CLIMATE RESEARCHClim Res

Vol. 43: 103–114, 2010doi: 10.3354/cr00869

Published online August 5

1. INTRODUCTION

Understanding the impact of human-driven climatechange in biological systems is one of the mostimportant challenges that researchers face during the21st century, with important repercussions in themedium and long term (IPCC 2007). It is currentlyclear that species are responding in various ways toglobal warming (Peñuelas & Filella 2001, Peñuelaset al. 2002, Walther et al. 2002, Root et al. 2003,Parmesan 2006, 2007 and references therein), yetpredicting how species and communities will respondto changes in environmental conditions on a globalscale remains considerably difficult. This is partlybecause these changes vary geographically as well as

due to the inherently complex relationship organismshave with their surrounding biotic and abiotic envi-ronment.

Recent studies have shown that predictions of organ-isms’ responses based solely on climatic variablescan be dramatically different to those that take intoaccount additional knowledge of biology. For example,although the impact of global warming is more pro-nounced at high latitudes, comparative studies on theranges of thermotolerance in ectotherms suggest thattropical species may be more vulnerable to increasesin mean environmental temperatures because theyalready encounter temperatures close to their physio-logical limits (Deutsch et al. 2008, Tewksbury et al.2008, but see Hoffmann 2010) or lack the appropriate

© Inter-Research 2010 · www.int-res.com*Email: enrico.rezende@uab.cat

Climate change and chromosomal inversions inDrosophila subobscura

Enrico L. Rezende1,*, Joan Balanyà2, Francisco Rodríguez-Trelles1, Carla Rego1, Inês Fragata3, Margarida Matos3, Luis Serra2, Mauro Santos1

1Departament de Genètica i de Microbiologia, Grup de Biologia Evolutiva (GBE), Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

2Departament de Genètica, Grup de Biologia Evolutiva (GBE), Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain

3Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisbon, Portugal

ABSTRACT: In natural populations, the large changes in chromosomal structure occurring throughchromosomal inversions show pronounced variations in frequency that often correspond to temporaland spatial climatic trends, which suggests that they may be employed to monitor the impact ofglobal warming. Here we review and update the evidence on the association between chromosomalinversions and climate in D. subobscura, which provides one of the best studied models in this con-text. Chromosomal inversion frequencies of D. subobscura populations vary predictably with lati-tude, and this association has evolved independently in Europe and South and North America. Theyalso exhibit clear seasonal trends that are consistent with temperature fluctuations. More importantly,latitudinal clines in chromosomal inversion frequencies seem to be responding to the global rise inmean temperatures in all continents. We analyze the relevance of these results in the light of climatechange, and discuss how a better understanding of the mechanisms underlying these patterns maycontribute to our knowledge on the impacts of global warming in biological systems.

KEY WORDS: Chromosomal inversions · Latitudinal clines · Thermal adaptation · Global warming

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Contribution to CR Special 21 ‘Climate and evolutionary physiology’

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genetic variation in key traits (Kellermann et al. 2009).This discrepancy illustrates how consideration of bio-logical data may drastically alter the outcome of pre-dictions on the impacts of climate change, and empha-sizes the necessity of more holistic approaches tomonitoring and investigating the effects of globalwarming on biological systems.

Long-term monitoring surveys capitalize on bioindi-cators or biomonitors for which comprehensive histori-cal records are available. In this sense, phenology (i.e.the study of changes in plant and animal life-stageevents, driven by environmental change) is becominga major component of climate change research, largelybecause long-term phenological changes are relativelyeasy to identify (see e.g. Sparks et al. 2009). Eventhough current DNA technologies make it possible toobtain information from archived samples (see Nielsen& Hansen 2008), time series of genetic data remainalmost nonexistent, with a few exceptions provided bya small number of Drosophila species that have beenregularly scored for chromosomal inversions. Here wepresent a brief overview (including some unpublishedresults) of what is known on the rich natural variationfor these genetic markers in D. subobscura, and dis-cuss the potential relevance of this model to under-stand: (1) how changes in climatic conditions might beaffecting the genetic composition of species; (2) whichenvironmental factors might be more relevant for pre-dictive purposes; and (3) how species may respond tothese environmental challenges.

1.1. Chromosomal inversionpolymorphisms

Chromosomal inversions were firstdetected from an alteration in the off-spring proportions derived from somegenetic crosses (Sturtevant 1917). Theuse of cytological techniques introducedin 1935 uncovered an almost bewilder-ing amount of inversion variation (poly-morphism) in natural populations ofDrosophila pseudoobscura and relatedspecies, which allowed the constructionof phylogenetic relationships (Dobzhan-sky 1937). Extensive surveys in southernCalifornia from 1939 to 1946 showedthat chromosomal inversion frequenciesof D. pseudoobscura presented markedseasonal cycles (Dobzhansky 1947),leading to a fundamental turning pointin evolutionary genetics because thesepatterns suggested that these polymor-phisms could be adaptive.

Succinctly, an inversion is a change in chromoso-mal structure in which a fragment of the chromo-some involving several genes rotates 180°, resultingin a region where the positions of these genes areinverted relative to the original sequence (Fig. 1).Cytological methods for detecting inversions havebeen predominantly employed in Diptera (flies, mos-quitoes, etc.) because these insects present special-ized cells that undergo repeated rounds of DNAreplication without cell division, forming giant chro-mosomes (polytenic chromosomes) that could thus bestudied at a greatly enhanced resolution. These chro-mosomes have characteristic banding patterns whichcan be used to identify structural changes, and oftenform an inversion loop in individuals containing 2different gene arrangements on the same chromo-some: one with and the other without the invertedfragment (heterokaryotypes; see Fig. 1). Another im-portant consequence of inversions in an evolutionarycontext is that they suppress recombination in hetero-karyotypes (i.e. suppress the reshuffling of genes be-tween chromosomes with inverted and non-invertedregions). As a result, genetic differences accumulatein genes associated with the inversions, which mayplay a significant role in population divergence andspeciation (reviewed in Hoffmann & Rieseberg 2008).

Following Dobzhansky’s pioneering works, many re-searchers studied chromosomal inversion polymor-phisms, gathering an extensive historical record of thedistribution of different inversions in Drosophila spe-cies worldwide. It was realized very soon that inversionfrequencies show conspicuous latitudinal clines in many

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Fig. 1. Chromosomal inversions can be detected in Drosophila by analyzing thebanding patterns or the inversion loops of polytene chromosomes. The picturesshow individuals that are homokaryotypic (top) and heterokaryotypic (bottom)for gene arrangements on the A chromosome (light micrograph). A schematicdiagram showing how inversions result in chromosomal loops in heterokaryo-

types is shown on the right

Rezende et al.: Chromosomal inversions in Drosophila subobscura

species (see Krimbas & Powell 1992). Together with thecyclical changes already reported by Dobzhansky inD. pseudoobscura, the idea that climatic factors favordifferent inversions at different latitudes and/or indifferent seasons is nowadays widely accepted.

2. THE INVASIVE SPECIES DROSOPHILASUBOBSCURA: A CASE STUDY

2.1. Historical records of clinal patterns

Drosophila subobscura is a native Palearctic specieswith a wide geographic distribution ranging fromScandinavia to Northern Africa (Fig. 2). This specieshas become particularly suitable for studies of experi-mental evolution on the adaptive role of inversionsbecause of its well-studied chromosomal inversionpolymorphism, which encompasses more than 90 genearrangements present in its 5 large acrocentric chro-mosomes. Naturally occurring inversions in D. subob-scura are nowadays distinguished as e.g. A1, J1 or O5,where the letter stands for the corresponding chromo-some and the subscripted number for the inverted seg-ment on that chromosome relative to a so-called stan-dard orientation).

The geographical distribution of gene arrangements inEuropean populations of Drosophila subobscura hasbeen studied extensively (see Krimbas 1992 for a re-view), and can be briefly summarized as follows. (1) Thefrequency of different gene arrangements varies dra-

matically between different populations, and severalarrangements that are frequent in some populations maybe absent in others. (2) There is a general correspon-dence between the genetic and geographical distancebetween populations, hence the genetic constitution ofdifferent populations becomes increasingly different astheir geographic distance increases. The effects of geo-graphic barriers are also evident in this context, and ge-netic differentiation between populations separated bysea straits and high mountains is generally higher thanexpected in the absence of geographic barriers (Prevostiet al. 1975). (3) Whereas the genetic constitution of north-ern populations is relatively homogeneous and includesfew inversions, southern populations present a consider-ably larger number of inversions that form complex genearrangements, including many that are apparentlyunique to specific locations. (4) The relative frequenciesof several gene arrangements present marked geo-graphical clines running in the northeast–southwest di-rection (Fig. 3).

Taken together, geographic patterns suggest thathistorical processes, migration and local adaptationhave been involved in shaping the distribution ofgene arrangements across Europe and northwestAsia. The most widely accepted explanation for thesepatterns proposes that separate populations of Dro-sophila subobscura remained isolated in southern re-gions of Europe during the last glaciation, eventuallybecoming genetically differentiated (from a numberof European case studies it appears that the Balkans,the Iberian Peninsula, Italy and Turkey contained

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Fig. 2. Drosophila subobscura. Global distribution, and inva-sion of the Americas, depicting the estimated population size

during the colonization events

Fig. 3. Drosophila subobscura. Latitudinal cline for the genearrangement OST observed in Europe. Lines representisoclines with similar frequencies of OST (from 0 to 100%).Note that OST is absent in southern populations and fixed innorthern populations, hence it is considered a ‘cold-climate’

arrangement

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refugia and were a source for post-glacial populationexpansions of many species; Hewitt 2000, 2001). Sub-sequently, D. subobscura from different refugia recol-onized the northern regions of Europe (Krimbas1992). This scenario explains the geographic structureand increased number of chromosomal arrangementsin southern populations, and partly accounts for themore homogeneous pattern observed in northernEurope. The latitudinal clines in several chromosomalarrangements provide circumstantial evidence thatselective processes might have also been involved,and whether historical processes alone can explainthe lower diversity in northern populations remains amatter of debate. Evidence that these clines are partlyadaptive has emerged from research on the rapidinvasion of the New World by D. subobscura (Fig. 2,Section 2.3).

2.2. Local seasonal variation

Seasonal changes in genetic markers can be eithererratic or cyclical. As mentioned previously, the obser-vation of seasonal changes—repeated over the years—in the frequencies of gene arrangements representedthe first piece of evidence in support of the adaptivevalue of chromosomal inversion polymorphisms (Dob-zhansky 1970, Lewontin 1974, Endler 1986). Seasonalcycling of chromosomal inversions has been recordedin a number of Drosophila species, including D. pseudo-obscura (Dobzhansky 1947), D. persimilis (Dobzhan-sky 1956), D. funebris (Dubinin & Tiniakov 1945), D.flavopilosa (Brncic 1972), D. robusta (Levitan 1973,Etges 1984), D. melanica (Tonzetich & Ward 1973), D.melanogaster (Stalker 1980, Sánchez-Refusta et al.1990) and D. mediopunctata (Ananina etal. 2004), highlighting the generality ofthis pattern across species with contrast-ing geographic distributions.

In the case of Drosophila subobscura, itsrich inversion polymorphism was initiallysupposed to be invariant, or to vary errat-ically with respect to the annual climaticcycle. This conclusion, however, wasbased on results from few studies in a lim-ited number of populations. This situationmay have favored the concealment of sea-sonal cycling of inversions if they cycleseasonally in some populations but notin others, such as has been reportedfor D. pseudoobscura (Dobzhansky 1962,Dobzhansky & Ayala 1973), D. robusta(Carson 1958, Dobzhansky 1962) and D.persimilis (Dobzhansky 1956, Dobzhan-sky & Ayala 1973). Seasonal cycling could

also have been neglected because the sampling peri-odicity and/or schedule may have missed the inver-sions’ seasonal cycle (e.g. Krimbas 1967, who collectedsamples in spring and autumn), and in generalbecause of the short length of the early monitoringstudies, generally spanning 1 yr (e.g. De Frutos & Pre-vosti 1984), and in no case longer than 2 consecutiveyears.

The early view that the chromosomal inversion poly-morphisms of Drosophila subobscura do not cycle withthe seasons was subsequently challenged by a long-term seasonal study (spanning 2 sampling periodswithin 16 yr), using time series methods, which focusedon the temporal variation of the O chromosomal poly-morphisms at Mount Pedroso, northwest of the IberianPeninsula (Rodríguez-Trelles et al. 1996). The studydisclosed a complex picture: the most frequent genearrangements exhibited pronounced seasonal cyclessuperimposed on long-term trends (Fig. 4). Althoughthe intensity and direction of the temporal patternsdiffered from one arrangement to the next, there wasa general agreement between seasonal, long-termand latitudinal trends. So-called cold-climate or cold-adapted gene arrangements (i.e. those gene arrange-ments that show a negative correlation coefficient withmaximum temperatures, or a positive correlation coef-ficient with latitude, such as OST; Menozzi & Krimbas1992) increased in frequency in winter and decreasedin summer. In addition, these arrangements also showeda decrease in frequency in the long-term (Fig. 4) thatcoincided with a mean increase in environmental tem-peratures within the 16 yr survey period (Rodríguez-Trelles & Rodríguez 1998).

How widespread are the seasonal cycles observed atMount Pedroso? Seasonal changes of inversions con-

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0.7

0.6

0.5

0.4

0.3

0.2

0.1

1976 1977 1978 1979 1980 1988 1989 1990 1991

Freq

uen

cy O3+4+7

O3+4OST

O3+4+8

Fig. 4. Drosophila subobscura. Seasonal fluctuations of gene arrangementfrequencies in the population of Mount Pedroso, Spain. Different arrange-ments in the O chromosome show seasonal cycles and long-term trends thatcoincide with temperature fluctuations and global warming: cold-climatearrangements such as OST decrease in frequency during summer and in thelong-term, whereas the opposite is true for warm-climate arrangementssuch as O3+ 4 (redrawn from Rodríguez-Trelles et al. 1996, Rodríguez-Trelles

& Rodríguez 1998)

Rezende et al.: Chromosomal inversions in Drosophila subobscura

sistent with those found in this population were alsoobserved at other locations (e.g. Burla & Götz 1965,Zivanovic 2007), although these studies lacked the res-olution to differentiate erratic from cyclic variation andresolve whether gene arrangement frequencies wereshifting in response to climate change in those loca-tions. In summary, the most extensive temporal surveyperformed in a single population and a single chromo-some of Drosophila subobscura does suggest that sea-sonal changes in gene arrangement frequencies canbe an important source of temporal variation, yet thegenerality of this pattern remains poorly studied.

2.3. Invasion of the Americas

The invasion of the Americas by Drosophila subob-scura about 30 yr ago provided a unique opportunity tostudy this colonization event from its inception, andsomewhat revitalized the use of this species in evolu-tionary studies. The first individuals of D. subobscurain South America were detected in Puerto Montt,southern Chile, in February 1978 (Brncic & Budnik1980). By then, researchers had been collecting Dro-sophila species in Chile on a yearly basis for almost 2decades, which strongly suggests that colonization wasindeed detected at its beginnings. Within a year, D.subobscura had been collected near Santiago, located1000 km north from Puerto Montt (Brncic et al. 1981,Prevosti et al. 1988). This strongly suggests that thespecies has huge dispersal ability. Also remarkablewas the discovery in 1982 of D. subobscura in NorthAmerica near Port Townsend, WA (Beckenbach & Pre-vosti 1986), because those 2 colonization events couldnow be considered a large-scale natural experimentwith 2 replicates (i.e. ‘a grand experiment in evolution’;Ayala et al. 1989).

Although it was initially unclear whether the arrivalin North America represented a second independentinvasion, South and North American flies are nowknown to share identical genetic markers (genearrangements, lethal genes, microsatellite loci, etc.)likely derived from a source population in southwest-ern Europe (Mestres et al. 2005, Pascual et al. 2007and references therein). Recent analyses using micro-satellite loci provide compelling evidence that thespread of Drosophila subobscura in the Americas oc-curred following 2 colonizing events: from Europe toSouth America, and from South to North America(Fig. 2). The initial propagule size was small (<15 ind.),but a substantially larger number of effective founders(around 100 to 150) colonized North America (Pascualet al. 2007). The genetic diversity of New World popu-lations was greatly reduced in comparison with nativeEuropean populations as a consequence of the initial

genetic bottleneck, hence it was initially unclearwhether D. subobscura would be able to adapt to thenew conditions (e.g. inbreeding can have importanteffects on thermal tolerance and fitness in D. melano-gaster; Joubert & Bijlsma 2010, this Special).

The regular tracking of the inversion polymorphismand body dimensions since the colonization of theAmericas has provided clear evidence that this speciescan evolve very fast. For instance, just 3 yr after theSouth American invasion, Prevosti et al. (1985) col-lected flies from 7 Chilean sites spanning 12° of lati-tude and found incipient clines for gene arrangementsthat were convergent with the long-standing clines innative Palearctic populations. Further collections fol-lowing the second invasion from 8 North Americansites spanning 13° of latitude revealed clines that wereagain congruent with those in Europe and SouthAmerica (Prevosti et al. 1988). Such a degree of concor-dance on all 3 continents (summarized in Balanyà etal. 2003) provided unambiguous evidence that selec-tion on the chromosomal inversion polymorphism ofDrosophila subobscura must be strong, and that envi-ronmental factors associated with latitude probablyplay an important role in this context.

Some contrasting patterns between Old and NewWorld populations are, however, likely due to the ini-tial bottleneck. About 2 decades after the Americaninvasion, Huey et al. (2000) collected flies from 11localities in North America and 10 localities in conti-nental Europe. Their aim was to determine whetherbody size clines had eventually evolved in the NewWorld since large-scale patterns of body size varia-tion, with genetically larger individuals encounteredat higher latitudes, are common in Drosophila (seeBlanckenhorn & Demont 2004 and references therein),including native Old World populations of D. sub-obscura that offered an evolutionary baseline as theseflies have evolved for millennia. Convergence of cli-nal variation in wing size (a proxy for body size)between Old and New World populations was de-tected (see also Gilchrist et al. 2001, 2004), but aninteresting observation was that such convergencewas achieved by latitudinal changes in the relativelength of different parts of the wing. Contingent,unpredictable evolution was suggested as an expla-nation for the different details involved in the other-wise parallel wing size clines between continents(Huey et al. 2000, Gilchrist et al. 2001). However,recent results suggest that the association betweeninversions and particular genes influencing wingtraits have changed as a consequence of the bottle-neck, and that variable evolutionary patterns mightbe predictable as long as the genetic makeup offounder populations is taken into account (Fragata etal. 2010; see Section 4.2).

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3. GLOBAL WARMING

3.1. Tracking the change in chromosomal inversions

The repeated evolution of latitudinal clines followingcolonization has allowed researchers to make large-scale comparisons to forecast unprecedented changes inecological systems due to global warming. Historical sur-veys suggest that warm-climate gene arrangements areincreasing in frequency in European populations ofDrosophila subobscura (Orengo & Prevosti 1996, Rodrí-guez-Trelles et al. 1996; Fig. 5), possibly due to the ongo-ing climate warming (Rodríguez-Trelles & Rodríguez1998, Rodríguez-Trelles et al. 1998, Solé et al. 2002, Bal-anyà et al. 2004). The same trend has been reported inpopulations of South and North America, indicatingwarm-climate inversions are increasing in frequency athigher latitudes in all continents (Balanyà et al. 2006).Analyses pooling data from European, South Americanand North American sites showed that shifts in chromo-somal arrangement frequencies reflect quite accuratelychanges in ambient temperature: in 22 of the 26 sampledsites, the shifts occur in the direction predicted from ob-served temperature trends (Balanyà et al. 2006).

This tendency is not limited to Drosophila subobs-cura. Surveys in populations of the cosmopolitan spe-cies D. melanogaster from eastern Australia show asimilar pattern (Umina et al. 2005). Although the mag-nitude of the response varies depending on the gene orchromosomal arrangement targeted, Umina et al.(2005) reported a latitudinal shift of nearly 800 kmwithin roughly 20 yr for one particular inversion. In

addition, studies performed in populations of D.robusta from eastern North America also suggestedlatitudinal shifts possibly associated with changingtemperatures (Levitan & Etges 2005, Etges et al. 2006,Etges & Levitan 2008). Chromosomal arrangementsin D. robusta populations have changed within 15 to20 yr, though responses in some populations were notalways consistent with expectations based on envi-ronmental trends (Etges & Levitan 2008). Long-termchanges in some chromosomal arrangements have alsobeen reported in North American populations of D.pseudoobscura (Anderson et al. 1991) and in Japanesepopulations of D. melanogaster (Inoue et al. 1984). How-ever, in these cases it is not clear how the observedpatterns correlate with climatic trends.

Although much of the evidence is fragmented andcomes from a few studies, the apparent generality ofsome patterns emphasizes the necessity of increasedmonitoring efforts. As briefly outlined in the previousparagraphs, the latitudinal shifts in inversion frequen-cies in Drosophila populations seems to be a wide-spread phenomenon that encompasses a great varietyof chromosomal arrangements, relatively distant spe-cies of drosophilids (e.g. whereas the robusta groupis currently classified within the subgenus Drosophila,the melanogaster and obscura groups are classifiedwithin the Sophophora subgenus, and both subgeneradiverged about 40 million years ago; Markow &O’Grady 2006), and a broad geographical range thatspans North and South America, Europe, Asia andOceania. The evidence pointing towards an associa-tion between these patterns and climate change is

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Fig. 5. Drosophila subobscura. The shift observed in the northern limit of warm-climate gene arrangements after comparing re-cent surveys with historical records. Isoclines were drawn separating populations (circles) with detected chromosomal arrange-ments from those for which they were apparently absent. The generality of the pattern suggests that southern populations areshifting towards higher latitudes, though the possibility that rare gene arrangements were not detected in historical samples

cannot be completely discarded

Rezende et al.: Chromosomal inversions in Drosophila subobscura

robust but still limited to a few species, and additionalwork is therefore necessary in this context.

3.2. What do these shifts indicate?

Results described above indicate that variation inchromosomal polymorphisms and other non-neutralgenetic markers may eventually be employed to moni-tor the impact of climate change on the genetic consti-tution of populations at different locations. Nonethe-less, little is known on the causal mechanisms behindsuch a pattern. A potentially serious problem consists ofwhat is the most appropriate period to update historicalrecords. Specifically, in species with short generationtimes relative to season length, the seasonal climaticcycle can induce constantly recurring seasonal changesin climate-related traits superimposed on long-termtrends (Section 2.2). To prevent confounding both pat-terns, collections may be updated using the same cal-endar dates of old samples. However, this relies on thepremise that the seasonal cycles have remained station-ary over the monitoring period in the populations beingcompared (cf. Rodríguez-Trelles & Rodríguez 2007,2010, Balanyà et al. 2007). However, another possibilityis that the latitudinal clines and observed shifts in genearrangement frequencies have evolved in response toseasonality. Bradshaw & Holzapfel (2008) suggestedthat changes in the length of the growing season mightbe more important than the direct effects of tempera-ture (this may be the case particularly in regions athigher latitudes with more marked seasonal variation;see Fig. 1 in Chown et al. 2004). Recent reports of shiftsin the timing of events such as reproduction or migra-tion, which are presumably associated with climatechange, partly support this alternative (Peñuelas &Filella 2001, Parmesan & Yohe 2003, Root et al. 2003).Interestingly, gene arrangement frequencies of thepopulation at Mount Pedroso exhibit both seasonal andlong-term trends (Fig. 4), emphasizing that these alter-natives are not mutually exclusive (Rodríguez-Trelles& Rodríguez 2010).

Latitudinal clines of polymorphic inversions in Droso-phila subobscura seem to emerge from the interactionbetween dispersal and local adaptation. This idea isreinforced by the fact that the dispersal ability of thisspecies is known to be very high (i.e. the expansionfrom Puerto Montt to Santiago within 1 yr points to adispersal rate of roughly 2.7 km d–1; see Section 2.3).Furthermore, no genetic differentiation between Euro-pean populations has been detected using microsatel-lite loci, which suggests that gene flow due to migra-tion is sufficiently high to homogenize the geneticconstitution of populations at putatively neutral mark-ers (Pascual et al. 2001). Gene flow prevents popula-

tions from optimally adapting to their environments(Ronce & Kirkpatrick 2001), but there is no clearamount of migration or gene flow at which point popu-lations can become effectively isolated with respect totheir evolutionary dynamics (Rice 2004). The rapidlyevolved inversion clines in the Americas, however,suggests that local adaptation is taking place in spite ofhigh gene flow (Kirkpatrick & Barton 2006). Therefore,chromosomal arrangements in D. subobscura likelyplay an important role in preventing the genetic ex-change between locally adapted alleles from otherpopulations or genetic backgrounds. The clinal shiftsin the frequency of chromosomal inversions correlatedwith current climate change might then reflect: (1)locally ongoing adaptation to global warming trends,and/or (2) invasion from more equatorial populationscarrying warm-climate gene arrangements that nowa-days encounter better conditions to flourish at higherlatitudes.

We took advantage of one of the most remarkablefeatures of Drosophila subobscura (i.e. its extremelyrich inversion polymorphism encountered in Palearcticpopulations; see Section 2.1) and asked the question:Can we now detect chromosomal arrangements thatwere formerly restricted to southern populations athigher latitudes? If this were the case, it would providecompelling evidence that migration is taking place,because overwhelming evidence favors the view thatchromosomal inversions in Drosophila are mono-phyletic (Powell 1997). In fact, the geographical distri-butions of some gene arrangements now appear atrelatively high frequencies in some northern locations(Fig. 5), where they were undetected in historical sur-veys (Solé et al. 2002, Balanyà et al. 2004, J. Balanyà etal. unpubl. data). A remarkable example is the genearrangement O3+4+8, the most frequent genetic con-figuration found in North Africa (Prevosti 1974), butwhose frequency is now 22.6% in Groningen, Nether-lands, and 2.7% in Sunne, Sweden. A potential caveatwith these data is that the gene arrangements couldhave been already present in historical times at thesame locations, but were not detected because theyhad very low frequencies. However, this pattern wasobserved repeatedly in several gene arrangements indifferent chromosomes (Fig. 5), which suggests thatthe overall trend probably does not reflect a samplingartifact.

To summarize, gene arrangement frequencies inDrosophila subobscura are shifting in response to cli-mate change in Europe and South and North America,but it remains unclear why. Although this does notexclude the use of chromosomal polymorphisms as bio-logical monitors of climate change, a better under-standing of the processes involved is certainly neces-sary. Determining which factors are more relevant in

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shaping the response of D. subobscura to global warm-ing is crucial to predicting how other biologicalsystems may be affected by—and may eventually re-spond to—climate change.

4. TESTING HYPOTHESES DERIVED FROMNATURAL PATTERNS

4.1. Phenotypic latitudinal clines

Several complementary approaches may be em-ployed to elucidate which factors ultimately influencethe evolution of latitudinal clines. Perhaps the mostintuitive approach involves determining which pheno-typic traits that are directly associated with Darwinianfitness (such as fecundity, developmental rates, heat orcold tolerance, etc.) show significant latitudinal clines.This is particularly challenging because measure-ments of this nature involve very large sample sizesand controlling for environmental effects that mightobscure real genetic trends. In addition, in the case ofDrosophila subobscura, the genetic bottleneck duringthe colonization of the New World (Section 2.3) hasresulted in somewhat different genetic compositions inAmerican and European populations, which is anotherconfounding factor that might obscure comparisonsacross continents.

Few studies have addressed whether latitudinal gra-dients exist at the phenotypic level in Drosophila sub-obscura (for phenotypic clines in other Drosophila spe-cies, see e.g. Hoffmann et al. 2003, Hoffmann & Weeks2007, Arthur et al. 2008, Turner et al. 2008 and refer-ences therein). A recent example is the analysis oflatitudinal patterns in desiccation- and starvation-resistance (Gilchrist et al. 2008). One would intuitivelyexpect that these phenotypic traits may be importantdeterminants of survival in the field. Their analysisinvolved 9 populations from Europe, 9 from NorthAmerica and 10 from South America, that were distrib-uted across a gradient of more than 15° of latitude oneach continent. Although some clinal patterns weredetected, results were not conclusive: starvation toler-ance did not vary with latitude on any continent, andthe latitudinal cline in desiccation resistance in SouthAmerica showed the opposite trend (i.e. an increasein resistance with latitude) than in Europe and NorthAmerica.

Conversely, convergent trends were observed acrossEurope and North and South America in the evolutionof larger body sizes at higher latitudes (Section 2.3).However, latitudinal variation in quantitative traitspotentially unrelated to climatic variables may occuras a correlated response to selection on other targets.This is particularly relevant because inversions involve

many genes (Section 1.1); hence different chromoso-mal gene arrangements from natural populations candiffer in their mean value for one or more phenotypictraits. For instance, Orengo & Prevosti (2002) uncov-ered a relationship between chromosomal polymor-phisms and wing size in a European population of D.subobscura (see also Fragata et al. 2010). In accor-dance with the clinal patterns, they found bigger sizesto be preferentially associated with gene arrange-ments whose frequencies increase with increasing lat-itude. Correlated clinal patterns in inversion frequen-cies and body size might therefore have a similarunderlying cause, although it is unclear how tem-perature mediates selection of body size in small ecto-therms (Blanckenhorn & Demont 2004). In summary,determining if observed clinal patterns result fromdirect selection of body size or as a correlated responserequires a mechanistic understanding of how variationin size translates into differences in Darwinian fitness,which is not possible with a strictly correlationalapproach. Researchers must then rely on differentapproaches to understand how these patterns emerge.

4.2. Experimental evolution

In an attempt to determine if differences in tempera-ture could account for the observed latitudinal patternsin chromosomal inversion frequencies, lines derivedfrom a South American population of Drosophila sub-obscura from Puerto Montt (the epicenter of the Amer-ican invasion) were maintained in the laboratory forseveral generations at 3 constant temperatures: 13°C(cold), 18°C (around the optimum temperature; seeKrimbas 1993, Rego et al. 2010) and 22°C (warm) (San-tos et al. 2004, 2005). If temperature was the mainfactor driving the evolution of latitudinal clines of genearrangements, one would expect chromosomal fre-quencies of laboratory lines subjected to colder tem-peratures to resemble those from natural populationsat increasing latitudes.

Succinctly, the evolutionary responses detected inthe laboratory could not account for most trends ob-served in nature (see also discussion in Chown et al.2010, this Special). Several gene arrangements quicklyresponded to the contrasting thermal regimes, butresponses often contradicted expectations based onthe clinal patterns. Phenotypic responses were alsodiscrepant: selected lines did not differ in wing size inspite of the conspicuous convergent clines observedacross continents, casting doubts on the adaptive valueof size variation across different thermal environ-ments (Section 4.1). Nonetheless, it remains debatablewhether the selection regimes in the laboratory, sub-mitting lines to constant temperatures, accurately

Rezende et al.: Chromosomal inversions in Drosophila subobscura

reflect the selective pressures in nature. In spite ofsome inherent limitations (see Huey & Rosenzweig2009 for a general discussion on the topic, and Rego etal. 2010 on this particular experiment), experimentalevolution under more well-established conditions canprovide valuable information on the mechanismsunderlying correlational patterns observed in nature.

4.3. Association between inversions andphenotypic traits

Another complementary approach involves deter-mining how chromosomal gene arrangements corre-late with variation at the phenotypic level. Thisapproach can shed light on the genetic basis ofobserved phenotypic variation with an unprecedentedlevel of resolution, and requires measuring the pheno-typic trait of interest in individual flies (preferentiallyafter removing environmental sources of variation) andthen determining their gene arrangements.

For instance, a recent study has shown that flies car-rying different gene arrangements differ in their ther-mal preferences and tolerances to high temperatures(Rego et al. 2010). Individual flies from a populationcollected near Lisbon, Portugal, and maintained forseveral generations in the laboratory were firstallowed to select their preferred ambient tempera-tures in a thermal gradient, and then were submittedto increasing temperatures in a water bath until theybecame unconscious. After analyzing the gene ar-rangements of each individual, it became clear thatflies carrying warm-climate arrangements chose andtolerated higher temperatures than their cold-climatecounterparts, which agrees with the expectation fromnatural patterns. Interestingly, thermal preferenceand thermal tolerance were genetically independent,being primarily associated with inversions on differ-ent chromosomes. Comparisons between temporalsurveys in the field and results from Rego et al. (2010)suggest that thermal tolerance is more important inthe evolution and dynamics of latitudinal clines thanthermal preference.

This approach has also been employed to study thediscrepant morphological clines observed in Europeand the Americas. As discussed above, latitudinal clinesin wing length were generally convergent but involvedchanges in different sections of the wing on each con-tinent. In other words, ‘the evolution of geographicvariation in wing length has been predictable, but themeans by which the cline is achieved is contingent’(Huey et al. 2000, p. 308). To test if this was the case,Fragata et al. (2010) estimated how wing length andshape (computed as a ratio of proximal wing length tototal wing length; Huey et al. 2000) were associated

with gene arrangements in a European (Lisbon) and aSouth American (Puerto Montt) population, and foundthat the genetic basis underlying wing morphologicalvariation was dramatically different (for comparisonson the genetic basis behind wing shape and size acrossDrosophila species, see Trotta et al. 2010, this Special).

It became apparent that the bottleneck during thecolonization of the Americas affected the associationbetween some gene arrangements and wing shape,raising the possibility that morphological responseswere not related to the evolution of wing size clines.This hypothesis was tested by reanalyzing the datareported in Pegueroles et al. (1995), who sampledDrosophila subobscura flies from 6 North Americanpopulations in 1986 (i.e. 4 yr after the first colonizersarrived). Whereas no latitudinal cline was detected inwing size at this early stage of colonization, wingshape clines were already present in both males andfemales (Santos et al. 2004) and matched the patternsreported for populations collected 11 yr later (Fig. 1Bin Huey et al. 2000, Gilchrist et al. 2001). Therefore,although the genetic composition of the populationthat invaded America was contingent on the bottle-neck effect, the wing shape cline that evolved sub-sequently in North America was not, highlightingthat contingent, unpredictable evolution might be lesswidespread than some evolutionary biologists assume(cf. Gould 1989, Conway-Morris 1998).

5. CONCLUDING REMARKS

Recent studies in natural populations of Drosophilahave shown that species can respond considerablyquickly to changes in environmental conditions, sug-gesting that genetic responses might provide relevantinformation on the impacts of climate change (Rodrí-guez-Trelles & Rodríguez 1998, Umina et al. 2005,Balanyà et al. 2006, 2009). Although observed pat-terns such as the latitudinal shifts in gene frequen-cies highlight their importance as biological monitors,a better understanding of the processes underlyingthese patterns is necessary. In this context, experi-ments in the field and/or in mesocosms may providecomplementary approaches to the ones outlined inSection 4. Recent field release studies have been suc-cessfully employed to test the adaptive role of accli-matory responses in D. melanogaster (Loeschcke &Hoffmann 2007, Kristensen et al. 2008) and the puta-tive role of local adaptation as a determinant of chro-mosomal frequencies in D. robusta (Levitan & Etges2009), and similar experiments might shed light onthe mechanisms underlying the geographical andtemporal changes in the chromosomal polymorphismof D. subobscura.

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A better understanding of the processes that underliethe evolution of latitudinal clines of inversion frequen-cies may allow researchers to determine which pheno-typic traits are being influenced by environmental con-ditions and to extrapolate results obtained in Drosophilato other systems. Extrapolations should be performedwith caution, however, because species differ in a varietyof ways that will ultimately determine how they respondto environmental challenges (Rose et al. 2005). One wayto partially circumvent this problem is to develop geneticand/or phenotypic markers in a wide range of modelspecies belonging to different groups. These model spe-cies should share 2 basic attributes: distribution rangesmust be large to minimize the confounding effects of lo-cal processes, and generation times must be short to en-sure that evolutionary responses are observed. The list ofpotentially suitable models in this context includes notonly other Drosophila species (a genus with over 1500species distributed across the globe; Markow & O’Grady2006), but also rodents (e.g. genera Mus and Pero-myscus), plants such as the cosmopolitan weed Arabi-dopsis and small invertebrates from aquatic systemssuch as Daphnia and Littorina. Ultimately, these biolog-ical monitors may prove useful to quantify the impact ofclimate change in an ecosystem, assess which taxa aremore vulnerable and determine which climatic variablesare more relevant in a given scenario.

Acknowledgements. We thank C. Pertoldi for the opportunityto present this overview, and 2 anonymous reviewers forhelpful comments on an earlier draft. E.L.R. is supported bya Ramón y Cajal contract from the Ministerio de Ciencia eInnovación (Spain). C.R. is supported by a fellowship(SFRH/BPD/39998/2007) from Fundação para a Ciência e aTecnologia (Portugal). I.F. was partially supported by anexchange grant (reference no. 2239) from the ESF Pro-gramme on ‘Thermal adaptation in ectotherms: linking lifehistory, physiology, behaviour and genetics’. This researchwas supported by the grants CGL2006-13423-01/BOS fromthe Ministerio de Ciencia y Tecnología (Spain), CGL2009-12912-C03-01 and BFU2009-07564 from the Ministerio deCiencia e Innovación (Spain) and 2005SGR 00995 and2009SGR 636 from Generalitat de Catalunya to the Grup deBiologia Evolutiva.

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Submitted: April 6, 2010; Accepted: April 30, 2010 Proofs received from author(s): June 10, 2010