Adaptation of Metabolism and Evaporative Water Loss Along An

Received 9 July 2002 Accepted 16September 2002 Publishedonline 10December 2002

Adaptationof metabolismand evaporative water lossalong anaridity gradient B.Irene Tieleman 1 * ,Joseph B.Williams 2 and Paulette Bloomer 3 1ZoologicalLaboratory, University ofGroningen, PO Box14, 9750 AA Haren,The Netherlands 2Departmentof Evolution, Ecology and OrganismalBiology, Ohio State University, 1735Neil Avenue,Columbus, OH43210,USA 3MolecularEcology and EvolutionProgram, Department of Genetics, University ofPretoria,0002 Pretoria, SouthAfrica Broad-scalecomparisons of birds indicatethe possibility ofadaptive modification ofbasal metabolic rate (BMR)and total evaporative water loss(TEWL) in speciesfrom desertenvironments, but these might beconfounded by phylogeny or phenotypic plasticity. This studyrelates variation in avian BMRand TEWLto a continuouslyvarying measureof environment,aridity. Wetestthe hypotheses that BMRand TEWLare reducedalong anaridity gradient within thelark family (Alaudidae),and investigate therole ofphylogenetic inertia. For 12 speciesof lark, BMRandTEWL decreased along agradient ofincreasing aridity, afinding consistentwith ourproposals. We constructeda phylogeny for 22 speciesof lark based onsequences of twomitochondrial genes,and investigated whetherphylogenetic affinity played apart in thecorrelation ofphenotype and environment. A testfor serial independenceof the data for mass- correctedTEWL and aridity showedno influenceof phylogeny onour findings. However, we diddiscover asignificant phylogenetic effectin mass-correcteddata for BMR,aresultattributable tocommonphylo- genetichistory or tocommon ecological factors.A testof the relationship betweenBMR andaridity usingphylogenetic independentconstrasts was consistent with ourprevious analysis: BMRdecreasedwith increasing aridity. Keywords: basal metabolic rate; total evaporative water loss;aridity; larks; Alaudidae;phylogeny

1. INTRODUCTION couldoperate onthese variables (Furuyama &Ohara 1993; Konarzewski& Diamond 1995). Terrestrial vertebrates continuouslyexpend energy to After correcting for body massand higher level taxo- carry outchemical processesnecessary to sustainlife, and nomicaffiliations, alarge variation in metabolism and constantlylose water through respiration, cutaneousevap- TEWLremains (Kleiber 1961; Crawford& Lasiewski oration andexcretion. Rates of energy expenditureand 1968; Dawson1982; McNab1988; Williams 1996). Dif- water lossvary considerably among andwithin vertebrate ferencesin BMRandTEWL have beenreported between taxa evenunder standard laboratory conditions.The speciesor populations differing in diet,altitude, latitude, reasonsunderlying this variation have beenthe focus of temperature andaridity (Dawson& Bennett1973; Wea- intensiveresearch and are still only partially understood thers1979; Nagy 1987; McNab1988; Nagy et al. 1999; (Kleiber 1961; Bartholomew &Cade1963; Crawford& Williams &Tieleman 2001), buta unifying environmental Lasiewski1968; Aschoff& Pohl 1970; McNab1986; characteristic that determinesthe metabolic physiology of Nagy 1987; Bennett1988; Williams 1996; Mueller& Dia- animals hasnot been identified. Mueller & Diamond mond2001). Basal metabolic rate (BMR),the minimum (2001) reportedthat primary productivity explains 88% energy expenditureof afastedendotherm in its restphase ofthevariation in BMRbetweenfive speciesof Peromyscus at thermoneutral temperatures(King 1974), integrates all mice,and suggested that foodavailability, acorrelate of catabolic energy transformations required for bodily main- primary productivity, might bea unifying explanatory tenance.BMR correlates with theenergy expenditureof variable for metabolic physiology. Onemight also predict free-living animals (Nagy 1987; Daan et al. 1990; Ricklefs that water availability is amajor factor explaining variation et al. 1996) andwith life-history attributes,such as growth in TEWL. andreproduction (Kleiber 1961; Bennett1988; Trevelyan Previous workon BMR andTEWL has compared spec- et al. 1990; Harvey et al. 1991; Hulbert &Else2000). iesfrom disparate environments,such as the tropics versus Total evaporative water loss(TEWL), the sum of respir- temperate or desertversus non-desert (Scholander et al. atory andcutaneous water losses,constitutes a significant 1950; Bartholomew &Cade1963; Dawson& Schmidt- proportion ofan animal’ s total water loss,up to70– 80% Nielsen1964; Dawson& Bennett1973; Weathers1979; in small birds whenmeasured at 25 °C(Lee& Schmidt- Dawson1984; Williams 1996; Tieleman &Williams Nielsen1971; Bartholomew 1972; Dawson1982; Willi- 2000). However,the use of adichotomouscategorization ams 1996). Both BMRandTEWL appear, in part, tobe ofenvironments might obscureuseful biological infor- genetically determined,suggesting that natural selection mation. Climatologists have long recognizedthat environ- mentsform acontinuumwith respectto meteorological parameters, andhave emphasizedthat, for example, the * Authorfor correspondence ([email protected]). environmentof a given desertdepends on the interaction

Proc.R. Soc.Lond. B (2003) 270, 207–214 207 Ó 2002 TheRoyal Society DOI10.1098/ rspb.2002.2205 208B. I.Tielemanand others Metabolismand evaporative water loss of larks ofa numberof variables, including temperature,amount National WildlifeResearch Center (NWRC), near Taif, Saudi andtiming ofrainfall, relative humidity andwind Arabia.Calandra larks ( Melanocoryphacalandra )fromIran were (Thornthwaite 1948; Meigs1953). To thebest of our transported to SaudiArabia and kept at the NWRC.Wecaught knowledge,our study is thefirst torelate variation in avian skylarks (Alauda arvensis )and woodlarks ( Lullulaarborea ) in the physiological variables toa continuouslyvarying measure provinceof Drenthe, The Netherlands,and kept them inout- ofenvironment, aridity. Aridity isdirectly related topri- dooraviaries at the ZoologicalLaboratory of the Universityof mary productivity (Emberger 1955) andprovides aproxy Groningen.We measuredBMR and TEWL ofallbirds in Saudi for theselection pressures that animals experiencewith Arabia and The Netherlands betweenJune and August of 1998 – increasing aridity, including decreasingwater andfood 2001.Birds were measured in the post-absorptive state during availability andincreasing air temperatures. theirnocturnal phase by standard flow-through respirometry The challenge in understandinginterspecific corre- and hygrometry methods (Williams& Tieleman2000; Tieleman lations betweenphenotype and environment is todis- et al. 2002).In addition, we useddata for spike-heeledlarks tinguish thecontributions of the various processesthat (Chersomanes albofasciata )fromKimberley, South Africa(C. underliethe pattern, including geneticadaptation, phylo- Brown, unpublisheddata), for short-toed larks( Calandrella geneticinertia (Gould& Lewontin1979; Westoby et al. brachydactyla )and lessershort-toed larks( Calandrellarufescens ) 1995; Hansen1997) andphenotypic responses(acc- fromthe North Caspian region,Russian Federation (Shishkin limatization). Our limited understandingof the time-scale 1980),for Stark ’s lark (Eremalaudastarki )and grey-backed at whichnatural selectionand other evolutionary pro- finchlarks( Eremopterixverticalis )fromthe Namib Desert cessesoperate has resultedin twomain approaches to (Willoughby1968) and for hornedlarks ( Eremophilaalpestris ) questionsin evolutionary biology, thephylogenetic com- fromNorth America(Trost 1972).For all these species,birds parative methodand the optimality approach. Compara- weremeasured during spring orsummer, in the post-absorptive tive methodsthat take intoaccount potential effectsof state and duringtheir rest phase. phylogeny emphasize thehistorical componentof adaptation (Gould& Vrba 1982) andimplicitly rule outthe (b) Environmental aridity possibility that atrait is maintained asan adaptation by Wecalculated an aridityindex as Q = P/((Tm a x stabilizing selection(Frumhoff &Reeve1994; Westoby et 1 Tm in )(Tm a x 2 Tm in )) ´ 1000, where P isthe average annual al. 1995; Hansen1997). By contrast,optimality studies precipitation(mm), Tm a x isthe meanmaximum temperature of assumethat traits with ample geneticvariation, suchas the hottest month ( °C) and Tm in isthe meanminimum tempera- mostquantitative characters (Houle1992; Lynch1988), tureof the coldestmonth ( °C)(Emberger1955). Although not are probably maintained by stabilizing selection(Hansen perhaps intuitivelystraightforward, this indexwas empirically 1997). Acomparative approach with anoptimality view- derivedto describeprimary productivity inarid and semi-arid point ofadaptation wouldassume that eachspecies is at areas (Emberger1955). The indexis low inhot dry desertsand anoptimum (Hansen1997). Oneway toreduce problems high incoolwet areas.We collected climatic data fromlocal or relating todifferences in phylogenetic history is tocom- nationalmeteorological institutes, from the literature(Walter & pare traits among closely related species(Coddington Lieth 1967;Williams 2001) and fromhttp:/ /www.worldclimate. 1988; Bennett1988; Price 1991; Leroi 1994). com/and http://www.onlineweather.com/(table 1). Because Q The family oflarks (Alaudidae) occursover awide increasesrapidly when environmentsbecome more mesic, we range ofenvironments and continents, with representa- avoidedunequal weighing of data for mesicspecies by using log tives in habitats ranging from hyperarid desertsto mesic Q inour analyses (table1). grasslands(Cramp 1988; Pa¨tzold 1994). Becauseall larks eat similar foods,a mixture ofinsects and seeds, diet is (c) Phylogeny oflarks nota confoundingvariable in ouranalyses. This family The geographical originsof the DNA samplesof alllarks in provides amodel toinvestigate physiological adaptation ourphylogeny areavailable on request from the authors. DNA toenvironment. was extracted fromblood or tissue samples using standard pro- This studytests the proposal that lower BMRsand tocols(50 mM of Tris,pH 7.6;100 mM of NaCl;1 mMof TEWLsin birds are correlated with increasing aridity, and ethylenediaminetetra-acetic acid (EDTA), pH 8.0;0.5% investigates therole ofphylogenetic inertia in shaping the sodiumdodecyl sulphate (SDS),1 mgml 2 1 of proteinaseK relationship betweenphysiology andenvironment. We and 0.1mg of RnaseA). Impuritieswere removed by phenol – constructeda phylogeny oflarks basedon twomitochon- chloroformextraction, and total genomicDNA was ethanol- drial genes,and investigated whetherphylogeny could precipitatedand elutedin sterile distilled water. explain thevariation in BMRandTEWL. We used con- Polymerasechain reaction (PCR) amplification(Saiki et al. ventional and,when appropriate, phylogenetically cor- 1988)followed standard protocols(Kocher et al. 1989). The rectedanalyses to examine therelationships between cytochrome b genewas amplifiedusing two primersets: L14990 aridity, BMRandTEWL. (shortenedprimer L14841 of Kocher et al. (1989))and H15696 (primerH15547 of Edwards et al. (1991));and L15245 (modifiedprimer CB4a-L of Palumbi et al. (1991))and H16064 2. MATERIALAND METHODS (locatedin the tRNA th r ).Aportion ofthe 16SrRNA genewas (a) Animals amplifiedusing primersL2313 and H4015(Lee et al. 1997). Wemist-netted hoopoe larks( Alaemon alaudipes) , Dunn’s Successfulamplicons were purified using aHigh PurePCR larks (Eremalaudadunni ),desertlarks ( Ammomanes deserti ), Purificationkit (RocheDiagnostics). black-crownedfinchlarks ( Eremopterixnigriceps )and crestedlarks WesequencedDNA using the fourPCR primersfor the cyto- (Galeridacristata )at various sitesin the west-centralArabian chrome b gene,and H4015and an internalprimer, L2925, Desert (table1), and housed them inoutdoor aviariesat the designedfor passerinesin our laboratory (5 9 AGCCATCAA-

Proc.R. Soc.Lond. B (2003) Metabolismand evaporative water loss of larks B.I.Tielemanand others 209

Table1. Geographical origin and environmental aridity for 14 speciesof lark. Theenvironmental aridity index ( Q)wascalculated following Emberger (1955), basedon precipitation ( P),maximum temperature ( Tm a x )and minimum temperature ( Tm in ).

species latitude longitude log Q P (mm) Tm a x (°C) Tm in (°C)

hoopoe larka 22°159 N 41°509 E 1.78 89.6 40.2 10.7 Dunn’s larka 22°159 N 41°509 E 1.78 89.6 40.2 10.7 desert larka 22°559 N 41°099 E 1.78 89.6 40.2 10.7 Stark’s larkb 23°349 S 15°039 E 1.76 57.2 33.0 10.0 grey-backed finchlark b 23°349 S 15°039 E 1.76 57.2 33.0 10.0 crested lark a 21°159 N 41°429 E 2.24 209.1 35.7 8.1 black-crowned finchlark a 21°159 N 41°429 E 2.24 209.1 35.7 8.1 calandra lark c ,d 35°009 N 51°009 E 2.26 250.0 37.0 22.4 horned larke 33°179 N/ 116°409 W/ 2.41 309.9 34.8 21.2 34°359 N 118°069 W short-toed lark c ,f 49°259 N 46°519 E 2.59 281.0 30.2 213.7 lesser short-toed lark c ,f 49°259 N 46°519 E 2.59 281.0 30.2 213.7 spike-heeled lark c ,f 28°489 S 24°429 E 2.60 420.4 32.6 2.7 woodlarkg 52°529 N 52°529 E 3.20 750.0 21.7 20.7 skylarkg 52°529 N 52°529 E 3.20 750.0 21.7 20.7

Climate datasources: a National Wildlife Research Center, Taif, Saudi Arabia; b Williams (2001), includes fog precipitation; c Walter &Lieth(1967); d http://www.onlineweather.com; e USNational Weather Service; f http://www.worldclimate.com; and g Koninklijk Nederlands Meteorologisch Instituut.

CAAAGAGTGCG3 9)for the 16SrRNA gene.Sequences of for serialindependence is moresuitable than other phylogenetic the light and heavy strands weredetermined using dye-termin- autocorrelationmethods for smalldatasets (Cheverud et al. ator cyclesequencing (Big Dye DNAsequencingkit, Applied 1985;Gittleman & Kot 1990;Martins & Hansen 1996; Biosystems)and an ABI377or ABI3100 automated DNA Abouheif1999). If nophylogeneticeffect exists, then incorpor- sequencer(Applied Biosystems). Sequences for eachtaxon were ating phylogeny instatistical methods isunnecessary proofread inSequenceNavigator, and completesequences were (Gittleman& Kot 1990;Bjo ¨rklund1997; Abouheif 1999). If a aligned in Clustal X (Thompson et al. 1997). phylogeneticeffect does exist, it may beattributable to phylo- Weanalysed the alignedsequences with P aup 4.0using mul- geneticconstraint orto ecologicalfactors, and correctionsfor tipleheuristic searches with defaultsettings (Swofford 1998). phylogeneticrelationships may ormay not beappropriate Weassessedthe resolutionof internalnodes using 1000boots- (Westoby et al. 1995). trap replicateswith randomreplacement (Felsenstein 1985 a). Phylogeneticsignal was determinedby evaluating the tree-length (e) Statistics distributionof 1000randomly generated trees (Hillis & Huel- Weperformed analyses of varianceand regressionanalyses senbeck1992). In addition to using unweighted parsimony with SPSS 10.0,and calculatedphylogenetic independent con- analysis,we attempted to reducehomoplasy by downweighting trasts (Felsenstein1985 b)with the PDT ree modulein the com- charactersbased ontheir consistency indices (Farris 1969). Pair- puter program PDAP(Garland et al. 1992).We calculated the wiseestimates of nucleotidesequence divergence were calcu- degreesof freedomas N 2 P u , where N isthe numberof inde- latedusing the HKY85model (Hasegawa et al. 1985). Gene pendentcontrasts and Pu isthe numberof unresolvedpolytom- sequencesare deposited in GenBank (accession numbers ies(Purvis & Garland1993). AY165123–AY165147(16S rRNA) and AY165148 –AY165172 (cyt b)). 3. RESULTS (d) Phylogenetic effect (a) Physiologyand environment Correlationsbetween species may bestatistically biased if sis- For 12 speciesof lark, BMRwasrelated tobody mass tertaxa tendto besimilar to oneanother as aresultof common aslog BMR(kJ day 2 1 ) = 0.225 1 0.901 log mass(g) 2 ancestry (Felsenstein1985 b; Cheverud et al. 1985;Harvey & (r = 0.53, d.f. = 11, s.e.s lo p e = 0.269, p = 0.007; table 2). Pagel1991). To evaluatewhether aphylogeneticeffect ( sensu Amultiple regressionanalysis with log BMRasthe depen- Grafen(1989) and Harvey &Pagel(1991)) exists among the dentvariable andlog massand log Q (aridity) asinde- larksin this study, we usedtests for serialindependence to deter- pendentvariables showedthat both body mass( t = 6.17, minewhether therewas asignificantpositive autocorrelation p , 0.0001) andaridity ( t = 6.08, p , 0.0001) had asig- for mass-correctedBMR, mass-correctedTEWL oraridity nificant effecton BMR: log BMR = 20.194 1 0.845 (Abouheif1999; Reeve & Abouheif1999). In each simulation log mass 1 0.208 aridity ( r2 = 0.91, d.f. = 11, p , 0.0001). the topology was randomlyrotated 2000times per iteration and The BMRoflarks increasedas the environment became the originaldata wereshuffled 2000 times to providethe null more mesic(figure 1 a). hypothesis sampling distribution(Reeve & Abouheif1999). We TEWLamong larks wasrelated tobody mass as calculatedmass-corrected BMR and mass-correctedTEWL by log TEWL(g day 2 1 ) = 20.814 1 0.816 log mass(g) ( r2 x dividingBMR and TEWL, respectively,by mass , where x is = 0.72, d.f. = 11, s.e.s lo p e = 0.162, p = 0.001; table 2). We the slopeof the allometricequations relatinglog BMRand log usedmultiple regressionanalysis toassess the effect of TEWL to log body mass in12 lark species (see § 3). The test aridity onTEWL: log TEWL = 20.903 1 0.684 log

Proc.R. Soc.Lond. B (2003) 210B. I.Tielemanand others Metabolismand evaporative water loss of larks

Table2. Basalmetabolic rate (BMR) and total evaporative water loss (TEWL)for 14 speciesof larks.

massBMR s.d. BMR massTEWL s.d. TEWL species (g) (kJ day2 1 ) (kJ day2 1 ) n (g) (g day2 1 ) (g day2 1 ) n

grey-backed finchlark a — — — — 15.1 1.31e — — Stark’s larka — — — — 15.6 1.31e — — desert lark 21.5 20.1 2.46 6 21.5 1.60 0.26 6 Dunn’s lark 20.9 24.7 2.61 22 20.5 1.69 0.49 16 hoopoe lark 36.9 32.8 4.45 21 36.9 2.59 0.62 21 black-crowned finchlark 15.2 16.5 1.10 6 15.2 1.34 0.36 6 crested lark 31.2 32.2 2.30 6 31.2 2.44 0.24 6 calandra lark 50.6 49.5 1.07 2 50.6 3.03 0.57 2 horned larkb 26.0 28.6 — — 26.0 2.08 — — lesser short-toed lark c 23.6 31.6 3.08 27 — — — — short-toed lark c 24.0 35.6 6.73 8 — — — — spike-heeled lark d 25.7 29.1 4.98 20 25.0 3.33 0.53 10 skylark 32.0 62.4 8.43 29 31.7 3.47 0.88 15 woodlark 25.6 49.4 9.96 20 25.5 2.41 0.70 14

Datasources: a Willoughby (1968); b Trost (1972); c Shishkin(1980); d C.Brown (unpublished data). e Day-timevalues.

a) asindependent variables. The modelswith thebest fit to 3.5 thedata (see § 3a) includedlog massand aridity, butnot )

5 latitude (BMR: t = 1.85, p = 0.10; TEWL: t = 1.17, 4 8 . 0 3.0 p = 0.28). Hence,aridity explained more ofthe variation – s s in BMRandTEWL than didlatitude after bodymass was a

m 2.5 takeninto account. 1 – d

k 2.0 (b) Phylogeny oflarks

R Cytochrome b (975 bases)and 16S rRNA (566 bp)

M 1.5 sequenceswere generated for 22 species.In addition to B thespecies for which ecophysiological data wereavailable, 1.0 weincluded other African speciesin an attempt to b) improve theresolution of the phylogenetic placementof 0.40 ourfocal taxa. Aheuristic searchof thecombined dataset

) yieldedtwo equally most-parsimonioustrees (length 1424 4 8 6 . 0.35 = = = 0 steps, CI 0.407, RI 0.492, g1 21.018, p , 0.001). – s

s Oneround of reweighting (usinga baseweight of1) a 0.30

m yieldeda single treeof 579.27 steps(figure 2;

1 – = =

d CI 0.490, RI 0.559). Out ofthe 378 parsimony-

g 0.25 informative characters,41 had aweight of1 and337 had

L aweight ofless than 1, reflecting homoplasy in thedataset. W 0.20 E Weperformed bootstrap analysis with 1000 iterations T usingthe hoopoe lark asan outgroup (figure 2). Several 0.15 highly supportedclades (more than 70% bootstrap 1.5 2.0 2.5 3.0 3.5 support)were consistently retrieved (also in separate aridity index log Q) analysesof the two genes, trees not shown): basal placementof spike-heeled lark clade;finchlarks/ desertlarks Figure 1. Mass-adjusted( a) BMR and (b) TEWL of 12 clade;singing bushlark/ monotonouslark clade;lesser speciesof lark asa function of environmental aridity. short-toed/Athi short-toedlark clade;and skylark/ Symbolsare speciesaverages. woodlark clade.Several currently recognizedgenera appear tobe polyphyletic (e.g. Ammomanes, Certhilauda , Mirafra).Alack ofresolution of some of the terminal mass 1 0.121 aridity ( r2 = 0.87, d.f. = 11, p , 0.0001). nodesmay becaused by therelatively shortinternal Massand aridity both had asignificant effecton TEWL branch lengths compared with thelong terminal branch (log mass: t = 5.50, p , 0.0001; aridity: t = 3.12, p = 0.011), lengths.The divergenceamong theselark cladesis also afinding consistentwith thehypothesis that larks have a indicatedby geneticdistances ranging from 7to19% lower TEWLwith increasing aridity (figure 1 b). basedon the cytochrome b geneand between 2.5 and8% Becausearidity wascorrelated with latitude (table 1; basedon the 16S rRNA fragment. r = 0.84, n = 9, p = 0.005), weperformed stepwise mul- Wedid not have DNA sequenceinformation for the tiple regressionanalyses with log BMRor log TEWLas calandra lark, hornedlark andshort-toed lark. To apply thedependent variable andlog mass,latitude andaridity thetest for serial independenceto all speciesfor which we

Proc.R. Soc.Lond. B (2003) Metabolismand evaporative water loss of larks B.I.Tielemanand others 211

12.588 23.294 100) black-crowned finchlark 15.263 16.037 100) grey-backed finchlark 24.365 10.532 99) bar-tailed desert lark 22.878 desert lark 15.120 6.175 92) crested lark 15.981 large-billed lark 14.790 10.515 88) skylark 2.137 52) 21.340 woodlark , 26.150 Temminck s horned lark 6.564 95) 6.735 15.869 , 3.594 63) 86) Stark s lark 19.361 , Sclater s lark 7.236 5.661 88) 14.307 90) lesser short-toed lark 19.017 Athi short-toed lark 2.000 8.207 88) 22.360 , Dunn s lark 100) 14.032 10.234 99) singing bushlark 21.383 monotonous lark 18.664 13.840 98) dune lark 21.547 sabota lark 23.588 6.430 96) spike-heeled lark , 10.955 100) 27.317 Gray s lark 24.838 long-billed lark 32.882 hoopoe lark

Figure 2. Phylogenetic tree of 22 speciesof lark basedon cytochrome b and 16SrRNA sequences, and analysed using maximum parsimony criteria. Numbers abovethe branches indicate reweighted branch lengths and, in parentheses, per cent bootstrap recovery in 1000 replications. had ecophysiological data,we placed the calandra lark in ity: t = 4.83, p = 0.001). Hence,the result of the phylogen- thepolytomy with dunelark/ sabota lark, the eticanalysis wasconsistent with that ofthe conventional finchlark/desertlark cladeand a third cladecontaining analysis: theBMR oflarks decreaseswith increasing arid- Galerida, Lullula, Calandrella andothers. We placed the ity. hornedlark assister species to the Temminck ’s horned lark. Weassumed that theshort-toed lark wasclosely 4. DISCUSSION related tothe Athi short-toedlark. The BMR (n = 12) andTEWL ( n = 12) of lark (c) Phylogenetic constraint onBMR andTEWL? decreasedalong agradient ofincreasing aridity, consistent Wefoundno significant phylogenetic autocorrelation in with ourproposal. Theseresults support the conclusions thedata for mass-correctedTEWL or aridity, butdid find ofprevious studiesthat foundreduced BMR andTEWL asignificant autocorrelation in thedata for mass-corrected in desertbirds compared with speciesfrom mesichabitats BMR(mass-correctedTEWL: p = 0.44; aridity: p = 0.09; (Dawson& Bennett1973; Weathers1979; Arad & mass-correctedBMR: p = 0.01). Therefore,the positive Marder 1982; Williams 1996; Tieleman &Williams associationbetween TEWL and aridity wasnot influenced 2000), butthe use of a continuousenvironmental classi- by phylogenetic constraint.The significant phylogenetic fication in this studymakes the argument more compel- autocorrelation in themass-corrected BMR data couldbe ling. Although bodymass alone explained 53% ofthe attributable tophylogenetic constraintor toecological fac- interspecificvariation in BMR,adding aridity increased tors,indistinguishable alternatives (Westoby et al. 1995). theexplained variance to91%. Similarly, bodymass Therefore,in addition toour conventional analysis, we explained 72% ofthe variance in TEWL,while adding testedwhether the relationship betweenmass-corrected aridity increasedthe explained variance to87%. Acombi- BMRandaridity couldbe confirmed using phylogenetic nationof low BMR andlow TEWL could be favourable independentcontrasts (Felsenstein 1985 b).Astepwise in birds from dry hot environmentsbecause it reduces multiple regressionanalysis with contrastsof log BMRas foodand water requirementsand minimizes heat pro- thedependent variable, andcontrasts of log massand of duction. aridity asindependent variables, showedsignificant effects The value ofcomparisons within agroup ofclosely oflog massand aridity onlog BMR( r2 = 0.88, d.f. = 9, related speciesis illustrated by thedeviations ofthe F = 32.36, p , 0.001; log mass: t = 6.07, p , 0.001; arid- allometries for BMRandTEWL of larks from thoseof all

Proc.R. Soc.Lond. B (2003) 212B. I.Tielemanand others Metabolismand evaporative water loss of larks birds.The equationrelating log body massto log BMR Theauthors thankP. Paillat, A.Khoja, P.Seddon, M.Shob- in larks deviatedfrom anallometry for all birds by 29% rak, S.Ostrowski, J.-Y. Cardona and theother staffat the for a15 glark andby 125% for a50 glark (Tieleman & National Wildlife Research Centre (NWRC), Taif,Saudi Ara- bia, for logistic support throughout thisstudy. Wildlife research Williams 2000). The BMRofdesert larks wasclose to programmes atthe NWRC are possible through thegenerous allometric predictions,in contrastto the BMR ofmesic support of HRHPrince Saud al Faisal and under theguidance larks, which far exceededpredictions. Predictions of of A.Abuzinada of theNational Commission of Wildlife Con- TEWLbased on thelark allometry werelower than those servation and Development. We are grateful toC.Brown and basedon anallometry including all birds (Williams 1996) K.Barnes for providing unpublished dataon thespike-heeled by 226% for a15 glark andby 212% for a50 glark. lark and Athi short-toed lark, respectively, and to A.Kuz- Although theTEWL of alark from thedesert was below menko for translating aRussian article. We are grateful to W. Delport for assistancewith DNA sequencing. G.Overkamp allometric predictions,as expected, larks in general had a and theanimal caretakers atthe Zoological Laboratory pro- lowTEWL. Hence, comparing theBMR or TEWLof a vided valuable help and advice. We thankS. Daan, R.Ricklefs single lark specieswith theall-bird allometry wouldhave and two anonymous referees for commenting on aprevious ledto erroneous conclusions about theadaptive signifi- draft. Financial support for thisstudy wasmade availableby canceof these traits. theSchuurman Schimmelvan Outeren Foundation, the The phylogeny oflarks wascharacterized by long ter- Schure Beijerinck Popping Foundation, theNational Wildlife Research Centre, theUniversity of Groningen, theOhio State minal branchesand short internal branch lengths (figure University and theNational Science Foundation (IBN- 2). This, combinedwith theoccurrence of larks in awide 0212092). array ofhabitats, may indicatethat lark speciesrapidly radiated into differentenvironments and lived for asignifi- REFERENCES cantpart oftheir evolutionary history in diversehabitats. Onemight predict that phylogenetic constraintsare small, Abouheif, E.1999 Amethod for testing theassumption of natural selectionhas had ample time toeliminate subopti- phylogenetic independence in comparative data. Evol. Ecol. mal phenotypesand the current trait values are ofadaptive Res. 1, 895–909. significance in thecurrent environment. The lack of Arad, Z.&Marder, J.1982 Comparative thermoregulation of phylogenetic effectin themass-corrected TEWL data four breeds of fowls ( Gallus domesticus )exposed to gradual increase of ambient temperatures. Comp.Biochem. Physiol. implies that phylogenetic relatednessis nota major evol- A 72, 179–184. utionary factor explaining interspecificdifferences in Aschoff, J.&Pohl, H.1970 Rhythmicvariations in energy TEWL.The positive phylogenetic autocorrelation in the metabolism. Fed. Proc. 29, 1541–1552. mass-correctedBMR data couldindicate either aphylo- Bartholomew, G.A.1972 Thewater economy of seed-eating geneticconstraint or closely related speciesexperiencing birds thatsurvive without drinking. In Proc.15th Int. Orni- commonecological factors(Westoby et al. 1995). The thol. Congr.,The Hague (ed. K.H.Voous), pp. 237 –254. correlation betweenBMR andaridity isnot the result of Leiden: E.J.Brill. phylogenetic constraint,because the results of the contrast Bartholomew, G.A.&Cade, T.J.1963 Thewater economy analysis are consistentwith thoseof theconventional stat- of land birds. Auk 80, 504–539. istics.Therefore, evolutionary forcesother than phylogen- Bennett, A.F.1988 Structural and functional determinates of metabolic rate. Am. Zool. 28, 699–708. eticconstraint are likely tounderlie the correlations Bjo¨rklund, M.1997 Are ‘comparative methods ’ alwaysneces- betweenBMR, TEWL and environmental aridity. sary? Oikos 80, 607–612. Interspecificphenotype –environmentcorrelations can Cheverud, J.M., Dow, M.M.&Leutenegger, W.1985 The indicateeither geneticdifferences brought about by natu- quantitative assessmentof phylogenetic constraints in com- ral selectionor phenotypically plastic responsesto parative analyses: sexual dimorphism in body weight among environmental conditions.Plastic responsesinclude primates. Evolution 39, 1335–1351. changesin adult phenotypedepending on the environ- Coddington, J.A.1988 Cladistic testsof adaptational hypoth- ment(acclimation) anddifferences between phenotypes eses. Cladistics 4, 3–22. resulting from developmental conditions(ontogenetic Cramp, S.1988 Handbook ofthe birds ofEurope, the Middle East plasticity). In aseparate study,we foundthat thedecrease and North Africa .Oxford University Press. in BMRandTEWL in larks along anaridity gradient can- Crawford, E.C.&Lasiewski,R. C.1968 Oxygen consumption and respiratory evaporation of theemu and rhea. Condor 70, notbe attributed toacclimation tothermal environment, 333–339. foodavailability orphotoperiod (Tieleman et al. 2003). Daan, S.,Masman,D. &Groenewold, A.1990 Avian basal In summary, decreasinglevels ofBMR andTEWL in metabolic rates: their association withbody composition and larks correlate with increasing aridity. Thesephysiological energy expenditure in nature. Am.J. Physiol. 259, R333– traits may have adaptive significance in thecurrent R340. environment,and natural selectionis alikely processto Dawson, W.R.1982 Evaporativelosses of water bybirds. explain ourfindings. Identifying evolutionary processes Comp.Biochem. Physiol. A 71, 495–509. that causecorrelative associationsbetween traits remains Dawson, W.R.1984 Physiological studies of desert birds: difficult (Leroi 1994), butwe have eliminated phylogen- present and future considerations. J.AridEnviron. 7, 133– eticconstraint and acclimation aslikely alternative 155. Dawson, W.R.&Bennett, A.F.1973 Roles of metabolic level explanatory processes.The resultsof this studytogether and temperature regulation in theadjustment of western with thedecrease in BMRin mice along anenvironmental plumed pigeons ( Lophophaps ferruginea )todesert conditions. gradient ofdecreasing primary productivity (Mueller& Comp.Biochem. Physiol. A 44, 249–266. Diamond 2001) suggestthat thedecrease in energy and Dawson, W.R.&Schmidt-Nielsen, K.1964 Terrestrial ani- water requirementswith decreasingfood and water avail- malsin dry heat: desert birds. In Handbook ofphysiology: ability is ageneral pattern foundin birds andmammals. adaptation to the environment (ed. C.G.Wilber, E.F.

Proc.R. Soc.Lond. B (2003) Metabolismand evaporative water loss of larks B.I.Tielemanand others 213

Adolph &D.B.Dill), pp. 481 –492. Washington, DC: Lee, P.&Schmidt-Nielsen, K.1971 Respiratory and American Physiological Society. cutaneous evaporation in thezebra finch: effecton water bal- Edwards, S.V., Arctander, P.&Wilson, A.C.1991 Mito- ance. Am.J. Physiol. 220, 1598–1605. chondrial DNAresolution of adeep branch in thegenealogi- Leroi, A.M.1994 Whatdoes thecomparative method reveal cal tree of perching birds. Proc.R. Soc. Lond. B 243, 99–107. about adaptation? Am. Nat. 143, 381–402. Emberger, L.1955 Afrique du Nord-Ouest. In Plant ecology: Lynch, M.1988 Therate of polygenic mutation. Genet. Res. reviews ofresearch (ed. UNESCO),pp. 219 –249. Paris, 51, 137–148. France: UNESCO. McNab,B. K.1986 Theinfluence of food habitson theener- Farris, J.S.1969 Asuccessive approximation approach to getics of eutherian mammals. Ecol. Monogr. 56, 1–19. character weighting. Syst. Zool. 18, 374–385. McNab,B. K.1988 Food habitsand thebasal rate of metab- Felsenstein, J.1985 a Confidence limits on phylogenies: an olism in birds. Oecologia 77, 343–349. approach using thebootstrap. Evolution 39, 783–791. Martins, E.P.&Hansen, T.F.1996 Thestatistical analysis Felsenstein, J.1985 b Phylogenies and thecomparative method. of interspecific data: areview and evaluation of phylogenetic Am. Nat. 125, 1–15. comparative methods. In Phylogenies and the comparative Frumhoff, P.C.&Reeve, H.K.1994 Using phylogenies to method in animal behavior (ed. E.P.Martins), pp. 22 –75. testhypotheses of adaptation: acritique of some current pro- NewYork: Oxford University Press. posals. Evolution 48, 172–180. Meigs, P.1953 Reviews ofresearch onarid zone hydrology . Paris, Furuyama, F.&Ohara, K.1993 Genetic development of an France: UNESCO. inbred rat strain withincreased resistance adaptation to a Mueller, P.&Diamond, J.2001 Metabolic rate and environ- hot environment. Am.J. Physiol. 265, R957–R962. mental productivity: well-provisioned animals evolved torun Garland Jr, T., Harvey, P.H.&Ives, A.R.1992 Procedures and idle fast. Proc.Natl Acad.Sci. USA 98, 12 550–12 554. for theanalysis of comparative datausing phylogenetically Nagy,K. A.1987 Field metabolic rate and food requirement independent contrasts. Syst. Biol. 41, 18–32. scaling in mammalsand birds. Ecol. Monogr. 57, 111–128. Gittleman, J.L.&Kot, M.1990 Adaptation: statisticsand a Nagy,K. A., Girard, I.A.&Brown, T.K.1999 Energetics of null model for estimating phylogenetic effects. Syst. Zool. 39, free-ranging mammals,reptiles and birds. A.Rev. Nutr. 19, 227–241. 247–277. Gould, S.J.&Lewontin, R.C.1979 TheSpandrels of San Palumbi, S.,Martin, A., Romano, S.,McMillan, W.O., Stice, Marco and thePanglossian paradigm. Acritique of theadap- L.&Grabowski, G.1991 The simple fool’s guide to PCR,ver- tationist program. Proc.R. Soc. Lond. B 205, 581–598. sion 2.Honolulu: Department of Zoology and Kewalo Mar- Gould, S.J.&Vrba, E.S.1982 Exaptation —amissing term ine Laboratory, University of Hawaii. in thescience of form. Paleobiology 8, 4–15. Pa¨tzold, R.1994 Die Lerchender Welt .Magdeburg, Germany Grafen, A.1989 Thephylogenetic regression. Phil. Trans.R. Die Neue Brehm-Bu ¨cherei, WestarpWissenschaften. Soc. Lond. B 326, 199–257. Price, T.1991 Morphology and ecology of breeding warblers Hansen, T.F.1997 Stabilizing selection and thecomparative along an altitudinal gradient in Kashmir, India. J. Anim. analysisof adaptation. Evolution 51, 1341–1351. Ecol. 60, 643–664. Harvey, P.H.&Pagel, M.D.1991 The comparative method in Purvis, A.&Garland Jr, T.1993 Polytomies in comparative evolutionary biology .Oxford University Press. analysesof continuous characters. Syst. Biol. 42, 569–575. Harvey, P.H., Pagel, M.D.&Rees, J.A.1991 Mammalian Reeve, J.&Abouheif, E.1999 Phylogenetic independence. metabolism and life histories. Am. Nat. 137, 556–566. Version 1.1. [1.1]. Department of Ecology and Evolution, Hasegawa,M., Kishino, H.&Yano, T.1985 Dating of the SUNYatStony Brook. human-apesplitting bythemolecular clock of mitochondrial Ricklefs, R.E., Konarzewski, M.&Daan, S.1996 The DNA. J.Mol.Evol. 22, 160–174. relationship between basalmetabolic rate and daily energy Hillis, D.M.&Huelsenbeck, J.P.1992 Signal, noise, and expenditure in birds and mammals. Am. Nat. 147, 1047– reliability in molecular phylogenetic analysis. J. Heredity 83, 1071. 189–195. Saiki, R.K., Gelfand, D.H., Stoffel, S.J., Scharf, S.J., Higu- Houle, D.1992 Comparing evolvability and variability of chi, R., Horn, G.T., Mullis, K.B.&Erlich, H.A.1988 quantitative traits. Genetics 130, 195–204. Enzyme directed enzymatic amplification of DNAwitha Hulbert, A.J.&Else, P.L.2000 Mechanismsunderlying the thermostable polymerase. Science 239, 487–491. cost of living in animals. A.Rev. Physiol. 62, 207–235. Scholander, P.F., Hock, R., Walters, V.&Irving, L.1950 King, J.R.1974 Seasonal allocation of time and energy Adaptation to cold in arctic and tropical mammalsand birds resources in birds. In Avian energetics (ed. R.A.Paynter Jr), in relation tobody temperature, insulation and basalmeta- pp. 4–85. Cambridge, MA:Nuttall Ornithological Club. bolic rate. Biol. Bull. 99, 259–271. Kleiber, M.1961 The fireof life: an introduction to animal ener- Shishkin, V.S.1980 Theenergetic estimation of trophic getics.NewYork: Wiley. relations of thelarks (Alaudidae) in theCaspian semi-desert. Kocher, T.D.,Thomas,W. K., Meyer, A., Edwards, S.V., Zool. Zh. 59, 1204–1216. Paabo, S.,Villablanca, F.X.&Wilson, A.C.1989 Dynam- Swofford, D.L.1998 Phylogenetic analysis usingparsimony, ver- ics of mitochondrial DNAevolution in animals: amplifi- sion 4.Sunderland, MA:Sinauer. cation and sequencing withconserved primers. Proc. Natl Thompson, J.D.,Gibson, T.J., Plewniak, T.F., Jeanmougin, Acad.Sci. USA 86, 6196–6200. F.&Higgins, D.G.1997 TheC lustal Xwindows inter- Konarzewski, M.&Diamond, J.1995 Evolution of basalmeta- face: flexible strategies for multiple sequence alignment bolic rate and organ massesin laboratory mice. Evolution 49, aided byquality analysistools. Nucleic Acids Res. 24, 1239–1248. 4876–4882. Lee, K., Feinstein, J.&Cracraft, J.1997 Thephylogeny of Thornthwaite, C.W.1948 An approach toward arational ratite birds: resolving conflicts between molecular and mor- classification of climate. Geogr. Rev. 38, 55–94. phological datasets. In Avian molecular evolution and system- Tieleman, B.I.&Williams, J.B.2000 Theadjustment of avian atics (ed. D.P.Mindell), pp. 173 –211. SanDiego, CA: metabolic rates and water fluxes to desert environments. Academic Press. Physiol. Biochem. Zool. 73, 461–479.

Proc.R. Soc.Lond. B (2003) 214B. I.Tielemanand others Metabolismand evaporative water loss of larks

Tieleman, B.I., Williams, J.B.&Buschur, M.E.2002 Physio- Williams, J.B.2001 Energy expenditure and water flux of free- logical adjustments to arid and mesic environments in larks living dune larksin theNamib desert: atestof there-allo- (Alaudidae). Physiol. Biochem. Zool. 75, 305–313. cation hypothesis. Funct. Ecol. 15, 175–185. Tieleman, B.I., Williams, J.B., Buschur, M.E.&Brown, Williams, J.B.&Tieleman, B.I.2000 Flexibility in basal C.R.2003 Phenotypic variation of larksalong an aridity metabolic rate and evaporative water loss among hoopoe gradient: are desert birds more flexible? Ecology (In the larksexposed to different environmental temperatures. J. press). Exp. Biol. 203, 3153–3159. Trevelyan, R., Harvey, P.H.&Pagel, M.D.1990 Metabolic Williams, J.B.&Tieleman, B.I.2001 Physiological ecology rates and life histories in birds. Funct. Ecol. 4, 135–142. and behavior of desert birds. In Currentornithology , vol. 16 Trost, C.H.1972 Adaptations of horned larks( Eremophila (ed. V.Nolan &C.F.Thompson), pp. 299 –353. NewYork: alpestris)tohot environments. Auk 89, 506–527. Plenum Press. Walter, H.&Lieth, H.1967 Klimadiagramm—Weltatlas . Jena: Willoughby, E.J.1968 Water economy of theStark ’s lark and VEBGustavFischer. grey-backed finch-lark from theNamib desert of South West Weathers, W.W.1979 Climatic adaptation in avianstandard Africa. Comp.Biochem. Physiol. 27, 723–745. metabolic rate. Oecologia 42, 81–89. Westoby, M., Leishman,M. R.&Lord, J.M.1995 On misin- terpreting the ‘phylogenetic correction ’. J. Ecol. 83, 531–534. Williams, J.B.1996 Aphylogenetic perspective of evaporative As this paper exceedsthe maximum lengthnormally permitted, the water loss in birds. Auk 113, 457–472. authors have agreedto contributeto production costs.

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