Resilience of plant-insect interactions in an oak lineage throughQuaternary climate change

Tao Su, Jonathan M. Adams, Torsten Wappler, Yong-Jiang Huang, Frédéric M. B. Jacques,Yu-Sheng (Christopher) Liu, and Zhe-Kun Zhou

Abstract.—Plant-insect interactions are vital for structuring terrestrial ecosystems. It is still unclear howclimate change in geological time might have shaped plant-insect interactions leading to modernecosystems. We investigated the effect of Quaternary climate change on plant-insect interactionsby observing insect herbivory on leaves of an evergreen sclerophyllous oak lineage (Quercus sectionHeterobalanus, HET) from a late Pliocene flora and eight living forests in southwestern China. Among themodern HET populations investigated, the damage diversity tends to be higher in warmer and wetterclimates. Even though the climate of the fossil flora was warmer and wetter than modern sample sites,the damage diversity is lower in the fossil flora than in modern HET populations. Eleven out of 18damage types in modern HET populations are observed in the fossil flora. All damage types in the fossilflora, except for one distinctive gall type, are found in modern HET populations. These results indicatethat Quaternary climate change did not cause extensive extinction of insect herbivores in HET forests.The accumulation of a more diverse herbivore fauna over time supports the view of plant species asevolutionary “islands” for colonization and turnover of insect species.

Tao Su, Frédéric M.B. Jacques, and Zhe-Kun Zhou*. Key Laboratory of Tropical Forest Ecology, XishuangbannaTropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, China. E-mail: zhouzk@xtbg.ac.cn

Tao Su. State Key Laboratory of Paleobiology and Stratigraphy, Nanjing Institute of Geology and Paleontology,Chinese Academy of Sciences, Nanjing 210008, China

Jonathan M. Adams*. College of Natural Sciences, Seoul National University, Gwanak-Gu, Seoul 151, Republicof Korea. E-mail: jonadams@snu.ac.kr

Torsten Wappler. Steinmann Institute for Geology, Mineralogy and Palaeontology, Division Palaeontology,University of Bonn, Nussallee 8, D-53115 Bonn, Germany

Yong-Jiang Huang and Zhe-Kun Zhou. Key Laboratory for Plant Diversity and Biogeography of East Asia,Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China

Yu-Sheng (Christopher) Liu. Department of Biological Sciences and Don Sundquist Center of Excellence inPaleontology, Box 70703, East Tennessee State University, Johnson City, Tennessee 37614-1710, U.S.A.

Accepted: 15 August 2014Supplemental materials deposited at Dryad: doi:10.5061/dryad.0hq44

Introduction

Climate change has long been considered adriving force in the evolution of ecosystems(Scholze et al. 2006; Blois et al. 2013). Plants andinsect herbivores are two major components ofterrestrial communities, and their interactionsplay an important role in structuring ecosys-tems (Schoonhoven et al. 2005). Consequently,there is considerable interest in the evolution ofplant-insect herbivore interactions in responseto climate change (Bale et al. 2002; Brooker2006; Futuyma and Agrawal 2009; Labandeiraand Currano 2013). Most work on the evolu-tion of plant-insect interactions has studiedonly the present-day state, using phylogeniesderived from DNA and/or morphology

(Nyman et al. 2012; Whitfeld et al. 2012). Thefossil record could potentially provide moredirect observations of past processes that haveshaped ecosystems (Louys et al. 2012). In recentyears, a series of studies have documentedplant-insect interactions in response to climatechange in geological timewith evidence of insectdamage on fossil leaves (Wilf 2008). Insectdamage on fossil leaves relates not only toplant-insect interactions, but also to the evolu-tion and diversity of the herbivorous insectfauna (Currano 2009; Carvalho et al., 2014).Fossil evidence of insect damage from differentregionsworldwide indicates that climate changehas shaped plant-insect interactions in thepast (Wilf and Labandeira 1999; Wilf et al.2001; Currano et al. 2008; Prokop et al. 2010;

Paleobiology, 41(1), 2015, pp. 174–186DOI: 10.1017/pab.2014.11

© 2015 The Paleontological Society. All rights reserved. 0094-8373/14/4004-0006

Knor et al. 2012). However, there remains afundamental disconnect from the present, interms of understanding how climate change ingeological time has shaped the present plant-insect interactions.The evergreen sclerophyllous oak (Quercus

section Heterobalanus [HET]) is at presentmainly distributed in southwestern China,occurring as the dominant species in manyforests of this region. HET has a long history,dating back to 15Ma in Namling, southernTibet (Li and Guo 1976). Forests dominated byHET have existed since the late Pliocene insouthwestern China. In this study, we wereable to document insect herbivore damage in a3-Ma flora in Yongping County of YunnanProvince, on fossil HET leaves subject toexceptional preservation. Because HET stillexists in southwestern China (Yang et al. 2009;Su 2010), we were able to compare the range ofherbivore damage types within a narrow plantlineage on an evolutionary time scale, in a waythat has never been possible before.Here, we mainly focus on the evolution of

plant-insect interactions in HET forests inresponse to Quaternary climate change. TheQuaternary (~2.588Ma to present) is a world-wide cool period marked by a series ofglaciations (Pillans et al. 2012). Owing to thechanges of global ice volume, southwesternChina experienced climate fluctuations duringthe Quaternary, generally characterized by thevariations of the monsoon intensity (An et al.2011). Both the Indianmonsoon and East Asianmonsoon converge in southwestern China,giving a pronounced winter dry season-summer wet season pattern (Wang 2006).Variations in the monsoon system during theQuaternary have had a significant influence onthe pattern of precipitation, and consequentlyon the pattern of temperature. Meanwhile, thecontinuous orogenic uplift of this region hasalso helped to shape the spatial distributionpatterns of both temperature and precipitationduring the Quaternary (Su et al. 2013).Climatic change during the Quaternary has

likely had a direct influence on the evolution ofmodern communities as well as whole ecosys-tems. It is therefore of great interest to havean ancient plant-herbivore system where thedirect descendant of the fossil plant taxon

remains extant and can be studied in detail inthe present-dayworld, and the differences overtime can be carefully compared.

Our detailed observations here allow thetesting of three hypotheses for the evolutionaryhistory of plant-insect interactions:

1. The climate bottleneck hypothesis. Repeated,dramatic climate fluctuations have sup-pressed damage diversity by causingpopulation bottlenecks of both the hosttree and its associated insect herbivores,resulting in herbivore extinctions (AdamsandWoodward 1989; Labandeira et al. 2002).Thus we may expect to see a reduction indamage diversity over time.

2. Accumulation of herbivores over evolution-ary time. As a plant species ages since its timeof origin, it will accumulate more species ofherbivores that successfully adapt to over-coming its defenses, and consequently anincrease of damage diversitywill be observed(Southwood 1973; Feeny 1976). According tothis hypothesis, an increase in damagediversity over time can be expected.

3. Null hypothesis (Coope 1979). Neithermechanism has been an overriding influence,resulting in stability of damage diversity andcomposition over time.

Based on the evidence of insect damagefrom the same vegetation type and within anarrowly defined taxon lineage, this studyfor the first time combines the fossil recordwith contemporary samples to test thesehypothetical scenarios stated above, providingimportant evidence for the resilience of ecosys-tems in response to long-term climate change,something that cannot be tested by ecologicalstudies from modern samples alone.

Materials and Methods

Evergreen Sclerophyllous Oak.—Our studymaterial is evergreen sclerophyllous oak(Quercus section Heterobalanus [HET]; Figs. 1, 2).This type of oak is mainly distributed in theQinghai-Tibet Plateau (Working Group onYunnan Vegetation 1987). Heterobalanus “sensulato” consists of various populations with minor

INSECT HERBIVORY AND PALEOCLIMATE CHANGE 175

morphological differences, and even thoughthese populations have sometimes beendivided off as separate species, it is clear thatHeterobalanus s.l. is monophyletic and of recentorigin, without a strong basis for distinguishingseparate species (Nixon 1993; Denk and Grimm2010). In this study, we treat the fossil species asQ. preguyavaefolia (Tao et al. 2000) and the extantspecies as Q. guyavaefolia. Fossil HETs haveleaves and cupules that are morphologicallyindistinguishable from the extant ones (Fig. 2).Forests dominated by HETs are one of the mainvegetation types ranging from 2000 to 4500m inaltitude in southwestern China (Working Groupon Yunnan Vegetation 1987). It is not until thelate Pliocene that fossil floras dominated byHETare found in sediments from southwesternChina (Tao et al. 2000). Among these floras,leaf fossils of Q. preguyavaefolia in the Longmenflora of Yongping, western Yunnan, are themost abundant and have the best qualityof preservation (Su 2010), allowing detailed

examination of damage type (DT) morphologieson fossil leaves.

The Late Pliocene Longmen Flora.—TheLongmen flora is situated in Yongping Countyof western Yunnan Province (25°31′N, 99°31′E;1715m a.s.l.; Table 1, Fig. 1). The plant-fossil-bearing sediment in this study belongs to theSanying Formation, which is widely distributedin western and northwestern Yunnan. Thegeological age of the fossil flora is about 3Maaccording to a recent paleomagnetic study(Li et al. 2013). Detailed information on thisstratum is summarized in Su et al. (2011).Fossil specimens in this study were uncoveredfrom the fine gray clay in the upper layer ofthis lacustrine sediment. The fossils are all fromthe same site with sample size spanning 0.5min depth, 2m in width, and 10m in length.It may have received leaves washed or blown infrom a wide area of forest around (Ellis andJohnson 2013). All specimens are depositedin Paleoecology Laboratory, Xishuangbanna

31°

30°

29°

28°

27°

26°

25°

24°

23°

22°

106°105°104°103°102°101°99°98°96° E

21°

100°97° 107°

N

1

2

34

5

6

78

Salw

een River

0 125 250km

Fossil site

Modern sample

N

site

Mekong River

Yangtze

R i ver

Sichuan

Guizhou

GuangxiYunnan

Tibet

India

Myanmar

Laos Vietnam

Lijiang

Kunming

Dali

Kangding

Yongping

Litang

Daocheng

Mangkang

Jinghong

Chengdu

Guiyang

Kaiyuan

Qujing

Chongqing

ChinaShangri-La

FIGURE 1. Localities of the late Pliocene Longmen flora and eight samples from living Quercus guyavaefolia forests.Information on each site is listed in Table 1.

176 TAO SU ET AL.

FIGURE 2. Fossil Q. preguyavaefolia and living Q. guyavaefolia in this study. A, C, Fossil Q. preguyavaefolia from the latePliocene Longmen flora. A, Leaf; C, Cupule. B, D, E, F, Living Q. guyavaefolia in southwestern China. B, Leaf;D, Cupule; E, Scene of an HET forest dominated by Q. guyavaefolia (trees in brown) in Mt. Xiaoxue, Shangri-La County,northwestern Yunnan Province; the elevation range is 3400–3500m; F, Fruit-bearing branch of Q. guyavaefolia.

INSECT HERBIVORY AND PALEOCLIMATE CHANGE 177

TABLE1.

Inform

ationon

position

san

dclim

ates

ofthelate

Pliocene

Lon

gmen

floraan

deigh

tmod

ernsamplingforests.Dataon

clim

atepa

rametersof

theLon

gmen

floraare

from

Suet

al.(20

13);presen

tclim

aticdatawereacqu

ired

from

aglob

alclim

atemod

elin

1-km

spatialresolution(H

ijman

set

al.2

005).M

AT=meanan

nual

tempe

rature;

WMMT=warmest-mon

thmeantempe

rature;C

MMT=coldest-mon

thmeantempe

rature;M

AP=meanan

nual

precipitation;

GSP

=totalp

recipitation

with

meanmon

thly

tempe

rature

beingmorethan

10ºC

;3-W

ET=precipitationduringthethreeconsecutivewettest

mon

ths;3-DRY=precipitationduringthethreeconsecutivedriestm

onths.

Datain

bold

aretheup

peran

dlower

limits

ofeach

clim

atepa

rameter

inmod

ernsamplesites.

Samplesite

Altitude

(m)

Latitud

e(N

)Lon

gitude

(E)

MAT

(ºC)

WMMT

(ºC)

CMMT

(ºC)

MAP

(cm)

GSP

(cm)

3-W

ET(cm)

3-DRY

(cm)

Lon

gmen

,Yon

gping,

Yun

nan(LM)

LatePliocene

1715

25°31′

99°31′

17.4±1.3

25.8±1.5

8.9±2.6

—17

3.6±21

.880

.0±13

.918

.5±4.1

Presen

t16

.721

.89.7

103.9

102.1

52.3

6.8

1Sh

uang

shao

,Kun

ming,

Yun

nan(SS)

2447

25º13'

102º45

'12

.517

.65.8

105.0

97.8

63.1

3.8

2Hutiaox

ia,S

hang

ri-La,

Yun

nan(H

TX)

2955

27º21'

99º54'

9.2

15.3

2.1

90.9

82.3

56.5

2.1

3Nixi,Sh

angri-La,

Yun

nan

(NX)

3055

28º01'

99º32'

10.5

16.8

2.9

83.3

76.7

49.3

2.5

4W

engshu

i,Sh

angri-L

a,Yun

nan(W

S)42

5628º34'

99º50'

3.6

10.8

−4.7

66.8

43.2

43.2

1.5

5Mt.Hon

gla,

Man

gkan

g,Tibet

(HL)

4220

29º15'

98º41'

4.0

11.3

−4.2

54.9

32.5

32.5

1.2

6Gatuo

,Man

gkan

g,Tibet

(GT)

4014

29º43'

98º37'

3.1

10.8

−5.4

52.2

22.9

31.8

0.8

7Kag

ong,

Litan

g,Sich

uan

(KG)

3836

30º16'

100º16

'3.7

11.3

−5.6

69.2

44.7

44.7

0.7

8Gao

'ershi,K

angd

ing,

Sich

uan(G

ES)

4058

30º03'

101º23

'0.7

8.5

−8.2

88.8

0.0

54.3

1.6

178 TAO SU ET AL.

Tropical Botanical Garden, Chinese Academy ofSciences.Contemporary Samples.—Eight primary HET

forests with low human activity insouthwestern China were chosen as samplesites to be comparable with the vegetation typeof the Longmen flora (Table 1). Samples werecollected during October and November in2011, after the rainy season had ended. Thesemodern sample sites cover almost the entirerange of climate and elevational distribution ofHET forests in southwestern China (Yang et al.2009). For each sample site, three replicatesweretaken in order to sample as wide a range ofherbivory types on leaves as possible within thesame forest. The distance between replicateswithin each site was 200 to 500m in horizontaldistance, but with an elevational difference ofno more than 100m. These replicates were allcollected in forests located more than 100maway from any main road to exclude theinfluence of human activity on herbivory. Foreach replicate site, we collected the fallen leavesof HET on the ground with rubber gloves alonga horizontal line at a distance of 100m. In oursampling transects, we stopped every fivemeters and randomly grabbed five handfuls offallen leaves. This process was done repeatedlyat 20 stops along the transect. All leaves from areplicate site were put in one plastic bag andlabeled with a number. In total, more than 5000leaves were collected from each replicate. Webelieve fallen leaves are a more representativesample than leaves sampled directly from partsof the canopy, because fallen leaves integratethe whole canopy. Also, this sampling strategyshould make the modern samples more closelycomparable to fossil samples, because all thefossil leaves are formed by fallen leaves. HET isnot deciduous during autumn; therefore,samples we collected might be fallen leavesfrom the current year or from the previous year.Climatic Settings.—The paleoclimate of the late

Pliocene Longmen flora has been quantitativelyreconstructed by leaf physiognomy-basedmethods in Su et al. (2013). It was concludedthat climate in western Yunnan at that timewas slightly warmer and much wetter than thepresent day (Table 1). These results aregenerally in agreement with other evidencesuch as modeling (Li et al. 1998) and isotope

records (Zachos et al. 2001). The climate data ofmodern sample sites could not be directlyobtained from weather stations, because HETforests with low human disturbance areusually far from the cities where most climatestations are set up in China. To infer climateparameters, we used a high-resolution globalclimate model with a scale down to one squarekilometer (Hijmans et al. 2005). Data werereferenced with the geospatial processingsoftware ArcGIS, version 9.3 (ESRI, Redlands,USA). Seven temperature and precipitationrelated parameters were calculated for thesemodern sample sites in order to keep theseconsistent with climate parameters calculatedfor the Longmen flora (Table 1).

Scoring of Insect Damage.—We used theDamage Type Guide (Labandeira et al. 2007)to assess insect damage types from the PlioceneLongmen locality and modern samples (Fig. 3).For the Longmen flora, we used fossil leaves ofQ. preguyavaefolia s.l. with very good qualityof preservation as the research materials toobserve the presence/absence of insectdamage. These were fossil leaves with at leasthalf of the blade preserved, and very clearanatomical detail of the leaf structure (e.g.,clearly defined venation, leaf margin, andpetiole). Damage types were identified undera dissecting microscope (Leica S8APO). Intotal, 1028 fossil leaves of Q. preguyavaefolia s.l.were checked and scored in the Longmenflora (Supplementary Table 1). For moderncollections, we scored 500 leaves for eachreplicate and three replicates were lumped asone forest sample. For each replicate, all leaveswere put into a box and gently mixed; leaveswere chosen randomly to score the insectdamage types. In this procedure, leaves thatwere not heavily decayed by microbes (i.e., notbearing fungal strands or falling to pieceswhen lightly pulled) were used. Both sides ofeach leaf were carefully checked to categorizedamage types. Eighteen distinctive anddiagnostic insect DTs are found in the totaldata set. The data set of insect damage types for1028 leaves from the Longmen flora and 12,000leaves from eight extant HET forests aredeposited in Supplementary Table 1. Thepercentage of each damage type for everysample is given in Supplementary Table 2.

INSECT HERBIVORY AND PALEOCLIMATE CHANGE 179

Data Analyses.—Insect folivory was examinedusing three damage-type metrics: frequency,diversity, and distribution. Damage diversity, orthe number of DTs present at each locality, wasnormalized for the number of leaves sampled, asin previous studies (Wappler 2010; Knor et al.2012). The percentage of explained variabilitywas computed by means of Nagelkerke pseudo-R2 measure as implemented (Harrell 2009) in thesoftware R, version 2.13.1 (R Development CoreTeam, 2011). Where necessary, overdispersionwas treated by refitting to the quasibinomialfamily of generalized linear models. Tounderstand the correlation between damagediversity/frequency and climate in living HETforests, the single linear regression was usedwith the software R. Data on the percentageof every damage type in each sample wereclustered with the package “vegan” (Oksanen2009) in the software R by using the method ofBray-Curtis distance.

Results

Indicators of Adequate Sample Quantity andQuality.—Bootstrap curves derived from thecensus data obtained in this study indicate thatthe modern and fossil leaf sample sizes usedhere are large enough to cover all damage

types (DTs) present at each sample site (Fig. 4).In each case, the bootstrap curve levels off wellbefore the total sample number of leaves hasbeen reached (Fig. 4). Furthermore, we havecarefully checked the morphology of eachdamage type under a dissecting microscope:all damage types present on modern leavescould be preserved if they existed in fossilleaves, judging by the morphology of eachdamage type and extremely good preservationof the fossils (Fig. 3, Supplementary Fig. 1). Forexample, DT 168 belongs to the piercing scar,which is similar to a fungal fruiting body.

FIGURE 3. Damage diversity on leaves of fossil Quercus preguyavaefolia (A–E) and living Q. guyavaefolia (F–J). DT01 (holefeeding), DT 12 (marginal feeding), DT36 (mining), and DT45 (mining) are found in both Q. preguyavaefolia andQ. guyavaefolia; DT 33 (galling on the main vein) is observed only in Q. preguyavaefolia, and DT163 (galling on thesecondary vein) is only in Q. guyavaefolia. See morphologies of all damage types in Supplementary Figure 1. Scale bar ineach figure, 2 mm.

16000 200 400 600 800 1000 1200 1400

18

02468

10121416

Number of specimens

Num

ber o

f dam

age

type

s SS / NXHTX

Modern sampleFossil sample

WS / HL

GTKG / GES

Longmen

FIGURE 4. Bootstrap curves of insect damage diversity forthe late Pliocene Longmen flora (LM) and eight modernforests. A total of 1028 leaves from the Longmen flora and1500 leaves from each modern forest were scored fordamage types. Abbreviation and detailed information oneach sample site are given in Table 1.

180 TAO SU ET AL.

However, the smooth and convex surface of DT168 along the adaxial side of the leaf blademakes it readily distinguishable from the fungalfruiting body. Besides, DT 168 could bepreserved as fossils judging by the prominentconvex surface (Supplementary Fig. 1AB).Patterns of Damage Diversity in Modern

Oak Populations.—In our extant evergreensclerophyllous oak (HET) forest samples, thediversity of damage types significantly correlateswith all the investigated temperature-related

parameters (Table 2), such as mean annualtemperature (MAT; r2= 0.73, p<0.05) andwarmest-month mean temperature (WMMT,r2= 0.71, p<0.05). Regarding precipitation-related parameters, the diversity of damagetypes significantly correlates only with growingseason precipitation (GSP, r2= 0.72, p<0.05).The damage frequency does not significantlycorrelate with any of the seven investigatedclimate parameters (Table 2), further supportingthe conclusion that the strongest signal in insect-folivore damage is for diversity, not frequency(Currano et al. 2008). The damage frequencyvaries widely among sites, with 46.1% of leavesdamaged in Wengshui (Site 4) to 96.1% inHutiaoxia (Site 2). The number of damage typesranges from 13 (Gatuo, Site 6) to 17 (Hutiaoxia,Site 2). Among the broad categories of damagetypes, hole-feeding and marginal feeding, bothbelonging to generalized feeding categories, arethe most abundant (Fig. 5). In the hole-feedingcategory, six damage types were found in total,and formarginal feeding therewere five damagetypes. Among all hole-feeding damage types,DT4 and DT5 could not be observed in sites 4-8.All modern samples show a lower percentage of

> 05

1015

20

25

3035

40

4550

55

60

Dam

age%

NA

1 SS

LM

2 HTX

3 NX

5 HL

7 KG

6 GT

4 WS

8 GRS

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

HF GAMF SK PSMI

DT1

DT2

DT3

DT4

DT5

DT7

DT1

2

DT1

3

DT1

4D

T15

DT8

1

DT1

6

DT3

6

DT4

5

DT1

95

DT3

3

DT1

63

DT1

68Host specificity

Generalized

Intermediate

Specialized

Height

FIGURE 5. Heat map and cluster analysis showing the composition and similarity of damage types (DTs) amongsample sites. The number and abbreviation of each sample site are given in Table 1. HF, hole feeding; MF, marginalfeeding; SK, skeletonization; MI, leaf-mining; GA, galling; PS, piercing-and-sucking.

TABLE 2. Single linear correlations among climate para-meters and number of damage types/damage frequencyin living Quercus guyavaefolia forests. See Table 1 forclimate parameter abbreviations.

No. of damagetypes

Damage frequency(%)

Climate parameter r2 p r2 p

MAT (ºC) 0.731 0.007* 0.412 0.086WMMT (ºC) 0.713 0.008* 0.408 0.088CMMT (ºC) 0.734 0.006* 0.396 0.095MAP (cm) 0.589 0.026* 0.649 0.016*GSP (cm) 0.721 0.008* 0.333 0.1343-WET (cm) 0.559 0.033* 0.604 0.023*3-DRY (cm) 0.565 0.031* 0.361 0.115

*Statistically significant at 0.05 level.

INSECT HERBIVORY AND PALEOCLIMATE CHANGE 181

leaf damage from likely host-specific damagecategories, occurring in less than 25% of leaves.Galling has the lowest frequency in all modernsamples (Supplementary Table 2).

Damage Diversity of the Late Pliocene LongmenFlora.—In the late Pliocene Longmen flora, thetotal damage frequency on HET leaves is58.9 %, which is within the range of moderndamage percentages in the sites we sampled(Supplementary Table 2). Twelve damagetypes in total are recognized in the Longmenflora, with three feeding types that are likely tobe host specific: two in mining and one ingalling (Fig. 5). Only one galling damage type(DT33 in Fig. 3E) occurs in the Longmen flora,characterized by circular to ellipsoidal gallsoccurring on primary veins of the leaf. Thistype is not observed in any modern sample,but it is abundant in the Longmen flora, beingpresent in 5.9% of leaves. This is much higherthan the percentage of other types of galling inany of the extant samples (Fig. 5).

Among all damage categories, leaf marginfeeding and hole feeding are the most abun-dant in the Longmen flora, with five and threedamage types, respectively at 39.4% and 17.4%in terms of frequency. Although the tempera-ture estimated by leaf physiognomy in theLongmen flora is the highest among all sam-ples (Table 1), the number of damage types isunexpectedly the lowest among all investi-gated samples (Fig. 5). Three damage typesbelonging to putatively host-specific categoriesare lacking in the Longmen flora, but found inmodern forests: DT195 (mining; Supplemen-tary Fig. 1Y), DT163 (galling; SupplementaryFig. 1AD), and DT168 (piercing-and-sucking;Supplementary Figs. 1AB and 1AC). Accordingto cluster analysis based on the abundance ofeach damage type, the sample from the Long-men flora falls within the scatter of modernsamples, and is the closest to samples fromHutiaoxia (Site 2) and Nixi (Site 3) in north-western Yunnan (Fig. 5), which are also theclosest in terms of spatial distance betweenmodern samples from the fossil locality.

Discussion

The saturation of the bootstrap curve fordamage diversity, in the large sample of leaves

studied here, makes a strong case that thelower damage diversity in the Longmen flora isnot merely an effect of taphonomy. Instead itappears to represent a genuine difference indamage diversity (Fig. 4). Comparison ofplant-insect interactions from widely scatteredmodern-day sites in our study makes it clearthat leaf damage diversity in HET forests ishigher in warmer climates (Table 2). This resultagrees with a pattern previously observedfrom other plant species (Hódar and Zamora2004) and whole forest communities (Adamset al. 2011) in the present-day world. Addition-ally, our result shows higher leaf damagediversity in wetter climates (Table 2). This isunsurprising, because temperature and preci-pitation have been recognized as the majorabiotic factors influencing the development,survival, abundance, and range of insects (Baleet al. 2002). Therefore, more leaf damagemorphologies are likely formed in regionsunder warmer and wetter climates, because ofthe higher herbivore diversity. Despite theevidence that the climate of the Longmen florawas warmer and wetter than the climates thatmodern-day HET populations inhabit (Su et al.2013), the damage diversity is unexpectedlylower than for the present day (Fig. 6).

We assume that in HET forests, the damagediversity is associated with the diversity of

0 10 1510

12

14

16

18

Mean Annual Temperature (MAT, oC)

Num

ber o

f dam

age

type

s (N

DT)

NDT = 13.1 + 0.3 * MAT

r2= 0.73, p = 6.78 * 10-3

SS

GRS

GT

WSKG

HL

HTX

NX

5

FIGURE 6. Correlation of the damage diversity andmean annual temperature among eight living Quercusguyavaefolia forests. Solid/hollow squares represent theestimated/observed damage diversity of the late PlioceneLongmen flora. See Table 1 for sample site abbreviations.

182 TAO SU ET AL.

insect herbivores. Among all 18 observeddamage types in this study, 11 of them belongto leaf-chewing damage types (SupplementaryTable 2). In the Damage TypeGuide (Labandeiraet al. 2007), most leaf chewing damage typesare described as generalized feeding, meaningthat many different types of insects couldmakethe same leaf-chewing damage type. However,there is still apparently a relationship betweenherbivore diversity and chewing damagediversity of leaves. Recently, Carvalho et al.(2014) studied leaf-chewing damage types in24 host-plant species at two lowland tropicalrainforest sites of Panama. They concluded thatthe number of leaf-chewing damage typeswithin one host plant species correlates closelywith the diversity of insect species feedingon leaves of the host plant (Carvalho et al.2014). Therefore, the number of leaf-chewingdamage types present on leaves of HET likelyreflects the diversity of damage makers. Otherforms of damage types in this study, i.e.,skeletonization, mining, galling, and piercing-and-sucking, require specific mouthpart struc-tures, and they tend to be created by particularinsect species.During the Quaternary, southwestern China

was affected by a series of colder and drierepisodes associated with glacial-interglacialcycles (An et al. 2011). The apparent increasein insect damage diversity over the last 3 Myrsince the Longmen flora was deposited (Fig. 5)therefore indicates that the climate fluctuationsof the Quaternary period did not have anoverwhelming effect on the damage diversitythrough causing massive extinctions amongthe insect fauna in HET forests of southwesternChina. It is always possible to argue that thedamage diversity might have risen still higherhad it not been for the Quaternary climateinstability, but one can at least conclude that noeffect is evident. It seems that in the HETlineage, factors other than climate-inducedextinction have dominated the trend.There is, however, one instance of extinction

of a common damage type present in the fossilflora, a distinctive type of gall (DT33; Fig. 3E).This type of gall is the most frequentlyoccurring (5.9%) among all specialized damagetypes in the Longmen flora, but it is not foundin any of the modern-day samples that span

the modern climate range of HET. We alsochecked more than 200 modern-day herbariumspecimens of Quercus guyavaefolia withoutfinding this galling type. Galls are often formedwith species-specific morphology and narrowhost-plant range (Stone et al. 2008; Knor et al.2012). Shifts in range and bottlenecks in boththe host species and insect caused by climatevariation might have led to this particular gall-making insect’s extinction. It is possible thatthe apparent shift in the climate range of thisoak, from a climate around MAT 17ºC in thePliocene to a maximum MAT around 14ºC inthe present day, has led to an evolutionaryniche shift toward cooler climates in thepresent day, perhaps induced by responses tothe trend of regional cooling during the latePliocene time.

Although there is no evident effect of theQuaternary climate change in depressingdamage diversity in this species, it is importantto note that southwestern China suffered muchless plant extinction during the Quaternarythan other temperate regions in the NorthernHemisphere such as Europe (Lang 1994).Because HET forests have existed and beenwidespread in southwestern China since thelate Pliocene (Tao et al. 2000; Yang et al. 2009;Su 2010), insects in HET forests would havebeen able to migrate to areas within the samevegetation, where the climate was suitable fortheir survival. Thus good opportunities mighthave occurred for survival and migration ofboth herbivores and host during theQuaternary.This would be plausible considering the gen-erally higher rainfall of southwestern China,and the presence of many relatively warmmountain valleys that would have providedrefugia enabling the resilience of both plantsand insects (Huang et al. 2008), as well aswhole ecosystems, under cooling and dryingof the Quaternary. The much more severecontraction of tree ranges that occurredin North America and Europe during theQuaternary (Comes and Kadereit 1998) mighthave resulted in more extinctions of associatedinsect herbivores and a substantial reductionin damage diversity. This hypothesis couldbe further tested in these regions, such asin North America by using California oaks(McElwain 2004).

INSECT HERBIVORY AND PALEOCLIMATE CHANGE 183

The fact that damage diversity in this oaklineage has increased over time is consistentwith the evolutionary accumulation hypothesisfor herbivore diversity. This could be throughthe process of evolutionary adaptation to thehost plant, analogous to that seen on a muchshorter time scale in the apple maggot fly ineasternNorth America (Bush 1969). In a sense, aplant speciesmay be seen as an “island” habitat,which can accumulate successful colonists overtime (Opler 1974), except through adaptationrather than dispersal (Berg et al. 2010). Viewedfrom this perspective, the diversity of herbi-vores attacking the plant may become a balancebetween new evolutionary arrivals and extinc-tion during population bottlenecks. Evolution-ary colonization, and lack of extinction, may beaided by the plant’s having a very large overallpopulation—with a wide climate range andhigh population density. This is effectively theconcept of “apparency” proposed by Feeny(1976). In the time since its earliest record in theQinghai-Tibet Plateau, the HET lineage appearsto have become more abundant in areas whereit occurs. At the earliest fossil localities from15Ma, the HET lineage was mixed with a largediversity of other tree species (Li and Guo 1976).Since the late Pliocene, HET has becomedominant, forming almost monospecific naturalforests (Tao et al. 2000; Su 2010). If the“apparency” of the HET lineage has increased,it is unsurprising that its damage diversity hasbeen increasing, reflecting a more diverse insectherbivore community.

As might be expected, if indeed a plant is an“island” to be colonized and occupied inevolutionary time, we find evidence of turn-over for insect damage in HET. A commongalling type in the late Pliocene Longmen florais now absent, presumably reflecting extinctionof this particular herbivore in the interveningperiod. Conversely, the increase of damagetypes in modern HET forests might reflect new“colonizations” of the host plant by herbivores.Our results indicate that overall insect folivory insouthwestern China was not heavily perturbedby the Quaternary climate cycles. Althoughturnover through extinction has apparently beenpresent, it has not prevented the damagediversity on the HET lineage from increasingover time.

Acknowledgments

We thank G.-F. Li, L. Wang, Q.-S. Yang,Y. Yang, W.-T. Yu, and F.-M. Zhang for theirassistance in sample collections; M. Sun andH. Xu for preparing the clearing leaves ofQuercus guyavaefolia; P. Wilf, M. T. J. Johnson,and M. Donovan for constructive suggestions;and E. D. Currano and B. Jacobs for reviewingthe manuscript. This work is supported byNational Natural Science Foundation of China(31100166, 41030212, and 41272007), the 973Project (2012CB821900), the Foundation of theState Key Laboratory of Paleobiology andStratigraphy (Nanjing Institute of Geologyand Paleontology, Chinese Academy ofSciences [CAS]) (123106, 143107), the WestLight Foundation of CAS, the CAS 135 pro-gram (XTBG-F01), the U.S. National ScienceFoundation (EAR-0746105), and a Heisenberggrant (WA1492/8-1) from the DeutscheForschungsgemeinschaft. This work is a con-tribution to Neogene Climate Evolution inEurasia (NECLIME).

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