2000 gen- 2004). erations. To ensure that the Markov chains reached a stable Application of this tree-based approach to mtDNA phy- equilibrium, ln-likelihood values for sampling points were logenies has an important strength for delimiting independent plotted against generation time. All samples prior to the point evolutionary lineages. Mitochondrial genes have an effective at which ln-likelihood values achieved stationarity were dis- population size one quarter that of nuclear genes. Thus, spe- carded as burn-in; remaining samples estimated the posterior- cies become reciprocally monophyletic for mtDNA haplo- probability distributions for the tree topology, branch lengths, types more quickly than for those of neutral nuclear genes and substitution parameters. To ensure that analyses were not (Moore 1995; but see Hoelzer 1997). However, for species trapped on local optima, ®ve replicate searches were con- in which gene ¯ow among populations is restricted by geo- ducted; independent analyses were considered to have con- graphic distance (isolation by distance), haplotypes from pop- DIVERSIFICATION OF DUSKY SALAMANDERS 2003

FIG. 1. Geographic sampling locations for the Desmognathus populations included in our phylogenetic study. Detailed locality information is listed in Appendix 1 (available online). 2004 KENNETH H. KOZAK ET AL. ulations may appear exclusive when sampling is sparse rel- sumption of equal speciation and extinction among lineages. ative to the dispersal abilities of the species (Hedin and Wood Incomplete taxon sampling at the species level tends to un- 2002; Irwin 2002; Templeton 2004). In these cases, failure derestimate the number of nodes toward the present and may to detect gene ¯ow among populations will cause overre- cause an apparent slowdown in the diversi®cation rate (Nee solution of species limits. Mitochondrial DNA gene trees and et al. 1992; Pybus and Harvey 2000). Our use of geographic species trees may also be discordant as a result of intro- patterns of haplotype coalescence to discriminate tokogenetic gression or lateral transfer mediated by interspeci®c hybrid- (within species) and hierarchical branching patterns (between ization (TegelstroÈm 1987; Good et al. 2003; Weisrock et al. species) in mtDNA phylogenies permitted a rigorous delim- 2005). Another potential problem is that mtDNA gene trees itation of putatively independent population-level lineages and species trees may be discordant because of differences included in our analyses. in male and female-mediated gene ¯ow (e.g., Jockusch and Wake 2002). We therefore conducted our lineage-accumu- Comparative Analyses lation rate analyses under minimum and maximum estimates We measured 16 morphological variables of the head, of the number of putatively independent evolutionary line- trunk, limbs, and tail, from 300 adult salamanders repre- ages. Because concordance of species limits inferred from senting 24 species/geographic lineages of Desmognathus.We unlinked markers often provides the strongest evidence of entered the ln-transformed morphological variables into prin- species status (Avise and Ball 1990; Sites and Crandall 1997; cipal component analyses using the covariance matrix. To Wiens and Penkrot 2002), we also examined whether inde- test whether lineages could be discriminated in morpholog- pendent evolutionary lineages inferred from the mtDNA data ical space according to a priori categories of microhabitat are con®rmed by published morphological and nuclear-en- use (Table 1), we conducted a multivariate analysis of var- coded allozyme data. iance (MANOVA). For each lineage we used the mean factor scores along each principal component axis as dependent Estimates of Lineage-Accumulation Rates variables and microhabitat category as the treatment effect. To test whether lineage-accumulation rates (speciation mi- Lineages showed highly signi®cant morphological differenc- nus extinction) remained constant, we transformed the Bayes- es based on their microhabitat use (Wilks' ␭ϭ0.0000151, ϭ Ͻ ian consensus phylogram using penalized likelihood (San- F48,9.7167 14.9250, P 0.0007). Discriminant-function derson 2002) and plotted the natural logarithm of the number analysis assigned all lineages to their designated categories of lineages against the branch-length distance from the root of microhabitat use except for D. brimleyorum, which was node, with the total tree depth scaled to a value of 1.0. We assigned to the stream microhabitat category. The range of tested for a signi®cant departure from the null hypothesis of this stream-edge species is geographically disjunct from all a constant rate of lineage accumulation by analyzing the dis- other desmognathines, suggesting that its large body size may tribution of relative divergence times among species using re¯ect release from competition with other congeners. Ex- survivorship models described by Paradis (1997, 1998). DIV- clusion of this species does not alter the ability of the analyses ERSI version 0.20.0 (Paradis 2000) was used to test three to discriminate all the categories of microhabitat use. alternative models of lineage accumulation in a maximum- Because overall body size (measured as the distance in likelihood framework. Model A speci®es a constant rate of millimeters from the tip of the snout to the posterior end of lineage accumulation, ␦, and represents the null hypothesis the cloaca) explained 94% of the morphological variation of no heterogeneity in rates through time. Model B speci®es (Fig. 2), we used ln-transformed snout-vent length (ln-SVL; a gradual change in the rate of lineage accumulation through Table 2) as a proxy for overall morphological divergence in time by estimating an additional parameter, ␤. Values of ␤ all subsequent analyses. The mean ln-SVL for each lineage Ͼ 1 indicate that ␦ has decreased through time; values of ␤ was used because it is not possible to incorporate variance Ͻ 1 indicate that ␦ has increased through time. Model C around the mean values with current comparative phyloge- speci®es two different rates of lineage accumulation before netic methods. ␦ ␦ ( 1) and after ( 2) a speci®ed break point in time. Because model A is nested within models B and C, a likelihood-ratio Phylogenetic tests of ecomorphological divergence test is used to ask whether the data reject the constant-rates Because lineages share parts of their evolutionary histories, model in favor of an alternative in which the rate of lineage they cannot be considered independent datapoints; therefore, accumulation changes over time. In the case of models B and comparative methods must correct for phylogenetic nonin- C, which are not nested, the Akaike information criterion dependence in statistical analyses (Felsenstein 1985). Due to (AIC; Akaike 1973) is used to ask whether one model is a lack of morphological data for some lineages, it was not signi®cantly better than the other. The model with the lowest possible to include all the lineages in the lineage-accumu- AIC is the one that best describes temporal variation in rel- lation rate analyses; however, because all missing lineages ative divergence times. are morphologically cryptic forms, our sampling captures the An interpretation that lineage-accumulation rates represent major trends of morphological evolution in Desmognathus. instantaneous rates of diversi®cation at past times requires We used phylogenetic analyses of variance (Garland et al. that speciation and extinction occur equally among lineages 1993) to test the hypothesis that morphological divergence during a given time interval, and that all extant species have among 24 lineages is correlated with microhabitat use in- been sampled. We used the relative-cladogenesis statistic dependent of phylogenetic effects (Glor et al. 2003). For these (Purvis et al. 1995; Rambaut et al. 1997) to test the as- analyses, we simulated body-size evolution under a gradual DIVERSIFICATION OF DUSKY SALAMANDERS 2005

FIG. 2. Scatterplot of principal component axes 1 and 2. Shapes indicate groups of microhabitat use. Mean principal component scores for each species/geographic lineage in Table 2 are depicted (excluding Desmognathus apalachicolae, which is included in all subsequent analyses based on the mean snout-vent length [SVL] reported in Titus and Larson 1996): 1, D. aeneus;2,D. wrighti;3,D. ocoee A; 4, D. ocoee F; 5, D. carolinensis;6,D. imitator;7,D. ocoee G; 8, D. ochrophaeus;9,D. orestes; 10, D. ocoee C; 11, D. santeetlah; 12, D. auriculatus; 13, D. conanti D; 14, D. fuscus B; 15, D. fuscus A; 16, D. monticola; 17, D. brimleyorum; 18, D. welteri; 19, D. quadramaculatus/ marmoratus A; 20; D. quadramaculatus/marmoratus C; 21, D. quadramaculatus/marmoratus B; 22, D. quadramaculatus/marmoratus E; 23, D. folkertsi. model (Brownian motion) of evolutionary change with the logenies sampled from the Bayesian posterior distribution. constraint that the mean and the variance of the simulated We then grouped the 24 sampled lineages into stream, stream- body sizes equal those observed for the real data. To account edge, seepage, and terrestrial categories of microhabitat use for uncertainty in the topology and branch lengths, we sim- and conducted an ANOVA on each of the 95,000 simulated ulated 100 datasets along each of the 950 post-burn-in phy- datasets. Using this procedure, we generated a null distri- bution for the F-statistic. If fewer than 5% of the simulated datasets had F-values greater than or equal to the F-value of TABLE 2. Twenty-four lineages of Desmognathus sampled for mor- phometric analyses. Lineage designations are identi®ed in Figure the real dataset, we considered the results statistically sig- 3. See Appendix 2 (available online only at http://dx.doi.org/ ni®cant. To determine whether all groups of microhabitat use 10.1554/05-024.1.s1) for exact geographic locations of specimens could be discriminated from each other, we repeated the pro- measured. cedure for each possible pair of microhabitat categories. These tests were implemented in software developed by L. Species/geographic Mean lineage sampled N SVL (mm) Ϯ S. E. J. Harmon (available upon request). D. wrighti 10 26.4 Ϯ 0.6 D. aeneus 7 25.8 Ϯ 0.2 Accumulation of ecomorphological disparity D. imitator 13 42.9 Ϯ 1.2 D. ocoee A 5 42.9 Ϯ 1.8 To quantify the tempo of ecomorphological divergence, D. ocoee C 10 44.6 Ϯ 0.8 we used relative disparity plots (Harmon et al. 2003). Dis- D. ocoee F 7 40.1 Ϯ 1.6 parity of a clade was calculated as the mean squared pairwise D. ocoee G 7 46.2 Ϯ 0.8 difference in body size between all pairs of terminal taxa. Ϯ D. carolinensis 12 45.6 0.8 We ®rst calculated the disparity for the entire Desmognathus D. orestes 10 38.9 Ϯ 1.2 D. ochrophaeus 12 40.4 Ϯ 0.7 clade and then for each subclade de®ned by a node in the D. apalachicolae * 42.7 phylogeny. Relative disparity of each subclade was stan- D. fuscus B 28 59.0 Ϯ 1.8 dardized by dividing a subclade's disparity by the disparity D. fuscus A 5 61.8 Ϯ 2.3 of the entire clade. To investigate the partitioning of eco- D. conanti D 24 56.8 Ϯ 1.4 D. santeetlah 10 45.2 Ϯ 0.9 morphological disparity among subclades of varying ages, D. auriculatus 8 51.1 Ϯ 0.3 we moved up the phylogeny from the root. At each depth in D. monticola B 23 58.9 Ϯ 1.7 the phylogeny, we calculated the mean relative disparity for D. brimleyorum 16 78.1 Ϯ 1.0 all subclades whose ancestral lineages were present at that Ϯ D. welteri 11 66.4 2.1 time. We then plotted these mean morphological disparities D. folkertsi 18 71.4 Ϯ 1.0 D. quad/marm A 6 70.2 Ϯ 2.6 across the full depth of the phylogenetic tree. D. quad/marm B 20 82.8 Ϯ 1.4 To evaluate how much the mean disparity differed from D. quad/marm C 20 77.1 Ϯ 1.4 that expected under a null hypothesis of unconstrained D. quad/marm E 19 69.4 Ϯ 1.4 Brownian motion, we conducted 1000 simulations on the * Data are from Titus and Larson 1996. ultrametric Bayesian consensus phylogram with the con- 2006 KENNETH H. KOZAK ET AL. straint that the mean and the variance of the simulated tip phological divergence promotes the coexistence of lineages. values be equal to those observed for the real data (Garland Second, a negative relationship is expected if lineages must et al. 1993). We then used the morphological disparity index be ecomorphologically similar to coexist. Third, no relation- (MDI) to compare the overall difference in relative disparity ship should occur if lineages have randomly assembled into of a clade compared with that expected under the null hy- communities with respect to ecomorphology. In groups show- pothesis (Harmon et al. 2003). The MDI calculates the area ing phylogenetic niche conservatism, the degree of ecomor- contained between the line connecting the actual relative dis- phological divergence among lineages is positively correlated parity points calculated from the data and the line connecting with their phylogenetic divergence (BoÈhning-Gaese and Ob- median relative disparity points of the simulations. We used errath 1999; Webb et al. 2002). Under such circumstances, the median relative disparity because some simulations pro- a signi®cant relationship between phylogeny and coexistence duce values that are not normally distributed and the median is expected also if community assembly has been nonrandom therefore provides a better measure of central tendency. Areas with respect to patterns of ecomorphological divergence. in which the observed values were greater than expected are Ecomorphological divergence also may be positively corre- assigned positive values; those below expected are given neg- lated with the geographic overlap of lineages independent of ative values. Positive MDI values indicate that the observed phylogenetic history. Such a relationship is expected if eco- disparity is mainly partitioned within subclades, whereas neg- morphs are evolutionarily labile and arose repeatedly during ative values indicate that it is partitioned mostly among sub- the assembly of ecological communities. clades. Our phylogenetic analyses of variance demonstrate To explore these potential relationships, we conducted that ecomorphological variation among lineages is greater three-way Mantel tests, which calculate the partial Mantel than expected by chance (see Results section), and these rel- correlation between two variables while holding the effect of ative disparity plots provide a visual depiction of the accu- a third variable constant (Legendre and Legendre 1998; mulation of ecomorphological disparity. All simulations and Smouse et al. 1986). We evaluated the relationship between calculations were implemented in software developed by L. the morphological divergence matrix and the geographic co- J. Harmon (available upon request). existence matrix while holding the effect of phylogenetic distance on these variables constant. We evaluated whether Phylogeny, ecomorphology, and community assembly the correlation between the matrices was statistically signif- icant using 9999 Monte Carlo randomizations. If fewer than We used Mantel tests to quantify the relationship between 5% of the randomizations had correlations greater than or matrices representing phylogenetic distance, ecomorphol- equal to the correlation in the real dataset, the results were ogical distance, and geographical coexistence among line- considered signi®cant. All Mantel tests were implemented in ages. Ecomorphological distances were calculated as the pair- the software package PASSAGE (Rosenberg 2002). wise difference in snout-vent length between lineages. Phy- logenetic distances were calculated as the path length sepa- RESULTS rating lineages along the ultrametric Bayesian consensus phylogram. To determine the pattern of spatial overlap among Phylogenetic Relationships lineages, we used information on Desmognathus community A total of 115 sequences representing 1581 aligned bases composition from the literature (summarized in Petranka is reported for 19 species of desmognathine salamanders and 1998) for nominal species that were monophyletic and/or two outgroup taxa. Absence of premature stop codons in the considered a single lineage. For other species we mapped the ND2 and CO1 protein-coding regions, functional stability of geographic distributions of monophyletic phylogeographic the ®ve tRNA genes, and strong bias against guanine on the subunits and determined whether their ranges overlapped. light strand indicate that the DNA sequences are from the Although some geographically overlapping Desmognathus mitochondrial genome and not nuclear-integrated copies of species differ in their mean elevational distributions, stream- mitochondrial genes (Zhang and Hewitt 1996). An indel from side communities at intermediate elevations contain both positions 967±972 in the ND2 gene precludes unambiguous high- and low-elevation forms (Hairston 1949; Organ 1961; alignment of these sites in some taxa; these sites are therefore Bruce 1991; Tilley 2000; Dodd 2004), suggesting that the excluded from phylogenetic analyses. Length variation in the geographical overlap of phylogeographic subunits is an ap- D-loop of the tRNAAsn gene (sites 1246±1259) and origin of propriate measure of the maximum number of species that light-strand replication (1289±1294) precludes unambiguous coexist in local assemblages. The pattern of coexistence alignment of those sites, which are excluded from phylo- among all lineages was then represented as a binary design genetic analyses. matrix (1, lineages coexist; 0, lineages do not coexist). This Bayesian phylogenetic analysis using the GTR ϩ I ϩ⌫ approach does not consider the magnitude of overlap between model produces a 50% majority-rule consensus tree with a lineages but potentially gives a more sensitive test of the mean ln-likelihood of Ϫ20,323.54 (SD ϭ 13.73) following relationship between phylogeny, ecomorphology, and geo- a burn-in of 100,000 generations (Fig. 3). Parsimony analysis graphic coexistence by retaining information on which lin- of 667 informative characters (613 within the ingroup) pro- eages coexist locally in at least some part of their geograph- duced 49 equally most parsimonious trees of 3788 steps. ical ranges (Barraclough et al. 1999). Because both analyses produced highly congruent phyloge- The relationship between ecomorphological divergence netic estimates, with all topological differences restricted to and geographic overlap of lineages may follow three possible nodes that are weakly supported (bootstraps Ͻ50% and pos- forms. First, a positive relationship is expected if ecomor- terior probabilities Ͻ95%), only the Bayesian tree is pre- DIVERSIFICATION OF DUSKY SALAMANDERS 2007

FIG. 3. Bayesian 50% majority-rule consensus phylogram. Posterior probabilities based on 950 post-burn-in trees (which had a mean likelihood score of Ϫ20,323.54; SD ϭ 13.73) are shown below the branches and nonparametric bootstrap/decay indices for the parsimony analysis above. Branch lengths represent means from the post-burn-in sample of trees. Black versus gray vertical bars designate alternative diagnoses of independent evolutionary lineages that were considered in the diversi®cation-rate analyses. 2008 KENNETH H. KOZAK ET AL. sented, with nonparametric bootstrap and decay indices from also included to test the robustness of our lineage-accumu- the parsimony analyses included for shared branches. lation rate analyses to possible errors of incomplete taxon Our phylogenetic analyses con®rm that Phaeognathus hu- sampling. The groupings of haplotypes corresponding to both brichti is the sister taxon to Desmognathus; D. wrighti is the schemes are denoted in Figure 3. sister taxon to a clade containing all other species of Des- Haplotypes sampled from D. folkertsi, D. marmoratus, and mognathus, and the lineage leading to D. aeneus diverged D. quadramaculatus form exclusive groups that are concor- early in the latter clade (Titus and Larson 1996; Rissler and dant with geography rather than current taxonomy. Desmog- Taylor 2003). The exact phylogenetic relationships among nathus folkertsi is known only from a single locality in the D. aeneus, D. imitator, and the D. folkertsi-marmoratus-quad- Georgia Piedmont where it shows complete reproductive iso- ramaculatus clade are not well supported. These lineages are lation from syntopic D. quadramaculatus (Camp et al. 2002). outside a clade containing all other species with aquatic larval Populations of D. marmoratus in the northern Atlantic Slope, periods, although the latter clade receives strong support only southern Atlantic Slope, and Tennessee River drainages are in the Bayesian analysis. Within the latter clade, compre- grouped with D. quadramaculatus haplotypes from the same hensive inter- and intraspeci®c sampling identi®es seven ma- drainage basins. These results are at least partly concordant jor evolutionary branches separated by short and poorly sup- with allozyme variation showing seven ®xed differences be- ported internodes (Fig. 3). These branches are (1) D. ocoee tween populations of D. marmoratus on opposite sides of the (populations 11±16) from the Great Smoky Mountains, Pis- eastern Continental Divide (Voss et al. 1995). An indepen- gah Ridge, Nantahala Mountains, and Tugaloo River; (2) D. dent study of the D. quadramaculatus-marmoratus complex brimleyorum; (3) D. orestes ϩ D. ochrophaeus; (4) D. welteri also reveals phylogeographic patterns corresponding to drain- ϩ D. fuscus ϩ D. auriculatus; (5) D. conanti ϩ D. santeetlah age basins rather than to named species (Jones et al. 2005). ϩ D. auriculatus; (6) D. carolinensis ϩ D. fuscus; and (7) D. We recognize D. folkertsi and ®ve strongly supported lineages apalachicolae ϩ D. monticola ϩ D. ocoee (populations 7± corresponding to major drainage basins (quad/marm A, B, 10) from the Tusquittee Mountains, Unicoi Mountains, and C, D, and E). lowland tributaries of the Tennessee River. Phylogenetic re- Desmognathus fuscus haplotypes are nonexclusive with re- lationships corroborated by analyses of nuclear-encoded al- spect to D. auriculatus and D. carolinensis. Geographic pat- lozymic loci and additional mitochondrial gene sequences terns of haplotype coalescence diagnose at least two inde- include the grouping of D. ochrophaeus and D. orestes as a pendent evolutionary lineages. Haplotypes from populations clade (Mead et al. 2001); the close relationship between D. in the Cumberland Plateau (fuscus A), and Ohio River Basin conanti and D. santeetlah (Tilley and Schwerdtfeger 1981; plus Atlantic Slope (fuscus B) form exclusive groups con- Titus and Larson 1996); the close relationship between D. cordant with geography. Six groups of haplotypes within the welteri and D. fuscus (Karlin and Guttman 1986); the close latter clade are also exclusive, but more inclusive groups of relationship between some populations of D. ocoee and D. these haplotypes have geographic ranges that overlap. This apalachicolae (Means and Karlin 1989); and the grouping of spatial and temporal phylogenetic pattern is suggestive of D. folkertsi, D. marmoratus, and D. quadramaculatus as a restricted gene ¯ow with isolation by distance (Wiens and clade (Titus and Larson 1996; Camp et al. 2002). Penkrot 2002; Templeton 2004). No topologies sampled from the Bayesian posterior dis- A small subset of D. fuscus haplotypes sampled from the tribution were compatible with monophyly of the stream, Atlantic Slope (populations 59, 64, 67) interdigitate with hap- stream-edge, seepage, or terrestrial groupings of microhabitat lotypes from sampled D. carolinensis. These haplotypes are use. sampled from within the range of D. fuscus, but they are Relative-rate tests demonstrate that a single lineage, P. geographically disjunct from the range of D. carolinensis, hubrichti, has accumulated nucleotide substitutions at a sig- making contemporary gene ¯ow an unlikely explanation for ni®cantly different rate than the remaining samples (P ϭ the discordance between the gene tree and current taxonomy. 0.029). One explanation for this pattern is that D. carolinensis is composed of multiple independent evolutionary lineages that Intraspeci®c Phylogeny and Delimitation of Independent extend beyond its currently known range in the Blue Ridge Evolutionary Lineages Mountains. However, a ®ne-scale study of allozyme variation in D. carolinensis suggests that isolation by distance has Haplotypes from D. brimleyorum, D. ochrophaeus, and D. shaped geographic patterns of genetic variation within this welteri form strongly supported monophyletic groups, and species (Tilley 1997), but that study did not include geo- these three lineages are diagnosable using available morpho- graphically proximate populations of D. fuscus. We therefore logical and/or allozyme data. These species exhibit shallow hypothesize that D. carolinensis is a single lineage, and that haplotypic divergences throughout their geographic ranges, discordance between the gene tree and species tree results suggesting that they do not harbor cryptic species. However, from mtDNA introgression facilitated by past geographic our comprehensive geographic sampling suggests that other contact with D. fuscus. nominal species of Desmognathus comprise phylogenetically Desmognathus conanti haplotypes are nonexclusive with and geographically distinct groups of haplotypes diagnosing respect to D. santeetlah and D. auriculatus from the Gulf multiple, independent evolutionary lineages. Below we dis- Coastal Plain. Geographic patterns of haplotype coalescence cuss diagnoses of 35 putatively independent lineages. A more diagnose four phylogenetically and geographically distinct extreme set of alternative diagnoses that likely exceed the evolutionary lineages. The phylogenetic split separating hap- maximum number of independent evolutionary lineages is lotypes of D. conanti populations in Atlantic Coastal Plain DIVERSIFICATION OF DUSKY SALAMANDERS 2009 drainages (conanti A) and Gulf Coastal Plain plus Lower sample multiple individuals from a population those haplo- Tennessee River drainages (conanti B ϩ C ϩ auriculatus)is types always form exclusive groups. These results are con- concordant with the allozymic results of Bonett (2002) in sistent with the allozyme study of Tilley and Mahoney (1996) diagnosing separate evolutionary lineages. Our results also in showing that D. ocoee populations from each major moun- suggest that populations of D. conanti from the Lower Ten- tain range in southwestern North Carolina might represent nessee River Basin (conanti D) are phylogenetically distinct different evolutionary species. from samples of D. conanti (conanti C) and D. auriculatus Geographic patterns of haplotype coalescence diagnose in Gulf Coastal Plain drainages. Our sampling of single hap- two exclusive lineages within D. orestes (orestes A and B). lotypes from the Mississippi Embayment (conanti B) and These lineages are also diagnosed by patterns of geographic Gulf Coastal Plain (conanti C) limits application of the Wiens genetic variation in allozymes (Tilley and Mahoney 1996). and Penkrot (2002) method. We anticipate that further ®ne- Hybridization occurs across their contact zone in the Blue scale study of genetic variation in these regions will corrob- Ridge (Mead et al. 2001). orate these phylogeographic patterns and uncover cryptic spe- A recent analysis of mtDNA and allozyme variation in D. cies corresponding to major drainage basins. We therefore wrighti demonstrates that populations distributed north recognize conanti A, B, C, and D as independent evolutionary (wrighti A) and south (wrighti B) of the Asheville Basin are lineages. distinct evolutionary lineages, and that isolation by distance Geographic genetic variation in D. auriculatus requires has shaped geographic patterns of genetic variation within much further study. An Atlantic Coastal haplotype of D. au- each of these lineages (Crespi et al. 2003). We therefore treat riculatus groups with eastern and northern D. fuscus haplo- D. wrighti haplotypes from these regions as independent evo- types, whereas a Gulf Coastal haplotype of D. auriculatus lutionary lineages. groups with haplotypes of D. conanti from this region. Geo- Desmognathus apalachicolae, D. imitator, and D. santee- graphic genetic variation in D. auriculatus has not been stud- tlah are diagnosable based on available morphological and ied. Karlin and Guttman (1986) also reported deep genetic allozyme data. These species have geographically restricted divergence between a Gulf Coastal Plain population of D. ranges and little geographic genetic variation (Tilley and auriculatus and D. conanti from Tennessee, but they did not Schwerdtfeger 1981; Karlin and Guttman 1986; Tilley 2000), compare populations of these species from the same region. so each is considered a single lineage. Genetic variation in Our study is the ®rst phylogenetic analysis to include geo- D. aeneus has not been studied. graphically separated populations of this form. The Atlantic Coastal Plain haplotype is nearly identical to northern and Diversi®cation Rates eastern haplotypes of D. fuscus; we consider these popula- tions conspeci®c until analyses demonstrate their evolution- The lineages-through-time plot indicates a burst of lineage ary independence. The Gulf Coastal haplotypes are exclusive, accumulation followed by deceleration in the accumulation and we consider these populations phylogenetically distinct of lineages (Fig. 4). To test whether the accumulation of from D. conanti for our analyses. Desmognathus lineages deviates from a constant-rates model The range of D. monticola is fragmented into geographic of diversi®cation, we ®t three alternative models of diver- isolates in the Alabama Piedmont (monticola A) and in the si®cation to our dataset using maximum likelihood. These Appalachian Highlands plus Alleghany Plateau (monticola tests assume that speciation and extinction are equally prob- B), and these isolates correspond to exclusive groups of hap- able among all lineages. lotypes. Haplotypes sampled throughout the range of this The relative cladogenesis statistic shows signi®cant dif- species are remarkably uniform and indicate that this species ferences in diversi®cation rates among Desmognathus line- has recently expanded its range throughout the Appalachian ages. In particular, the ancestral node uniting lineages with Highlands and Alleghany Plateau. These patterns of phylo- aquatic larval periods has experienced a higher rate of di- geographic divergence con®rm results of a recent study of versi®cation relative to the ancestral lineages leading to D. mtDNA variation (Rissler and Taylor 2003); we therefore wrighti and D. aeneus (P ϭ 0.016). At least some of this rate treat these two groupings as independent evolutionary line- difference is likely attributed to a lack of data on geographic ages. genetic structure within D. aeneus; however, this species Desmognathus ocoee is nonexclusive and comprises at least would have to harbor many cryptic species for rates of lineage two independent evolutionary lineages. A recent allozyme accumulation to be equal among direct-developing and meta- study also shows D. ocoee to be nonmonophyletic (Anderson morphosing lineages. This asymmetry in lineage-accumula- and Tilley 2003). Haplotypes sampled from populations in tion rates also remains signi®cant in a pruned dataset that the Great Smoky Mountains (ocoee A), Pisgah Ridge (ocoee treats each of the formally recognized species of Desmog- B), Nantahala Mountains (ocoee C), and lowland tributaries nathus as a single lineage (P ϭ 0.02). We therefore followed of the Tugaloo River in the Georgia Piedmont (ocoee D) form Pybus and Harvey (2000) and conducted these analyses on a strongly supported monophyletic group. The remaining both the full dataset and a subset containing only species haplotypes sampled from populations in the Tusquittee with aquatic larvae. Regardless of which dataset was used, Mountains (ocoee E), Unicoi Mountains (ocoee F) and the the survivorship analyses reject a constant-rates model of Alleghany Plateau in Alabama (ocoee G) and Tennessee lineage accumulation in favor of ones that specify declining (ocoee H) are grouped in a clade with D. apalachicolae and rates of diversi®cation (Table 3). D. monticola. Despite the highly restricted area from which Overdispersed taxon sampling may cause spurious rejec- D. ocoee populations are sampled, in all cases where we tion of a constant-rates model of diversi®cation in favor of 2010 KENNETH H. KOZAK ET AL.

FIG. 4. (A) Ultrametric Bayesian consensus phylogram for the 35 species/geographic lineage dataset. (B) Semilogarithmic plot of lineages through time for the ultrametric tree shown in (A); time is expressed as a proportion of the total time after the ®rst cladogenetic event inferred for the taxon; estimated absolute timing based on alternative fossil-based ages (28 vs. 47 million years; Chippindale et al. 2004) for the most recent common ancestor of Phaeognathus ϩ Desmognathus is shown below in parentheses. one showing lineage-accumulation rates to decrease over time some lineages that have become evolutionarily independent (Pybus and Harvey 2000; Shaw et al. 2003). Our phylogeo- too recently to have evolved diagnostic differences for our graphic methods permit more precise delimitation of puta- molecular markers could have been missed by our method tively independent population-level lineages; nevertheless, of lineage delimitation. Model C speci®es an abrupt shift in DIVERSIFICATION OF DUSKY SALAMANDERS 2011

TABLE 3. Tests of the null hypothesis that the rate of lineage accumulation (speciation minus extinction) was constant during the phylogenetic history of Desmognathus. Model A speci®es a constant lineage-accumulation rate. Model B speci®es a gradual decrease in the lineage-accumulation rate. Model C speci®es different lineage-accumulation rates before and after the most recent split in the mtDNA phylogeny that was diagnosed as leading to the formation of independent evolutionary lineages. Model C is expected to ®t the data best if a decrease in the lineage-accumulation rate is an artifact of the lag time needed for lineages to evolve diagnostic molecular differences.

Phylogeny n Model A lnL (AIC) Model B lnL (AIC) Model C lnL (AIC) 1. All Desmognathus 35 Ϫ148.08 (298.17) Ϫ140.621 (285.25) Ϫ142.79 (289.57) 44 Ϫ179.40 (360.80) Ϫ175.161 (354.32) Ϫ176.42 (356.84) 2. Biophasic Desmognathus only 32 Ϫ131.69 (265.38) Ϫ119.971 (243.93) Ϫ126.17 (256.34) 41 Ϫ162.23 (326.47) Ϫ155.211 (314.43) Ϫ159.00 (321.99) 1 The data favor model B, which has the highest ln-likelihood. A likelihood-ratio test is used to compare model A versus B, and model A versus C because they are nested hypotheses (Paradis 1998). Models B and C are not nested, so the Akaike information criterion (AIC; Akaike 1973) is used to compare their ln-likelihoods (Paradis 1998). Models that specify decreasing lineage-accumulation rates ®t the data signi®cantly better than model A (model A vs. B: ␹2 ϭ 8.48±14.92, df ϭ 1, P ϭ 0.01±0.001; model A vs. C: ␹2 ϭ 5.96±10.58, df ϭ 1, P ϭ 0.025±0.01). For all datasets, model B has the lowest AIC score and ®ts the data better than model C. the rate of diversi®cation following the most recent lineage- mognathus exhibit lower average disparity than expected un- splitting event that we diagnosed as leading to independent der the null hypothesis of unconstrained divergence by evolutionary lineages. This model would be expected to ®t Brownian motion (Fig. 5). This result is corroborated by a the data best if a constant-rate model is rejected purely as an MDI value of Ϫ0.18, which indicates that ecomorphological artifact of the lag time for lineages to evolve diagnostic mo- variation is partitioned mainly among older subclades of Des- lecular differences. Model B, which speci®es a gradual de- mognathus. cline in the diversi®cation rate, ®ts the data signi®cantly bet- ter than Model C (Table 3), suggesting that rejection of the DISCUSSION constant-rates model is not an artifact of the resolution of our molecular markers or methods of lineage delimitation. Classic models of adaptive radiation link the buildup and maintenance of species richness with the evolution of eco- Comparative Analyses morphological disparity. Ecomorphological divergence is hy- pothesized to play a key role in the diversi®cation, long-term Phylogenetic analyses of variance strongly support the hy- persistence, and coexistence of lineages by reducing the pothesis that lineages form distinct groupings in morpholog- strength of ecological interactions among species (Simpson ical space according to their microhabitat use (Table 4). Be- 1953; Schluter 2000). As a corollary, the accumulation of cause we conducted our trait simulations on samples from lineages and ecomorphological disparity may be intimately the Bayesian posterior distribution for the topology and linked to the persistence of species associations over evo- branch lengths, our results are robust to uncertainty in these lutionary time, because ecological interactions among co- parameters. existing lineages in¯uence opportunities for subsequent di- Mantel tests demonstrate a positive correlation between versi®cation (Stanley 1973; Schluter 2000; Harmon et al. morphological divergence and phylogenetic distance (Table 2003). We present a novel, synthetic analysis of phylogeo- 5). Lineages that coexist tend to show greater morphological graphic history, rates of ecomorphological evolution and spe- divergence and phylogenetic divergence than parapatric or cies accumulation, and community assembly in an adaptive allopatric lineages. The partial Mantel test indicates that the radiation. degree of morphological divergence among coexisting line- Methods that use relative branching times in molecular ages is no greater than would be expected from their phy- phylogenies to detect changes in diversi®cation rates (Paradis logenetic relationships alone. 1997, 1998; Pybus and Harvey 2000) rely critically on a The relative disparity plot shows that subclades of Des- complete sampling of extant independent evolutionary lin- eages (Pybus and Harvey 2000; Pybus et al. 2002; Shaw et TABLE 4. Phylogenetic analyses of variance for body size between al. 2003). Most studies that have used molecular phylogenies pairs of microhabitat categories (see Table 1). A signi®cant result of extant taxa to make inferences about diversi®cation pat- indicates that the paired categories differ in mean body size after terns consider only a single representative of each described correcting for phylogenetic relatedness. species (Barraclough and Nee 2001). Given the ubiquity of Observed historically isolated, cryptic phylogeographic units within Phylogenetic ANOVA F-statistic Simulated P1 many taxonomic species (Avise 2000), a rigorous character- Full dataset 73.46 0.000011 ization of geographic patterns of genetic variation among Stream vs. terrestrial 218.69 0.000011 populations is necessary in many taxonomic groups to delimit Stream vs. seepage 200.38 0.000011 the evolutionary units most appropriate for analysis. Our geo- Stream vs. stream-edge 11.73 0.003726 graphic sampling of genetic variation within many of the Stream edge vs. terrestrial 55.95 0.000074 Stream edge vs. seepage 36.43 0.000021 formally recognized species of Desmognathus suggests that Seepage vs. terrestrial 124.41 0.000011 the current taxonomy of the group underestimates extant spe- 1 P is the probability of obtaining an F-statistic equal to or greater than cies-level diversity. The geographic limits of many of these the observed value by chance. lineages are generally congruent with those diagnosed by 2012 KENNETH H. KOZAK ET AL.

TABLE 5. Mantel tests for association between morphological distance, phylogenetic distance, and coexistence among 19 lineages (Appendices 3±5, available online only at http://dx.doi.org/10.1554/05-024.1.s1). Signi®cance of Mantel correlation coef®cients (r)is evaluated using 9999 Monte Carlo randomizations.

Matrices tested t1 rP2 Morphological distance, phylogenetic distance 3.45 0.52 0.0001 Phylogenetic distance, coexistence 1.69 0.26 0.0562 Morphological distance, coexistence 1.79 0.21 0.0367 3Phylogenetic distance, morphological distance, coexistence 0.89 0.08 0.1774 1 Student's t. 2 Probability that a value of t this large or larger would result from chance alone. 3 Effect of phylogeny on morphological distance and coexistence was removed using a three-way Mantel test. strong allelic-frequency differences in allozymes, although that this major life-history shift was followed by a high rate we emphasize that some of our diagnoses are based solely of lineage accumulation that then gradually decreased until on mtDNA variation and should be con®rmed using nuclear approximately 1±2 million years ago (the minimum separa- DNA markers before they receive formal taxonomic recog- tion needed for independent evolutionary lineages to accu- nition (Glor et al. 2004). mulate diagnostic differences for our molecular markers). Recent molecular phylogenetic analyses of the salamander The diversi®cation of Desmognathus has important impli- family Plethodontidae provide strong support for the hy- cations for understanding the high species richness and phe- pothesis that the ancestral life-history mode in desmogna- notypic disparity that characterize eastern North American thines is direct development (Titus and Larson 1996) and plethodontid salamanders. Ecological access to terrestrial suggest that aquatic larvae re-evolved relatively early in the adaptive zones has likely been limited by competition from evolutionary history of dusky salamanders (Chippindale et codistributed species of the genus Plethodon, which are al. 2004; Mueller et al. 2004). Our diversi®cation-rate anal- among the most abundant vertebrates in terrestrial ecosys- yses are based on relative divergence times; nevertheless, the tems (Davic and Welsh 2004), and have occupied and di- absolute time course for the radiation of desmognathines can versi®ed within a terrestrial adaptive zone for approximately be roughly approximated. Based on molecular-rate calibra- 30 million years (Highton 1995; Larson et al. 2003). Our tions (Larson et al. 2003) and fossil-based dating methods inference of an increased rate of diversi®cation associated (Chippindale et al. 2004), the origin of a biphasic life history with a shift from direct development to a biphasic life history occurred 10±17 million years ago (Fig. 4). Our study shows provides strong support for the hypothesis that a major life-

FIG. 5. Relative disparity plot for Desmognathus compared to the expected disparity from phylogenetic simulations of unconstrained, gradual morphological divergence. Average extant disparity at each depth in the tree is the average disparity of subclades whose ancestral lineages were present at that time relative to the disparity of the entire taxon. The higher the value of relative disparity, the greater the average volume of morphological space occupied by subclades relative to the morphological disparity of the taxon as a whole. Time is expressed as a proportion of the total time after the ®rst cladogenetic event inferred for the taxon. The solid line is the actual disparity for the clade; the dashed line is the expected disparity derived from the phylogenetic simulations. At each depth in the phylogeny subsequent to the root, subclades show less average disparity than expected. This pattern suggests that ecomorphological divergence occurred early in the radiation of extant Desmognathus lineages. Because nested clades tend to contain less morphological disparity than the more inclusive clades to which they belong, average disparity generally decreases as time approaches the present. DIVERSIFICATION OF DUSKY SALAMANDERS 2013 history reversal promoted the radiation of Desmognathus in through the geographical overlapping of disparate ecomor- the southern Appalachians by allowing lineages to subdivide phologically stable lineages. Therefore, independent evolu- an aquatic adaptive zone (Chippindale et al. 2004). tion of ecomorphs, which is expected to erase the imprint of The peak period of lineage accumulation in Desmognathus phylogenetic history on community structure (Webb et al. coincides with inferred evolutionary partitioning of lineages 2002), has a limited role in the evolutionary assembly of with aquatic larvae into seepage, stream-edge, and stream Desmognathus communities. microhabitats. Our morphometric results demonstrate a Concentration of lineage accumulation and morphological strong correlation between morphology and microhabitat divergences disproportionately early in the history of the Des- ecology independent of phylogenetic effects, which supports mognathus radiation suggests that ecomorphological diver- the hypothesis that natural selection played an important role si®cation permitted coexistence of disparate forms as early in the buildup and maintenance of phenotypic disparity in as 6 million years ago, by which time ancestral lineages lead- Desmognathus (Miles and Dunham 1993; Schluter 2000). ing to clades of ecomorphologically stable terrestrial, seep- Variation in moisture predictability among microhabitats age, stream-edge and stream ecomorphs appear to have orig- along the aquatic-terrestrial gradient probably elicited adap- inated (Fig. 4). Geographic isolation induced by climatic ¯uc- tive divergence in length of the larval and/or juvenile period, tuations (Highton 1995; Tilley and Mahoney 1996) and shift- producing body-size divergence among Desmognathus spe- ing drainage divides (Voss et al. 1995) likely prevailed in cies (Bruce 1990, 1996; Camp et al. 2000). the multiplication of ecomorphologically similar species Although our molecular phylogenetic analyses reject hy- since that time. Although some populations of Desmognathus potheses that ecomorphs form monophyletic groups, our mor- have undergone localized range expansions at northern lat- phological-disparity analysis suggests that ecomorphological itudes (Rissler and Taylor 2003), the deep phylogeographic changes occur disproportionately early in the radiation of structure that we show within many codistributed ecomorph Desmognathus. Thus, the overall evolutionary pattern is one clades suggests long-term persistence and stability of endem- of early adaptive divergence followed by long-term persis- ic lineages and communities through multiple climatic cycles tence of ecomorphologically distinct lineages (Barraclough (Watts 1980; Webb and Bartlein 1992; Webb et al. 1995). et al. 1999). This temporal pattern of ecomorphological di- More generally, Desmognathus contrasts with other conti- vergence is found in other ancient vertebrate adaptive radi- nental evolutionary radiations where lineages diversi®ed in- ations (Streelman et al. 2002; RuÈber et al. 2003; Stephens dependently of each other in the absence of long-term evo- and Wiens 2003) and contrasts with young adaptive radia- lutionary stability in community structure (Barraclough et al. tions where most speciation events are associated with adap- 1999; Webb et al. 2002). tive ecological divergence (Baldwin and Sanderson 1998; In a study of adaptive radiations in iguanian lizards Har- Sato et al. 1999). Therefore, the Desmognathus radiation mon et al. (2003) found that groups whose lineage accu- lends support to a recent hypothesis that adaptive divergence mulation occurs disproportionately early in their evolutionary becomes less important in driving speciation in older adaptive histories partition ecomorphological changes among major radiations where lineages have persisted long enough to ®ll clades rather than among closely related species. This pattern available ecological and geographical space (Stephens and characterizes dusky salamanders, Caribbean Anolis, and Aus- Wiens 2003). tralian agamids, whereas the iguanid genus Liolaemus shows Our analyses of Desmognathus community structure show a steady increase in lineage accumulation through time, and that ecomorphological divergence promotes the coexistence ecomorphological disparity is common among relatively of lineages, a result that provides evidence for strong ongoing closely related species. Our inferred geographic history of ecological interactions among different ecomorph types in desmognathine evolution con®rms a prediction by Harmon the evolutionary assembly of communities. We hypothesize et al. (2003) that early evolution of microhabitat structuring that ecomorphological divergence promotes lineage coexis- among species and geographic expansion of codistributed tence in several ways. First, lineages with smaller body sizes species throughout the group's long-term geographic range have short larval periods (or direct development) and early produces long-term persistence and stability of lineages and maturation (Bruce 1991; Camp et al. 2000); these traits in- morphologies. The deep coregional history that we show for crease survivorship in semiterrestrial microhabitats that are many desmognathine lineages and evidence that ecomor- unsuitable for long-term occupation by large, predatory con- phological divergence promotes their coexistence supports geners with protracted aquatic larval periods and delayed the hypothesis that ecological interactions among lineages maturity (Werner 1986; Rowe and Ludwig 1991; Stearns have restricted opportunities for diversi®cation. The opposite 1992; Camp et al. 2000). Second, competition appears to limit pattern illustrated by Liolaemus appears when geographic ex- the distributional overlap of similar-sized species (Means pansion persists throughout most of a group's evolutionary 1975; Hairston 1986). Furthermore, ecomorphological di- history, and where microhabitat partitioning evolves inde- vergence may be associated with the evolution of reproduc- pendently after new areas are colonized by single lineages; tive isolation; disparity in body size reduces insemination Liolaemus is inferred to have experienced repeated episodes success during plethodontid salamander courtship (Beachy of dispersal and vicariance across the Andes throughout its 1996). evolutionary history (Schulte et al. 2000), in strong contrast The lack of a signi®cant relationship between ecomor- to patterns of diversi®cation in Desmognathus. phological divergence and lineage coexistence independent The Desmognathus pattern of early community assembly of phylogenetic history supports the hypothesis that des- through ecomorphological divergence followed by long-term mognathine communities have formed almost entirely evolutionary stasis of ecological communities and their lin- 2014 KENNETH H. KOZAK ET AL. eages is probably a common one. We speculate that this Bernardo, J. 2000. Early life histories of dusky salamanders, Des- community structure provides strong stabilizing selection on mognathus imitator and D. wrighti, in a headwater seepage in Great Smoky Mountains National Park, USA. Amphib.-Reptilia each of its component lineages. Although evolutionary tran- 21:403±407. sitions among ecomorphs in Desmognathus should be accom- BoÈhning-Gaese, K., and R. Oberrath. 1999. Phylogenetic effects on plished easily by changes in length of the larval period and morphological, life-history, behavioural, and ecological traits of age at maturity, these forms show enormous phylogenetic birds. Evol. Ecol. Res. 1:347±364. Bonett, R. M. 2002. Analysis of the contact zone between the dusky stability. We predict that the historical pattern of diversi®- salamanders Desmognathus fuscus fuscus and Desmognathus fus- cation shared by Anolis, Australian agamids, and dusky sal- cus conanti (Caudata: Plethodontidae). Copeia 2002:344±355. amanders is common in adaptive radiations in which an evo- Bremer, K. 1994. Branch support and tree stability. Cladistics 10: lutionarily stable community structure constrains diversi®- 295±304. cation of the codistributed lineages. Continental refugial ar- Bruce, R. C. 1990. An explanation for differences in body size between two desmognathine salamanders. Copeia 1990:1±9. eas in southeastern North America and the large islands of ÐÐÐ. 1991. Evolution of ecological diversi®cation in desmog- the Greater Antilles provide two examples of such regions. nathine salamanders. Herpetol. Rev. 22:44±45. Comparative studies of other evolutionary radiations using ÐÐÐ. 1996. Life-history perspective of adaptive radiation in des- the phylogeographic, morphometric, and community-ecolog- mognathine salamanders. Copeia 1996:783±790. Camp, C. D., J. L. Marshall, and R. M. Austin. 2000. The evolution ical analyses presented here for Desmognathus can test the of adult body size in black-bellied salamanders (Desmognathus generality of this evolutionary pattern and its ecological and quadramaculatus complex). Can. J. Zool. 78:1712±1722. biogeographic explanations. Camp, C. D., S. G. Tilley, R. M. Austin, and J. L. Marshall. 2002. A new species of black-bellied salamander (genus Desmogna- thus) from the Appalachian Mountains of northern Georgia. Her- ACKNOWLEDGMENTS petologica 58:471±484. We thank C. Camp, E. Casey, K. Irwin, L. Irwin, R. Ma- Campbell, J. A. 1999. Distribution patterns of amphibians in Middle America. Pp. 111±210 in W. E. Duellman, ed. Patterns of dis- kowsky, J. Marshall, L. Mead, D. B. Means, W. Savage, R. tribution of amphibians. 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