Novel Insect Leaf-Mining after the End-Cretaceous Extinction and the Demise of Cretaceous Leaf Miners, Great Plains, USA

Abstract

Plant and associated insect-damage diversity in the western U.S.A. decreased significantly at the Cretaceous-Paleogene (K-Pg) boundary and remained low until the late Paleocene. However, the Mexican Hat locality (ca. 65 Ma) in southeastern Montana, with a typical, low-diversity flora, uniquely exhibits high damage diversity on nearly all its host plants, when compared to all known local and regional early Paleocene sites. The same plant species show minimal damage elsewhere during the early Paleocene. We asked whether the high insect damage diversity at Mexican Hat was more likely related to the survival of Cretaceous insects from refugia or to an influx of novel Paleocene taxa. We compared damage on 1073 leaf fossils from Mexican Hat to over 9000 terminal Cretaceous leaf fossils from the Hell Creek Formation of nearby southwestern North Dakota and to over 9000 Paleocene leaf fossils from the Fort Union Formation in North Dakota, Montana, and Wyoming. We described the entire insect-feeding ichnofauna at Mexican Hat and focused our analysis on leaf mines because they are typically host-specialized and preserve a number of diagnostic morphological characters. Nine mine damage types attributable to three of the four orders of leaf-mining insects are found at Mexican Hat, six of them so far unique to the site. We found no evidence linking any of the diverse Hell Creek mines with those found at Mexican Hat, nor for the survival of any Cretaceous leaf miners over the K-Pg boundary regionally, even on well-sampled, surviving plant families. Overall, our results strongly relate the high damage diversity on the depauperate Mexican Hat flora to an influx of novel insect herbivores during the early Paleocene, possibly caused by a transient warming event and range expansion, and indicate drastic extinction rather than survivorship of Cretaceous insect taxa from refugia.

Full text

Novel Insect Leaf-Mining after the End-Cretaceous
Extinction and the Demise of Cretaceous Leaf Miners,
Great Plains, USA
Michael P. Donovan
1
*, Peter Wilf
1
, Conrad C. Labandeira
2,3
, Kirk R. Johnson
4
, Daniel J. Peppe
5
1Department of Geosciences, Pennsylvania State University, University Park, Pennsylvania, United States of America, 2Department of Paleobiology, National Museum of
Natural History, Smithsonian Institution, Washington, District of Columbia, United States of America, 3Department of Entomology and BEES Program, University of
Maryland, College Park, Maryland, United States of America, 4National Museum of Natural History, Smithsonian Institution, Washington, District of Columbia, United
States of America, 5Department of Geology, Baylor University, Waco, Texas, United States of America
Abstract
Plant and associated insect-damage diversity in the western U.S.A. decreased significantly at the Cretaceous-Paleogene (K-
Pg) boundary and remained low until the late Paleocene. However, the Mexican Hat locality (ca. 65 Ma) in southeastern
Montana, with a typical, low-diversity flora, uniquely exhibits high damage diversity on nearly all its host plants, when
compared to all known local and regional early Paleocene sites. The same plant species show minimal damage elsewhere
during the early Paleocene. We asked whether the high insect damage diversity at Mexican Hat was more likely related to
the survival of Cretaceous insects from refugia or to an influx of novel Paleocene taxa. We compared damage on 1073 leaf
fossils from Mexican Hat to over 9000 terminal Cretaceous leaf fossils from the Hell Creek Formation of nearby southwestern
North Dakota and to over 9000 Paleocene leaf fossils from the Fort Union Formation in North Dakota, Montana, and
Wyoming. We described the entire insect-feeding ichnofauna at Mexican Hat and focused our analysis on leaf mines
because they are typically host-specialized and preserve a number of diagnostic morphological characters. Nine mine
damage types attributable to three of the four orders of leaf-mining insects are found at Mexican Hat, six of them so far
unique to the site. We found no evidence linking any of the diverse Hell Creek mines with those found at Mexican Hat, nor
for the survival of any Cretaceous leaf miners over the K-Pg boundary regionally, even on well-sampled, surviving plant
families. Overall, our results strongly relate the high damage diversity on the depauperate Mexican Hat flora to an influx of
novel insect herbivores during the early Paleocene, possibly caused by a transient warming event and range expansion, and
indicate drastic extinction rather than survivorship of Cretaceous insect taxa from refugia.
Citation: Donovan MP, Wilf P, Labandeira CC, Johnson KR, Peppe DJ (2014) Novel Insect Leaf-Mining after the End-Cretaceous Extinction and the Demise of
Cretaceous Leaf Miners, Great Plains, USA. PLoS ONE 9(7): e103542. doi:10.1371/journal.pone.0103542
Editor: Andrew A. Farke, Raymond M. Alf Museum of Paleontology, United States of America
Received February 28, 2014; Accepted July 3, 2014; Published July 24, 2014
Copyright: ß2014 Donovan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a Geological Society of America Student Research Grant, a Paleontological Society of America Student Research Grant,
and the P.D. Krynine Memorial Fund, Penn State Department of Geosciences (MPD). Additional funding was provided by the American Philosophical Society, a
David and Lucile Packard Fellowship, and NSF Grant DEB-0919071 (PW). The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: Co-author PW is an editor for PLOS ONE. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.
* Email: mpd187@psu.edu
Introduction
One of the largest and most sudden mass extinctions in Earth
history occurred at the end of the Cretaceous period (66.0 Ma),
triggered by an asteroid impact at Chicxulub, Mexico [1–5]. The
paleobotanical record from the Western Interior United States
shows a ca. 30% (pollen) to ca. 57% (southwestern North Dakota
macrofossils) reduction in diversity at the Cretaceous-Paleogene
(K-Pg) boundary [6–9]. The richness of insect-feeding damage on
fossil leaves also decreased ca. 42% across the K-Pg boundary in
North Dakota [10,11]. Mines and galls, typically representing
host-specific interactions, were disproportionally affected in the
extinction [10,11].
Paleocene floras of the Western Interior U.S.A. typically had
low diversity and were dominated by widespread species [12–14].
For ca. 10 million years following the K-Pg extinction, floral
diversity was depressed compared to the latest Cretaceous and
finally began to increase during the latest Paleocene and early
Eocene [15–17]. Insect damage diversity was also low during most
of the Paleocene, but it increased to levels similar to the latest
Cretaceous during late Paleocene warming, preceding the increase
in plant diversity [10,11,16,18–20].
Insect diversity generally correlates with plant diversity today
[21]. Comparably, low diversity paleofloras with low diversity
insect damage have been observed at all regional early Paleocene
localities previously examined for insect damage, with the
exception of two unusual localities: Mexican Hat (southeastern
Montana) and Castle Rock (north-central Colorado) [10,11,19].
Castle Rock has a rainforest-like flora with high plant diversity and
low insect damage diversity [19,22,23]. Mexican Hat has a typical,
low diversity flora, but uniquely, this is paired with highly diverse
insect damage across host plants [19]. Wilf et al. [19] hypothesized
that these two anomalous localities represent decoupled plant and
insect diversity resulting from broken food webs in the wake of a
mass extinction.
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Besides Mexican Hat’s anomalous insect damage diversity, the
locality also marks the earliest evidence for both a plant and an
insect family, providing clear examples of novelty following the K-
Pg extinction. The earliest well-dated evidence for Juglandaceae
(walnut family) is the co-occurrence at Mexican Hat of Polyptera
manningii fruits and Juglandiphyllites glabra leaflets [24,25]. A
leaf mine occurring on Platanus raynoldsii at Mexican Hat,
Phytomyzites biliapchaensis, represents the earliest evidence for
Agromyzidae and, by extension, the encompassing Schizophora
clade of leaf-mining flies [26].
Despite Mexican Hat’s singular importance for understanding
the recovery of plant-insect ecosystems after mass extinction, its
insect damage is only partially described [26,27], aside from an
unpublished thesis examining one plant species [27], and has not
been rigorously compared to the well-preserved damage from
nearby exposures of the latest Cretaceous Hell Creek Formation
[19]. The purpose of this study is to determine whether the insect
damage at Mexican Hat is more likely to represent Cretaceous
insects from refugia or novel Paleocene taxa, and to use this
information for a reassessment of the overall extinction of leaf-
mining insects across the K-Pg boundary. To provide the first
detailed documentation of this spectacular ichnofauna, we first
describe all insect damage at Mexican Hat by host plant, and we
then focus analytically on leaf mines for comparison with local and
regional Cretaceous and Paleocene floras because mines com-
monly preserve various morphological details useful for species-
equivalent comparisons. We also focus on well-sampled surviving
plant families, including Platanaceae and Cercidiphyllaceae, to
determine if they provided a refuge for Cretaceous insects. Links
between the diverse mining fauna at Mexican Hat and local and
regional Cretaceous sites currently are unknown, and determining
if there are connections to the high damage diversity at Mexican
Hat will allow us to reassess the regional severity of leaf miner
extinction across the K-Pg boundary.
Materials and Methods
Ethics Statement
This study was done entirely using previously collected materials
that were permanently deposited in major natural history
museums, as detailed below. No new permits were required for
the described study, which complied with all relevant regulations.
The Mexican Hat locality is a sombrero-shaped, isolated butte
approximately 51 kilometers east of Miles City, southeastern
Montana, in the Hogan Creek Quadrangle at 46.43139uN,
105.24117uW. The site is in the northeastern portion of the
Powder River Basin, in the Lebo Member of the Fort Union
Formation. The Lebo Member locally overlies the Tullock
Member, which lies conformably above the Cretaceous Hell
Creek Formation. The Lebo Member represents an early
Paleocene meandering channel system that deposited sediments
from channels, point bars, crevasse splays, and floodplains [28].
The early Paleocene transgression of the Cannonball Sea in the
Western Interior USA may have raised base level inland, causing
more flooded landscapes with increased soil water concentration
and leading to deposition of mire and ponded-water facies and the
fossilization of swamp and riparian forest communities [6]. The
fossiliferous sediments at Mexican Hat are 2.5 m thick and are
composed mostly of mudstones and thin coals, with fine
sandstones at the base, all deposited in levee and floodplain
environments [27,28]. The flora most likely represents a riparian
forest community adjacent to a meandering channel.
Regarding the age of the flora, there are no paleomagnetic or
radiometric data from the outcrop itself; a possible volcanic ash
collected there by PW did not yield useful phenocrysts (M.E.
Smith, personal communication 2005). However, Belt et al. [29]
reported 40 Ar/39 Ar dates of 64.0–64.7 Ma from two nearby
outcrops in the Lebo Member [29]. The Mexican Hat fossil site is
located approximately 15 m above the contact between the
Tullock and Lebo Members (ES Belt, personal communication
2012). At Signal Butte, Montana, located 40 km west of Mexican
Hat, the magnetic polarity reversal between chrons C29n and
C28r occurs approximately 10 m above the Tullock-Lebo contact
[30]. Because of potential differences in sedimentation rates and
topography, the sediments at Mexican Hat could have been
deposited either during C29n or C28r, whose reversal boundary is
currently calibrated to 64.95 Ma [31]. This is consistent with the
previous 40 Ar/39 Ar ages for the Lebo Member [29] in light of
current calibration constants [32]. Thus, in the absence of more
precise geochronologic constraints, we estimate the age of the
Mexican Hat fossil assemblage to be approximately 65 Ma (1 m.y.
after the K-Pg boundary).
Williams [28] collected the first fossil plant specimens from
Mexican Hat in 1988 for a senior thesis study of depositional
environments of Paleocene plant fossil localities in southeastern
Montana. Her collection consists of ca. 400 specimens and is
curated at the Yale University Peabody Museum of Natural
History (YPM PB. 05250 and PB. 01790). In 1996, Lang [27]
collected Zizyphoides flabella leaves from Mexican Hat for his
doctoral thesis, which analyzed plant-insect associations in the
Western Interior USA ranging from the Late Cretaceous to
Eocene. From Lang’s collection, the 78 specimens curated at the
Denver Museum of Nature and Science were examined (DMNH,
localities 1251 and 1252). Williams and Lang did not perform
quantitative field censuses of the fossils at Mexican Hat, which was
later done by PW and CCL, June 22 to July 2, 2004, resulting in
the largest collection of Mexican Hat plant fossils. In this field
census, 2219 leaves and leaflets were scored for presence or
absence of insect feeding damage types (DTs), and 513 voucher
specimens and 82 fragmentary or indeterminate specimens were
deposited at the Smithsonian Institution National Museum of
Natural History (USNM, locality 42090) [19]. Damage types were
assigned using the ‘‘Guide to Insect (and Other) Damage Types on
Compressed Fossil Plants’’ [33]. Damage types are morphotypes
defined by characters, such as size, shape, tissue alteration,
damage pattern, and placement on the leaf, that are used to
unambiguously categorize insect damage so that it can be
quantitatively analyzed [34]. The number of insect DTs is
positively correlated with the diversity of herbivorous insects that
made the DTs across host plants in modern tropical rainforests in
Panama, which supports the use of DTs for interpreting insect
herbivore richness in the fossil record [35]. We reexamined all
three Mexican Hat collections listed above for this study, and we
rescored the USNM collection for damage (see Tables S1 and S2).
The Mexican Hat flora is dominated by Platanus raynoldsii
(Platanaceae), Juglandiphyllites glabra (Juglandaceae), Zizyphoides
flabella (Trochodendraceae), and Cercidiphyllum genetrix (Cerci-
diphyllaceae), which together comprise ca. 92% of the specimens
[19]. There are 17 documented dicotyledonous angiosperm leaf
species overall (Table 1), and one unknown monocot leaf species.
The most common leaf species at Mexican Hat also are
widespread throughout the Western Interior USA during the
early Paleocene [14]. Fruits include the aforementioned Polyptera
manningii;Joffrea speirsii (Cercidiphyllaceae), the presumed fruits
of the local C. genetrix [36]; Nordenskioldia (Trochodendraceae),
which has been correlated with Zizyphoides leaves [37]; and two
unidentified fruits. Non-angiosperm taxa include leafy branches of
the swamp redwood Glyptostrobus europaeus (Cupressaceae),
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which are very abundant but were not censused, and leaves of the
fern Onoclea hesperia (Woodsiaceae).
Mexican Hat insect damage morphology and composition were
compared to those of the Cretaceous Hell Creek and early
Paleocene Fort Union Formation collections from the nearby
Williston Basin in southwestern North Dakota, the Powder River
Basin in southeastern Montana, and from early late Paleocene
(58–59 Ma) collections available from the Bighorn Basin in north-
central and the Greater Green River Basin in southwestern
Wyoming (Table 2). From North Dakota, KRJ sampled fossil
floras intensively across the K-Pg boundary in a 183 m composite
section representing ca. 2.2 million years of deposition, as detailed
in several papers [6–8,38]. Subsequently, a total of 13,441 of the
resulting museum specimens, from 106 stratigraphic levels, were
scored for insect damage [11], and these are re-examined here,
9292 from the Cretaceous and 4149 from the Paleocene. These
specimens are housed at DMNH and YPM. Collections made by
DJP from early to middle Paleocene strata in the Williston Basin in
North Dakota (2916 specimens at YPM [14,39]; 66.0–58.2 Ma,
calculated with time scale from [31] and sedimentation rates from
[40]), as well as fossils from 14 early Paleocene sites in the Powder
River Basin in Montana (1280 specimens at DMNH and YPM
[39]; 65.1–61.8 Ma, calculated with time scale from [31] and
sedimentation rates from [30]) were examined for insect damage
for the first time in this study. For broader regional and temporal
context, the leaf fossils from early late Paleocene localities in
Wyoming (59.0–57.5 Ma) collected by PW and CCL and scored
for insect damage in [19] were also reexamined. Voucher
specimens preserving all insect damage types found on all host
plants from field-census tallies at these Wyoming sites are curated
at USNM and include 648 specimens from the Greater Green
River Basin (USNM localities 41687 (Persites Paradise), 41691
(Kevin’s Jerky), and 41694 (Haz-Mat)), and 440 specimens from
Polecat Bench in the Bighorn Basin (USNM localities 42041
(Skeleton Coast) and 42042 (Lur’d Leaves)), as described in
reference [19].
We described all insect damage at Mexican Hat by host plant
(except for previously described agromyzid mines [26]) and
focused on leaf mines in the analysis, because they preserve
morphological features that allow for detailed comparisons. These
morphological features include [41,42] 1) overall shape, size and
length of the mine; 2) oviposition site structure; 3) changes in mine
and frass trail width; 4) position of the mine on the leaf; 5) overall
mine trajectory; 6) features of the mine border where it contacts
unconsumed leaf tissue; 7) degree of restriction of the mine based
on vein rank; 8) frass type, whether fluidized or particulate, and if
particulate, pellet structure; 9) frass-trail continuity, including the
presence of sap-feeding (fluidized frass) and whole-tissue con-
sumption (particulate frass); 10) type of foliar tissue mined; and 11)
size, shape, and structure of the terminal chamber, including
whether an emergence hole or slit is present. Generalized DTs,
such as hole feeding and margin feeding, are made by a variety of
insect orders and cannot usually be attributed to specific insect
herbivores; thus, their low extinction across the K-Pg is not likely
to be biologically meaningful [11], although Carvalho et al. [35]
found a decrease in host-specificity for these damage types. Gall-
inducing insects are typically host-specialized, but morphological
characteristics of galls on compression fossils are sometimes
obscured, often because their three-dimensional structure is
flattened, making detailed comparisons difficult. Mines are made
by four orders of living insects: Coleoptera, Diptera, Hymenop-
tera, and Lepidoptera, and they can often be attributed to order,
or even family [26,41,43–45]. In this study, the morphologies of
leaf mines from all examined collections (Table S3) were
compared to determine whether they were unique, among known
fossil collections, to the Mexican Hat leaf-mining fauna.
We also focused on well-sampled surviving plant families to
determine whether they provided refuge for insects after the K-Pg
extinction. We compared insect damage on twenty-four Platana-
ceae species from the Hell Creek Formation (1397 specimens) to
that on Paleocene Platanus raynoldsii (933 specimens; 724 from
local early Paleocene; 214 from Mexican Hat, 41 from late
Paleocene, WY). Also, we compared insect damage on five
Cercidiphyllaceae species (323 specimens) from the Hell Creek
Formation to Paleocene Cercidiphyllum genetrix (556 specimens;
Table 1. Angiosperm leaf species at Mexican Hat ranked by abundance in 2004 census [19].
Family Species Abundance in 2004 census Percentage
Platanaceae Platanus raynoldsii Newberry 1207 53.4%
Juglandaceae Juglandiphyllites glabra Brown ex Watt 396 17.5%
Trochodendraceae Zizyphoides flabella (Newberry) Crane, Manchester and Dilcher 231 10.2%
Cercidiphyllaceae Cercidiphyllum genetrix (Newberry) Hickey 214 9.5%
Lauraceae Lauraceae sp. 2 Wilf et al. 2006 87 3.9%
Unknown affinity ‘‘Populus’’ nebrascensis Newberry 85 3.8%
Unknown affinity Dicot 1 Wilf et al. 2006 12 ,1%
Nyssaceae Browniea serrata (Newberry) Manchester and Hickey 12 ,1%
Unknown affinity ‘‘Ficus’’ artocarpoides Lesquereux 4 ,1%
Unknown affinity cf. Ternstroemites aureavallis Hickey 2 ,1%
Lauraceae Lauraceae sp. 1 2 ,1%
Unknown affinity Paleonelumbo macroloba Knowlton 2 ,1%
Unknown affinity Paranymphaea crassifolia Newberry 2 ,1%
Unknown affinity Dicot 2 Wilf et al. 2006 1 ,1%
Unknown affinity Dicot 3 Wilf et al. 2006 1 ,1%
Unknown affinity Dicot 4 Wilf et al. 2006 1 ,1%
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374 from local early Paleocene; 52 from Mexican Hat, 130 from
late Paleocene, WY).
Macrophotographs were taken using a Nikon D90 camera at
DMNH, USNM, YPM, and the Paleobotany Laboratory,
Pennsylvania State University. Microphotography was performed
in the Paleobotany Laboratory, Pennsylvania State University,
using a Nikon DS-Ri1 camera mounted on a Nikon SMZ 1500
binocular microscope. Images were processed using Nikon NIS
Elements v. 3 software. The Photoshop CS5 and CS6 Align and
Blend functions were used to vertically composite images of fossils
with uneven surfaces as needed. Composite images were carefully
checked for artifacts. Adobe Camera Raw Editor was used to
reversibly adjust white balance, temperature, contrast, etc. as
needed on whole images.
The Mexican Hat Insect Damage Fauna
Insect damage is presented by host plant, in decreasing rank
leaf-abundance order as shown in the 2004 field census [19]
(Table 1).
Platanus raynoldsii
Insect damage for this host plant is illustrated in Figures 1–3,
excepting previously described agromyzid mines [26]. External
foliage feeding on P. raynoldsii includes hole feeding, margin
feeding, skeletonization, and surface feeding (Fig. 1). Circular holes
(DT1, DT2 of [33]) range in diameter from 0.67–4.85 mm
(Fig. 1B). Oval holes (DT4) average 7.56 mm long by 2.50 mm
wide and are bounded by tertiary veins (Fig. 1C). Polylobate holes
(DT3, DT5) range in length from 3.60–8.95 mm and 2.10–
6.15 mm in width (Fig. 1E). Elongate holes include curvilinear,
parallel-sided slots, which thin to a point (DT8; 1.15–2.55 mm
L60.21–0.36 mm W; Fig. 1D); reaction rims are typically
0.07 mm wide. Margin feeding damage is also present, including
semicircular incisions across primary veins (DT12), ranging from
7.20–12.63 mm wide and 1.30–4.61 mm deep (Fig. 1E), and
incisions extending towards the midvein (DT15) measuring 1.20–
3.59 mm in width and 11.5 mm deep (Fig. 1A). Reaction rims
associated with margin feeding range in width from 0.30–
0.95 mm.
Platanus raynoldsii has five skeletonization DTs, typically in
circular or oval patterns with no reaction tissue (DT16; Fig. 1F).
When reaction tissue is present along the edges of skeletonized
areas, it averages 1.16 mm in width (DT17; Fig. 1G). Rectangular
patches of skeletonized tissue (DT19; Fig. 1H) typically cross major
veins, but some are also bounded by major veins (between the
midvein and two secondary veins). These rectangular patches of
skeletonized tissue resemble damage made by extant leaf-rollers or
tiers [46]. Skeletonized tissue that crosses primary veins at the base
of the leaf is also present (DT56; Fig. 1F). Fourth and even fifth
order venation is typically preserved in skeletonized areas of P.
raynoldsii (Fig. 1F,G, H). Circular surface feeding marks measure
0.77–2.22 mm in diameter, with reaction rims ranging from 0.16–
0.39 mm wide (DT29; Fig. 1I). A pattern of thin millimeter-wide
lines alternating in color from light to dark is present on some
specimens (DT23; Fig. 1J). The cause of this damage is unknown,
but it may represent viral damage [47], fungal marks, or parallel
surface feeding traces.
Circular piercing and sucking marks with central depressions
(DT46; Fig. 1K) on P. raynoldsii range in diameter from 0.08–
0.17 mm and are clustered in circular groups and curvilinear rows.
Three gall DTs are present on P.raynoldsii. Circular galls with
thickened outer rims surrounding unthickened tissue (DT11;
Fig. 1L) are positioned on intercostal regions and also adjacent to
primary and secondary veins. The DT11 gall diameters range
Table 2. Collections and repository information.
Formation Basin State Age (Ma) Localities Specimens Repository Citation
Fort Union Bighorn Basin WY 59.0 and 57.5 2 440 USNM Wilf et al., 2006
Fort Union Greater Green River Basin WY 59.0 3 648 USNM Wilf et al., 2006
Fort Union Powder River Basin MT 65.1–61.8 14 1280 DMNH, YPM Peppe, 2009; Peppe et al. 2011
Fort Union Powder River Basin MT 65 1 1073 DMNH, USNM, YPM Williams, 1988; Lang, 1996; Wilf et al., 2006
Fort Union Williston Basin ND 66.0–58.2 102 7065 DMNH, YPM Johnson, 2002; Peppe, 2010
Hell Creek Williston Basin ND 67.0–66.0 106 9292 DMNH, YPM Johnson, 2002
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between 1.59–2.37 mm, and the unthickened inner tissue ranges
from 0.50–0.75 mm. Other DTs include indistinct, dark, circular
galls positioned on intercostal tissue and primary veins (DT32,
DT33; Fig. 1A,M); these galls lack the unthickened inner region
that characterizes DT11 and range in diameter from 0.84–
1.50 mm. A three-dimensionally preserved circular gall (DT32;
Fig. 1N) is positioned at the intersection of a primary vein and a
secondary vein, and it measures 4.3 mm in diameter.
At least three and probably four mine DTs appear on P.
raynoldsii. The previously described Phytomyzites biliapchaensis
[26] is an Agromyzidae (Diptera) leaf mine characterized by an
irregular to serpentine or linear path with a central or more
typically an alternating frass trail composed of fluidized specks or
Figure 1. Non-mining insect damage on
Platanus raynoldsii
at Mexican Hat, early Paleocene, Montana. A: Margin feeding at leaf apex to
midvein (DT15) and dark, circular galls on interveinal tissue (DT32; arrow expands to M) and adjacent to secondary veins (DT33; USNM 560105). B:
Circular holes (DT2) and skeletonized areas (DT16) with well-developed reaction rims and minimal tertiary venation preserved (USNM 560106). C: Oval
(DT4) and polylobate (DT3) holes bounded by tertiary veins (USNM 560107). D: Parallel-sided slots (DT8), which thin to a point (USNM 560108). E:
Multiple margin feeding events characterized by shallow, semicircular incisions (DT12) and a large polylobate hole with possible secondary feeding
event (arrow) on upper right side of the hole (DT5; USNM 560109). F: Skeletonized areas lacking reaction tissue (DT16) and crossing primary veins at
the leaf base (DT56; USNM 560110). G: Skeletonized tissue with reaction rim adjacent to secondary veins and preserving fifth order venation (DT17;
USNM 560111). H: Rectangular skeletonized area (DT19) bounded by primary and secondary veins (USNM 560112). I: Circular surface-feeding areas
(DT29) with darkened reaction rims (USNM 560113). J: Parallel-sided, alternating light and dark lines of unknown origin; possibly viral damage, parallel
lines of surface feeding, or fungal damage (DT23; USNM 560115). K: Curvilinear rows of dark, circular piercing and sucking marks with central
depressions (DT46). Detail of piercing and sucking marks provided in inset (USNM 560114). L: Galls with thickened outer rims surrounding
unthickened tissue (DT11) positioned on intercostal tissue and adjacent to secondary veins (USNM 560116). M: Detail of galls in (A) (DT32; USNM
560105). N: Three-dimensionally preserved gall near intersection of primary and secondary veins (DT32; USNM 560117).
doi:10.1371/journal.pone.0103542.g001
Figure 2. Association between
Platanus raynoldsii
and a lepidopteran leaf miner (DT91) at Mexican Hat. A: Near-complete leaf mine
between two secondary veins (arrow expands to B; USNM 560118). B: Detail of terminal frass trail in (A) showing ellipsoidal pellets. C: Detail of frass
trail in (D) showing ellipsoidal pellets packed near the margin of the expanded terminal mine. D. Complete mine with initial, gradually widening trail
and expanded terminal path (USNM 560119; arrow expands to C). E. Mine delimited by secondary and tertiary venation (USNM 560120; arrow
expands to F). F. Detail of frass trail in (E), showing ellipsoidal frass pellets. G: Mine with tightly coiled path and blotch-like appearance with ellipsoidal
frass adjoining the mine margins (USNM 560113). H. Partially-preserved mine showing gradual widening during the early phase (USNM 498156).
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Figure 3. Sawfly (Tenthredinidae) blotch-mines (A–D; DT36) and a probable lepidopteran mine (E) on
Platanus raynoldsii
.A: Two
blotch mines near the leaf base (USNM 498155). B: Closeup of blotch mine showing dispersed frass pellets (USNM 560121). C: Closeup of blotch mine
on probable young leaf, showing increase in frass size from right to left (USNM 560122). D. Detail of frass pellets from the left blotch mine in (A). Each
box depicts a different frass size, starting from the smallest (1
st
instar; upper left box) to largest (4
th
instar; lower right box) (USNM 498155). All four
pictures are at the same scale for size comparison. E: Probable serpentine mine with parallel-sided frass trails (DT282; YPM 65939A).
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segments, whose only, and abundant, occurrence is at Mexican
Hat on Platanus leaves.
The second mine DT (Fig. 2) was previously assigned to DT91
[19]. The mines are characterized by a serpentine path with a
widened terminal section and ellipsoidal frass pellets. Frass
typically increases in size through the mine’s course, ranging from
15–130 mm length and 10–75 mm width. The initial portion of the
mine is serpentine, usually arising in intercostal tissue. It ranges in
width from 0.32–0.37 mm, and frass fills the central 41–71%
(Fig. 2). The frass pellets are initially spheroidal and measure
15 mm in diameter (min = 11, max = 20). The serpentine path
then continues to widen, reaching 0.66–1.59 mm with frass filling
the middle 65%. In most specimens, the mine increases greatly in
width during the terminal phase (Fig. 2A, D). The width increase
takes two forms: a winding path between tertiary veins bounded by
primary or secondary venation (Fig. 2A, E) or a large expansion
into a blotch-like mine, with clusters of frass deposited near the
mine margins (Fig. 2D, G). Mine paths of the first terminal phase
type (a winding pattern between tertiary veins) range from 1.53–
2.16 mm in width and are controlled by tertiary venation. Frass
fills the center 38–77% of the mines, and frass trails are 0.83–
1.19 mm wide. Frass pellets are ellipsoidal and average 0.095 mm
in diameter (min = 0.069, max = 0.13 mm) by 0.052 mm (min
= 0.038, max = 0.073). The larvae that produced the winding
paths between tertiary veins removed tertiary venation at crossing
points (Fig. 2F). Mine paths of the second terminal phase type
(elongate blotch-like mine) are 2.22–7.89 mm wide. Secondary
veins bound the widest portions (Fig. 2D, G). The frass trails in
serpentine sections of the mines are usually continuous but
discontinuous in the wider, blotch-like portions (Fig. 2D, G). Frass
usually is deposited in clusters near the mine margin (Fig. 2C).
Mine courses are strongly influenced by primary, secondary, and
tertiary venation throughout their length. Early portions of the
mines tend to turn with primary and secondary veins and cross
these major veins only where they are thin. Veins delimit the
margins of mines, giving them a wavy appearance. There is no
reaction tissue on any of the examined specimens.
The third mine DT on P. raynoldsii is a blotch mine assigned to
DT36 (Fig. 3) [19]. There are five specimens with this association,
including a large leaf with two mines, one on each side of the
midvein and bordering the leaf margin (Fig. 3A), another large leaf
with one mine that is positioned between the midvein and a first
secondary vein, crossing a lateral primary vein (Fig. 3B), and a
small, possibly young leaf with one mine crossing the midvein and
present throughout a majority of the leaf blade (Fig. 3C). Frass
pellets are ellipsoidal and dispersed throughout the mines. There
are four discrete frass sizes presumed to represent instar growth
phases, and similar sizes are clustered together (Fig. 3D). The
smallest frass pellets are spheroidal (51 mm in diameter, standard
deviation of 66mm) and are packed close together. The second,
third, and fourth sizes are ellipsoidal and measure 13369mm
L67467mm W, 174612 mmL695610 mm W, and 239629 mm
L6148615 mm W, respectively. The general path of the larvae
can be observed by following the width increases, and the distance
between frass pellets tends to increase with frass pellet size. Major
veins tend to control mine margins, but some portions end in
interveinal tissue. There is no reaction tissue along the mine
margins.
A probable mine is found on a poorly preserved, fragmentary
leaf fossil of P.raynoldsii and is defined by a serpentine path with
darkened margins, representing either reaction tissue or frass
(DT282; Appendix S1; Fig. 3E). The mine varies in width from
1.10–1.80 mm, and there is no apparent widening trend. The
darkened marginal areas vary in width from 0.33–0.84 mm and
comprise around 70% of the overall mine width. Mine path and
margins are not influenced by venation except for short sections
along the midvein, which suggests that the mine was embedded in
epidermal tissue. The beginning and end of the mine are not
preserved.
Juglandiphyllites glabra
Insect damage for this host plant is illustrated in Figures 4–6.
Because Polyptera manningii and Juglandiphyllites glabra at
Mexican Hat together represent the oldest reliable evidence for
Juglandaceae [24,25], this study likewise documents the earliest
evidence for herbivory on Juglandaceae. Juglandiphyllites glabra
includes the greatest number of distinct mine associations (at least
four and probably five; all probable lepidopteran mines) of any
plant species at Mexican Hat.
External foliage feeding on J. glabra (Fig. 4) includes hole
feeding, margin feeding, and skeletonization. Circular and oval
holes (DT1, DT2, DT4; Fig. 4B) range in diameter from 0.50 to
13.48 mm, with reaction rim widths averaging 0.17 mm. Poly-
lobate holes (DT3, DT5; Fig. 4C) include one measuring 3.79 mm
in length and bounded by primary, secondary, and tertiary
venation. One specimen has elongate, parallel-sided slots with
smooth margins (DT8; Fig. 4D); the slots vary in width from 0.20–
0.57 mm and are partly influenced by secondary and tertiary
venation. Margin feeding was only found on one specimen of J.
glabra (Fig. 4A) and is characterized by two adjacent shallow,
semicircular incisions along the margin (DT12) measuring around
10 mm wide by 4.5 mm deep, with a 0.09–0.60 mm wide reaction
rim. Only two specimens were found to have skeletonization: one
with patches of skeletonized tissue, with no reaction rim and fifth
order venation preserved (DT16; Fig. 4E), and another specimen
exhibiting a rectangular, elongate skeletonized area (DT19;
Fig. 4F) following the midvein. The skeletonized area decreases
in width apically, starting at 11.76 mm and decreasing to
2.52 mm, before a sudden width increase to 7.34 mm. The
portion closest to the leaflet apex is not preserved. Wide, elongate
skeletonization is typical of lepidopteran leaf-roller or tier larvae
[46], and the changes in width may be related to instar
development.
Piercing and sucking marks are found throughout two leaflets
(Fig. 4G). The marks are dark and circular with a central
depression (DT46) and are randomly distributed. They range in
diameter from 0.18–0.24 mm.
Two J. glabra leaflets were found with galls. One gall is circular
with an outer rim of thickened tissue (DT11; Fig. 4H) measuring
0.50 mm in diameter. Leaflet venation is visible in the center of
the gall. The gall measures 0.33 mm in diameter. The second
specimen (Fig. 4I) has a single, dark, circular gall (DT32)
measuring 1.0 mm in diameter. The outer rim is black and
surrounds an inner gray area. Within the gray area in the center of
the gall is a black region, possibly representing the chamber,
similar in color to the rim. This gall is positioned on interveinal
tissue. The galls on J. glabra are superficially similar to some galls
on modern Carya (Juglandaceae) made by gall midges (Cecido-
myiidae) [48].
Among the lepidopteran-type mines on J. glabra, one was
previously assigned to DT105 [19] and occurs on a single
specimen (Fig. 4J). The mine is serpentine and characterized by
parallel sides, angular turns, a semicircular terminus, and a lack of
frass. The miner may have been a sap-feeder, i.e. a consumer of
cell protoplasts and not the cell walls that frequently are processed
as fecal pellets. The oviposition site is next to a basal secondary
vein (Fig. 4J, arrow). The initial portion is 0.19 mm wide and
follows a serpentine path before following the basal secondary
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Figure 4. Insect-feeding damage on
Juglandiphyllites glabra
at Mexican Hat. A: Shallow, semicircular margin feeding (DT12; arrow; USNM
560123). B: Oval hole (DT4) delimited by secondary and tertiary veins (USNM 560124). C: Polylobate hole (DT5) bounded by primary, secondary, and
tertiary veins (USNM 560125). D: Parallel-sided, elongate slot feeding, influenced by major veins (DT8), or possibly an abiotic tear (DMNH 35833). E:
Patches of skeletonized tissue lacking reaction rim (DT16; USNM 560126). F: Rectangular skeletonized area (DT19; USNM 560127). G: Piercing and
sucking marks with central depressions (DT46). Detail of marks in inset (USNM 560128). H: Gall with thickened outer rim and possible exit hole at the
center (DT11) (USNM 560129). I. Gall on interveinal tissue (DT32) characterized by a darker outer rim and center (USNM 560130). J: Serpentine mine
lacking frass, with a gradual width increase and strong association with major veins (DT105). Arrow marks the oviposition site (USNM 498159). K:
Probable mine with parallel frass trails (DT282; YPM 65939).
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Figure 5. Additional mines on
Juglandiphyllites glabra
at Mexican Hat. A: Intestiniform mine with initially tightly packed frass trail and
subsequent looser deposition of spheroidal frass pellets (DT91). Box indicates area expanded in the inset, showing detail of the frass trail (USNM
560131). B: Sinusoidal mine with loosely packed frass trail (DT91; USNM 560132). C: Linear mine with loosely packed frass trail (YPM 65888). D:
Serpentine mine with sparse, spheroidal frass pellets and gradual width increase (YPM 65848). Detail of frass trail from counterpart in inset E:
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vein. It crosses the basal secondary vein near the primary vein,
follows the primary vein, and then turns along the next secondary
vein. The mine then crosses the secondary vein and extends
linearly across interveinal tissue. Finally, the mine meets the next
secondary vein, and follows it to termination. There is no major
width increase or evidence of a pupation chamber, so the mine
may have been aborted. The mine path is greatly affected by
major but not tertiary veins, and it usually turns and follows the
veins at first contact. There is a steady width increase throughout
the mine length, reaching 0.95 mm in width by the terminus.
Mine margins are smooth, and there is a thin reaction rim
measuring 0.15 mm surrounding the mines.
A second mining association on J. glabra, found on four
specimens (Fig. 5A–D), was previously assigned to DT91 [19]. The
mines are characterized by a serpentine path with loosely packed,
and locally sparse, spheroidal frass pellets. The initial portion of
the mines are serpentine or intestiniform and may have
overlapping sections whereby the earlier path is occupied by later
stages. The earliest frass trail is 0.21 mm wide on one specimen
(Fig. 5A), but overall mine width in this portion is unknown
because of overlapping mine paths. The earliest frass trail is also
not preserved on the other specimens. Mine width increases very
gradually throughout the course of the mine (min = 0.38 mm;
max = 2.33 mm), but the width increase is most dramatic near the
oviposition site (Fig. 5D). The subterminal frass trail varies from
0.29–1.13 mm wide. Frass trails are composed of individual
spheroidal frass particles, filling 25–70% of the mine width and
often adjoining the outside mine margin at turns. Frass does not
change in size through the length of the mine (early frass diameter
=5369mm; latest frass = 5168mm). The terminal 2–3 mm of
the mines contain no frass. All sections of the mine have wavy
borders and no reaction tissue. Borders are controlled by tertiary
venation in some areas, but the mines cross over both secondary
and tertiary veins (Fig. 5A–D).
A third mining association on J. glabra was assigned to DT92
[19] (Fig. 5E–F). It is preserved on a fragmentary leaflet, although
the mine is nearly complete. The mine is serpentine and fully
packed with spheroidal frass pellets. It increases in width
throughout, starting at 0.16 mm wide and terminating at
1.58 mm wide. Frass pellets fill the width of the mine, although
frass is more densely packed near the margins than the center
(Fig. 5F). The densely packed, marginal frass trails comprise about
25% of each side of the mine. Frass increases in diameter through
the length of the mine (from 0.11 to 0.18 mm). Tertiary veins do
not guide the mine, but it does follow the margin and the primary
and secondary veins. The mine margin is smooth, and there is no
reaction rim. A mine on another specimen of J.glabra (Fig. 5G)
was also assigned to DT92, and it may represent a poorly
preserved example of the same association (Fig. 5G). The early
portion of the mine is serpentine, and it then follows a secondary
vein in the direction of the leaf base. The mine then crosses the
secondary vein and reverses direction, following the same vein
distally. Frass pellets are spheroidal and measure 0.0760.01 mm
in diameter. No frass size difference is observed through the length
Sinusoidal mine packed with spheroidal frass pellets (DT92; arrow expands to F; USNM 498157). F: Detail of frass trail in (E), showing densely packed
frass along the margins, and slightly looser frass in the center of the mine. G: Mine following secondary venation, with small, spheroidal frass pellets
packed along mine margins (DT92; USNM 560124).
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Figure 6. Damage type 42 mines from Mexican Hat and the latest Cretaceous of North Dakota, USA. A: Dramatically expanding mine,
following primary and secondary venation, with ragged margins on Juglandiphyllites glabra at Mexican Hat (USNM 560133). B: Short, probable mine,
with initial linear phase and expanded terminal chamber on Liriodendrites bradacii (Battleship – DMNH loc. 900; DMNH 7317). C: Probable mine with
gradual width increase and fairly smooth margins on HC32 (Terry’s HCIIa Site – DMNH loc. 2097; DMNH 19984).
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of the mine. There is no reaction rim along the margins. Like the
specimen in Fig. 5E–F, frass is packed densely on both sides of the
mine. The mines differ in that there is no frass packed in the
middle of the mine in the specimen shown in Figure 5G.
A fourth mine DT on J. glabra, assigned to DT42 [19], also
lacks frass but differs from DT105 in that both tertiary and major
venation affect the mine path and margins (Fig. 6A). The
oviposition site is adjacent to the midvein. The mine dramatically
widens to 0.72 mm and turns away from the primary vein,
following the first intersecting secondary vein for the rest of the
preserved portion, widening throughout to a maximum observed
width of 3.83 mm. The mine terminus was not recovered. The
mine margin located in the intercostal area is wavy and delimited
by quaternary veins. There is no reaction tissue preserved.
A probable fifth mine DT on J. glabra is characterized by a
serpentine path with two dark lines along the margins, represent-
ing reaction tissue or frass (DT282; Appendix S1; Fig. 4K). The
mine is around 0.85 mm wide throughout, and there is no major
width increase. The dark areas along the margin range in size
from 0.19–0.37 mm, taking up 22–44% of the overall width of the
mine. Leaf venation does not influence mine path, which suggests
that the mine traveled through epidermal tissue. The proximal and
terminal portions of the mine were not preserved.
Zizyphoides flabella
Insect damage for this host plant is illustrated in Figures 7–8.
We note that Lang (1996) censused insect damage on 295
specimens of Z. flabella at Mexican Hat and noted high levels of
overall herbivory (48.5% leaves damaged), skeletonization
(31.9%), and mining (3.1%). In the present study, external foliage
feeding on Z. flabella (Fig. 7A–E) includes hole feeding, margin
feeding, skeletonization, and surface feeding. Circular holes on Z.
flabella range in diameter from 0.33–1.48 mm (DT1, DT2;
Fig. 7E), with reaction rims ca. 0.18 mm wide. Polylobate holes
range from 1.74–6.94 mm long by 2.22–4.21 mm wide (DT3,
DT5; Fig. 7C, E, F), and they include one hole with uneaten veinal
stringers (Fig. 7C). An elongate hole lacking parallel sides (DT7;
Fig. 7E, arrow) appears adjacent to a primary vein, measuring
5.5760.45 mm. Two margin feeding specimens are characterized
by a polylobate incision into the margin (DT12), and an incision
extending to a secondary vein measures 3.26 mm wide by
4.37 mm deep (DT15; Fig. 7E).
Zizyphoides flabella is associated with three skeletonization
damage types. The most common of these are large skeletonized
areas lacking reaction tissue (DT16; Fig. 7A), with third to fourth
order venation intact. The veins of some specimens appear darker
and thickened in skeletonized areas, suggesting a reaction to
feeding (DT17). Skeletonization is also present at the bases of
leaves (DT56; Fig. 7A, B), crossing all venation. Small patches of
surface feeding (DT29; Fig. 7D) bound by tertiary venation are
also present, with fourth order venation intact.
Piercing and sucking marks on Z. flabella are positioned in
curvilinear rows. The marks are dark and circular and have
central depressions (DT46; Fig. 7F). They measure 0.13 mm in
diameter.
Four gall damage types are found on Z. flabella (Fig. 7G–H),
including circular galls with dark, thickened tissue surrounding a
lighter colored, circular area at the center (DT11), possibly
representing exit holes. The internal area for these examples of
DT11 measures 0.29–0.71 mm in diameter, and the overall gall
diameter is 0.84–1.26 mm. Many galls are positioned close
together with their thickened, outer tissues overlapping (Fig. 7G),
and may actually be compound galls, consisting of multiple
chambers within the same, structurally confluent gall. Black
circular galls lacking a distinct rim are found on interveinal tissue
(DT32) and against primary (DT33) and secondary veins (DT34);
these galls have diameters of 0.34–0.71 mm. (Fig. 7H).
Zizyphoides flabella has two mining associations. The first was
assigned to DT91 [19], and there are six specimens of this
interaction (Fig. 8A–E). The mine is serpentine and typically
oviposited near a primary vein. It follows the vein and loops
around to the adjacent primary vein (Fig. 8A, D, E). Initial width is
0.45–0.60 mm, with a centered frass trail taking up 35–50% of the
mine. Individual frass pellets are spheroidal–ellipsoidal and
0.045 mm in diameter. The width of the mine increases
throughout, ending at 1.1–1.9 mm. The percentage of the mine
taken up by frass tends to decrease with width, ranging from 30–
40%, although the ratio does not change in some specimens. Frass
size increases towards the end of the mine, ranging in length from
0.05–0.09 mm and width from 0.041–0.082 mm (average
0.05760.01; Fig. 8B, C). The frass trail terminates at the end of
the mine. The paths of the mines are affected by primary and
tertiary venation, and veins border mine margins. Tertiary
venation causes the mine margins to be wavy, and the width of
the mine decreases when crossing tertiary veins (Fig. 8B). Reaction
tissue width outside the mine margins measures 0.09 mm.
The second mine association on Z. flabella was assigned to
DT41 [19], and only one specimen with this association is known
(Fig. 8F–H). The mine is serpentine and initially threadlike,
averaging 0.10 mm in width (Fig. 8H). It expands in width
throughout its length, ending at 0.24 mm wide. The mine typically
borders or crosses primary and secondary veins. The frass is non-
particulate and fills the width of the mine, although frass appears
to have been lost during preservation in some parts of the mine
(Fig. 8H). The frass trail loosens near the leaf margin and widens
to 0.95 mm (Fig. 8G), but it thins out again to 0.24 mm at the end.
The margins are smooth throughout the length of the mine. The
early, threadlike part of the trail is depressed from surrounding leaf
tissues, suggesting that it was probably a full depth mine, with all
the tissue between the upper and lower epidermises removed by
the leaf miner. We note that Lang ([27]; his Figure 6.1.4 A–C)
figured an apparently distinct blotch mine with ellipsoidal frass
pellets on Z. flabella. Because we did not examine this specimen, it
is not included in data analyses.
Cercidiphyllum genetrix
Insect damage for this host plant is illustrated in Figures 9–10.
External foliage feeding on C. genetrix includes hole feeding,
margin feeding, and skeletonization (Fig. 9). Circular holes range
in diameter from 0.65–3.36 mm (DT1, DT2; Fig. 9A). A single
three-lobed hole measures 2.84 mm between the two most distant
points (DT3; Fig. 9A). Reaction rims for all holes measure ca.
0.18 mm width. Margin feeding associations include a semicircu-
lar, shallow incision (DT12; Fig. 9B) measuring 8.62 mm wide and
3.43 mm deep, and a deep incision extending towards the midvein
(DT15; Fig. 9C) measuring 6.76 mm wide and 21.25 mm deep.
Skeletonized tissues include circular areas with no reaction
tissue (DT16) that are located between primary veins at the leaf
base (DT56; Fig. 9D). Tertiary venation is visible, but higher
venation was either removed by the insect’s feeding or by
taphonomic processes. Surface feeding with reaction tissue occurs
at the intersection between a primary and secondary vein and
across a primary vein (DT30; Fig. 9E). The damaged area on the
primary vein is 1.56 mm in diameter and is surrounded by a
polylobate reaction rim measuring 0.12 mm wide. There are also
curved, parallel swaths that travel from the leaf base to the apex
and diverge in opposite directions along a primary vein (DT23;
Fig. 9F). The alternating light and dark lines measure 1 mm in
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width. The lighter lines have a white film on the grains, and some
individual grains have both white and brown coloration. The
cause of these marks is unknown, but they may represent viral
damage [47], parallel surface feeding, or fungal damage.
The piercing and sucking marks on C. genetrix are typically
dense and spread throughout leaf. They are characterized by
circular punctures with a central depression, filled in with dark
carbonized material (DT46; Fig. 9D). There are a variety of
puncture sizes, ranging from 0.09–0.44 mm in diameter.
Circular galls characterized by a darkened outer rim, surround-
ing a lighter inner area representing unthickened tissue (DT11;
Fig. 9G), are positioned on secondary veins and interveinal leaf
tissue. The total diameter ranges from 0.80–1.13 mm, and the
diameters of the inner area range from 0.13–0.24 mm. Indistinct,
Figure 7. Non-mining insect damage on
Zizyphoides flabella
at Mexican Hat. A: A nearly completely skeletonized leaf lacking reaction tissue
(DT16; USNM 560134). B: Skeletonization crossing primary veins at the leaf base (DT56; USNM 560135). C: Polylobate hole (DT5) delimited by tertiary
venation and exhibiting uneaten veinal stringers (USNM 560136). D: Surface feeding (DT29) delimited by tertiary veins (USNM 560137). E: Polylobate
holes (DT3, DT5) and an elongate hole lacking parallel sides (DT7; arrow) near the base. Also, note the small margin feeding incision (DT15) on the left
side (USNM 560138). F: Curvilinear swaths of piercing and sucking marks with central depressions (DT46). Arrow indicates area expanded in the inset,
showing detail of the piercing and sucking marks (USNM 560139). G: Galls with thickened outer rims and possible exit holes at the centers (DT11;
USNM 560140). H: Dark, circular galls on interveinal tissue (DT32), primary veins (DT33), and secondary veins (DT34) (USNM 560141).
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black circular galls are also observed on C. genetrix. The galls are
positioned on interveinal tissue (DT32), primary veins (DT33), and
secondary veins (DT34). They range in diameter from 0.45 to
1 mm (Fig. 9C).
Cercidiphyllum genetrix has two mining associations. The first is
a serpentine mine known from one specimen, with a steady width
increase from oviposition to terminus, previously assigned to
DT91 [19] (Fig. 10A–D). The oviposition site was not preserved
because a later portion of the mine passed through it. The mine is
initially serpentine and affected by tertiary and higher-order
venation. The earliest preserved stage of the mine is 0.28 mm
wide, and the middle 62% of the mine is filled with sparse,
spheroidal frass pellets in a lighter colored frass matrix (Fig. 10D).
As the mine widens to 0.90 mm, individual spheroidal frass pellets
are visible but spread far apart (Fig. 10C). The visible frass pellets
average 33.064mm in diameter. The individual pellets are
surrounded by a lighter colored frass matrix, possibly fluidized
frass, which is similar in appearance to the early frass trail. The
percentage of the mine filled by frass decreases to 43% in the
widest portions of the mine (Fig. 10B). The end of the frass trail is
Figure 8. Leaf mines on
Zizyphoides flabella
at Mexican Hat. A: Serpentine mine delimited by primary veins, with gradual width expansion and
centered frass trail packed with ellipsoidal pellets (DT91; arrow expanded in B; USNM 560142). B: Detail of later frass trail in (A), showing increase in
frass size possibly associated with instar growth. C: Detail of frass trail in (D), showing tightly-packed, ellipsoidal frass pellets. D: Leaf mine with a
central frass trail composed of ellipsoidal pellets (DT91; arrow expanded in C; USNM 560136). E: Leaf mine looping between primary veins, with
central frass trail (DT91; USNM 560143). F: Threadlike mine (DT41), with gradually expanding width. Frass is initially densely packed and lacks
individual pellets (lower arrow, expanded in H), but subsequently loosens into a wider trail (upper arrow, expanded in G) (USNM 498160). G: Detail of
loosened frass trail in (F). H: Detail of early, threadlike path, with densely packed frass trail in (F).
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semicircular, and the last 22 mm of the mine contains no frass,
except for that left over from earlier phases of the mine that were
overlapped. Early portions of the mine are smooth-sided and
parallel. Quaternary veins delimit later portions of the mine,
making the margins wavy (Fig. 10C). Primary and secondary veins
also bind the mine margins in some areas. The mine does not cross
primary or secondary veins, but it does cross tertiaries. Darkened
reaction tissue, measuring up to 0.87 mm in width, borders the
outside margins of the wider portion of the mine.
A second leaf mine association, assigned to DT41 [19], is
positioned between two primary veins at the leaf base of one
specimen (Fig. 10E–G). The early mine phase averages 0.27 mm
Figure 9. External foliage feeding on
Cercidiphyllum genetrix
at Mexican Hat. A: Circular (DT2) and polylobate holes (DT3) with thin reaction
rims (USNM 560144). B: Semicircular margin feeding near leaf apex (DT12; USNM 560145). C: Deeply trenched margin feeding towards the midvein
(DT15; possibly an abiotic tear) and dark, circular galls on interveinal tissue (DT32), primary veins (DT33), and secondary veins (DT34; USNM 560146).
D: Skeletonized area at the leaf base (DT56) and circular piercing and sucking marks with central depressions spread throughout the leaf (DT46).
Arrow indicates area expanded in the inset, showing detail of the piercing and sucking marks (USNM 560147). E: Surface feeding delimited by two
secondary veins and crossing the midvein. Surface feeding crossing the midvein has polylobate reaction rim (DT30; USNM 560148). F: Alternating
light and dark lines of unknown origin; possibly viral damage, parallel lines of surface feeding, or fungal damage (DT23; USNM 560149). G: Galls with
thickened outer rims surrounding unthickened inner tissue (DT11; USNM 498161).
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in width and widens to 0.92 mm by the latest preserved portion.
The mine path is serpentine, and frass fills 32–65% of the width of
the mine (Fig. 10F). The frass trail is broken into irregular pieces,
which may be an artifact of preservation, possibly caused by
cracking and fragmentation of the frass trail upon desiccation of
the leaf. The mine margins are smooth, and there is no reaction
tissue along their outside. The mine does not cross primary veins
and does not appear to be directed by higher venation. On the
counterpart, the frass trail appears to expand to the entire width of
the mine, but the preservation is poor (Fig. 10G). The complete
mine path is not preserved because of fracturing. The base of the
leaf is preserved on the part, and the counterpart preserves the
base and apex. Both are missing the center portion of the leaf.
Lauraceae species
Insect damage for Lauraceae species 1 is illustrated in Figure 11.
This host has numerous piercing and sucking marks. The marks
are dark and circular with a central depression (DT46; Fig. 11A–
B) and are clustered in a small group. They range in diameter from
0.15–0.24 mm.
Insect damage for Lauraceae species 2 is illustrated in Figure 12.
Lauraceae species 2 exhibits hole feeding, margin feeding, surface
feeding, piercing and sucking, and galling. No leaf mines have
been recorded. Circular holes are 0.69–2.47 mm in diameter
(DT1, DT2; Fig. 12B) with reaction rims ranging in width from
0.07–0.18 mm. Polylobate holes range in length from 1.81–
5.24 mm long (DT3, DT5; Fig. 12I) with 0.14–0.22 mm wide
reaction rims. There are also parallel sided, curvilinear slot feeding
marks (DT8; Fig. 12C), ranging in length from 3.10 to 9.39 mm
and in width from 0.06 to 0.30 mm. The width of the DT8
reaction rim is 0.99 mm. One elongate hole is 3.08 mm long by
0.49–0.86 mm wide and tapers to one side (DT7; Fig. 12D). It has
a pronounced reaction rim, with a slightly darkened area directly
outside the hole (width = 0.32 mm) that grades to a thin, dark rim
(width = 0.14 mm). Margin feeding on Lauraceae sp. 2 includes
shallow, semicircular feeding (DT12; Fig. 12A) ranging in depth
from 0.98–6.42 mm, feeding at the apex through the midvein
(DT13; Fig. 12A), and deeper incisions proceeding from the
margin toward the midvein (DT15; Fig. 12E), measuring up to
3 mm wide and 5 mm deep. Circular surface feeding marks
(DT29; Fig. 12F) with well-developed reaction rims are inter-
spersed with similarly sized holes. Surface feeding marks measure
0.16–0.78 mm in diameter, with a 0.01 mm reaction rim.
A piercing and sucking association on this host plant is
characterized by elliptical and ovoidal puncture marks (DT48;
Fig. 12G, H). The marks range from 0.25 mm to 0.5 mm in
diameter. The centers of the marks are depressed and infilled with
a carbonized material. The marks are spread throughout the leaf,
but many are produced in close succession, within 1 mm from
primary and secondary veins (Fig. 12H).
A host-specific piercing and sucking pattern is found on four
specimens of Lauraceae sp. 2 (Fig. 12I–K); circular punctures with
central depressions, filled in with dark carbonized material, are
Figure 10. Leaf mines on
Cercidiphyllum genetrix
at Mexican Hat (A–G) and late Paleocene Wyoming localities (H–K). Mines on E–K
(DT41) are interpreted as belonging to the same species of lepidopteran leaf miner, representing at least a 6 million year association between the
host and miner. A: Mine characterized by overlapping trail, gradual width increase, spheroidal pellets in a frass matrix, and lack of frass in terminal
portion (DT91; USNM 498161). B: Closeup of mine in (A). C: Closeup of frass trail in (A) and (B), showing spheroidal frass pellets in a darkened matrix.
D: Detail of initial frass trail in (A) and (B). E: Serpentine mine characterized by initial threadlike phase arising from the base of a multi-veined leaf,
widening path, smooth margins, and packed frass (DT41; arrow expanded in F; USNM 560150). F: Detail of frass trail in (E), showing tightly packed
frass. G: Detail of counterpart of (E), showing packed frass in early portion of the mine. H: Threadlike mine with increasing width positioned at the
base of the leaf (DT41) (Haz-Mat; USNM 560151). I: Threadlike mine with increasing width positioned at the base of the leaf between two primary
veins (DT41) (Haz-Mat; USNM 560152). J: Tightly coiled mine with packed frass (DT41) (Skeleton Coast; USNM 560153). K: Poorly preserved mine
positioned at the base of the leaf between two primary veins interpreted as DT41 (Skeleton Coast; USNM 560154).
doi:10.1371/journal.pone.0103542.g010
Figure 11. Insect damage on Lauraceae sp. 1 at Mexican Hat. A:
Piercing and sucking marks with central depression (DT46; arrow
expanded in B; USNM 560155). B: Closeup of cluster of piercing and
sucking marks (DT46) on (A). Arrow indicates area expanded in the
inset, showing detail of piercing and sucking marks.
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arranged in an elliptical or circular pattern (DT281; Appendix 1;
Fig. 12J). Marks range in shape from circular to ellipsoidal or
comma shaped (Fig. 12K). The individual marks range in
diameter from 0.19 to 0.43 mm and tend to increase in size
towards the center of the pattern, with the largest marks touching
major veins. The marks are mostly positioned around primary or
secondary veins, but they are also found on interveinal areas.
Lauraceae species 2 has two associated gall DTs. Circular to
ellipsoidal galls with thickened tissue surrounding unthickened
inner tissue (DT11, Fig. 12L) are positioned near secondary veins
and on interveinal tissue. The galls range in length from 1.66–
4.23 mm and in width from 1.16–3.16 mm. The thickened tissue
ranges from 0.50–1.50 mm in width. The second gall DT is
characterized by dark, circular tissue on interveinal tissue (DT32)
and next to secondary veins (DT34; Fig. 12M). There are two
groups of four galls arranged together in a quadrangular
deployment (Fig. 12M), but most galls are randomly distributed.
Their average diameter is 0.41 mm.
‘‘Populus’’ nebrascensis
Insect damage for ‘‘Populus’’ nebrascensis is illustrated in
Figure 13. External foliage feeding on ‘‘P.’’ nebrascensis includes
hole feeding and skeletonization. Circular holes are 0.68–1.91 mm
in diameter, and oval holes are 0.70–5.76 mm long and 0.41–
2.80 mm wide (DT1, DT2, DT4; Fig. 13B). Holes are also found
at the angular intersections of primary and secondary veins
(DT57; Fig. 13B, C) and range from 2.16–4.71 mm in length
(measured from the vein intersection to the distalmost hole
margin). Reaction rims typically measure 0.18 mm wide. A
skeletonized area with reaction tissue (DT17; Fig. 13A) is mostly
bounded by primary veins, although small patches cross outside
this area. Tertiary venation is preserved, but some tertiary veins
were removed during feeding or through taphonomic processes.
Small patches of preserved tissue within the skeletonized area are
darker than the rest of the unskeletonized leaf, suggesting a
reaction to feeding. Reaction tissue surrounding the skeletonized
patches outside the primary vein boundaries measures 0.33 mm
wide.
Possible circular piercing and sucking marks measure 90 mmin
diameter (DT46; Fig. 13A, inset).
‘‘Populus’’ nebrascensis has two gall DTs. One probable gall is
circular, with dark, thickened tissue surrounding light-colored
central tissue (DT11; Fig. 13D). The gall is 0.66 mm in diameter,
and the darkened tissue is 0.17 mm wide. There is also a dark,
circular gall positioned on a secondary vein (DT34). The gall is
2.38 mm in diameter, and the outer 0.25 mm is darker than the
center of the gall (Fig. 13E).
A single mine assigned to DT91 [19] has been found on one
specimen of ‘‘P.’’ nebrascensis (Fig. 13F, G). The mine follows a
linear path, initially against the midvein, but it veers off slightly
into interveinal tissue before returning to the midvein (Fig. 13F,
arrow and inset). The width of the early portion of the mine is
0.33 mm wide and is completely filled with a loosely sinusoidal
frass trail. The mine widens to 0.69 mm at the first intersection
between the midvein and a secondary vein and follows the
secondary vein until termination. At the intersection, the frass trail
fills 80% of the mine but is positioned close to the guiding
secondary vein. The mine increases in width along the secondary
vein, reaching 0.85 mm wide with frass filling the center 60% of
the mine. Finally, the mine turns at a tertiary vein and follows the
lateral primary vein toward the base until termination. The final
4.5 mm of the mine is free of frass and was probably formed prior
to pupation by the last larval instar. Frass pellets are spheroidal,
increasing in average diameter from 0.042 mm to 0.077 mm
along the secondary vein (Fig. 13G, see inset). The mine does not
cross primary or secondary veins, but it does cross tertiary veins.
The width of the mine decreases slightly at its intersections with
each tertiary vein, giving the margin a wavy appearance (Fig. 13G).
Some areas along the margin show darkening, which suggests that
the plant reacted to the physical damage caused by feeding.
Browniea serrata
Insect damage for Browniea serrata is illustrated in Figure 14.
Browniea serrata (formerly Eucommia serrata and Dicotylophyllum
anomalum [49]) features a variety of holes, margin feeding,
skeletonization, and galling. Circular holes range from 0.75–
2.80 mm in diameter, with darkened reaction rims measuring
0.10 mm wide (DT1, DT2; Fig. 14A, B). All other holes are
polylobate (DT3, Fig. 14C), with widths varying from 1.52–
2.57 mm and lengths ranging from 2.72–4.17 mm. A 0.10 mm
wide reaction rim surrounds the holes (Fig. 14A, B, C). Besides
circular and polylobate holes, B. serrata also exhibits evidence of
slot feeding (DT8; Fig. 14A, arrow and inset). The single slot is
rectilinear, parallel sided, and measures 6.4 mm long by 0.44 mm
wide. Its reaction rim measures 0.14 mm across. There is one
specimen with margin feeding, with a small incision into the leaf
margin terminating at a secondary vein (DT15; Fig. 14A). The
excision is 1.5 mm wide at the leaf margin and tapers to 0.5 mm
wide, meeting a secondary vein at a depth of 3.5 mm. There is a
thin reaction rim surrounding the cut area.
Browniea serrata is also associated with circular piercing and
sucking marks with central depressions, filled with dark carbonized
material (Fig. 14D). The marks range in diameter from 0.17 to
0.36 mm. They are spread throughout the leaf, but commonly
found near secondary veins.
Browniea serrata has one gall association. The galls are
darkened circular areas representing thickened tissue and are
positioned next to secondary veins (DT34) as well as on interveinal
tissue (DT32; Fig. 14C). The average diameters of the galls range
between 0.98–1.46 mm.
There is a mine on one specimen of Browniea serrata.Itis
serpentine and filled with what appears to be miniscule, spheroidal
frass pellets (DT91; Fig. 14E), although preservation is poor. The
preserved mine gradually increases in width from 0.10 to
0.88 mm, but portions of the mine were not recovered. The
preserved path of the mine suggests that it crosses secondary veins,
Figure 12. Insect damage on Lauraceae sp. 2 at Mexican Hat. A: Shallow, semicircular margin feeding along the sides of the leaf (DT12) and at
the apex through the midvein (DT13) (USNM 560156). B: Circular hole (DT2) near secondary vein (USNM 560157). C: Elongate, parallel-sided hole (DT8)
positioned at the intersection between the primary and secondary veins (USNM 560158). D: Elongate and tapering hole (DT7), with well-developed
reaction tissue (USNM 560159). E: Margin feeding incision along a secondary vein towards the midvein (DT15; USNM 560160). F: Surface feeding areas
(DT29) with well-developed reaction rims (USNM 560161). G: Elliptical and ovoidal piercing and sucking marks (DT48) associated with major veins and
leaf margins (box expanded in H; USNM 560162). H: Detail of piercing and sucking marks in (G), showing linear association with secondary veins. I:
Elliptical groupings of piercing and sucking marks (DT281) associated with secondary veins (arrow expanded in J; USNM 560163). J: Closeup of a
piercing and sucking cluster in (I), showing an increase in the size of individual marks towards the vein (arrow expanded in K). K: Detail of piercing and
sucking marks in (I) and (J). L: Galls with thickened outer rims surrounding unthickened tissue (DT11; USNM 560164) M: Circular galls clustered in
groups of four and dispersed randomly on interveinal tissue (DT32) and adjacent to secondary veins (DT34; USNM 560157).
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probably near the margin of the leaf. There is no reaction tissue
along the mine margins.
‘‘Ficus’’ artocarpoides
Insect damage on ‘‘Ficus’’ artocarpoides is illustrated in
Figure 14F–H, comprising hole feeding and one mine. Holes are
polylobate (DT3, DT5), varying in length from 1.95 to 5.54 mm
and in width from 0.97 to 2.77 mm (Fig. 14F, H). The mine is a
blotch with an internal serpentine frass trail, assigned to DT37
(Fig. 14F, G). The preserved portion is serpentine and sinusoidal.
There is a noticeable increase in frass size throughout the trail,
increasing from 0.08 to 0.14 mm in diameter. The portion of the
frass trail with small frass pellets (,0.08 mm) is 0.46 mm wide and
follows a linear path with continuous frass deposition. The portion
with larger pellets (,0.14 mm) varies in width from 0.18 to
0.40 mm with a curved path and discontinuous frass deposition.
The overall width of the mine varies from 2.70–4.26 mm, and
secondary veins delimit the widest portions. The mine does not
cross secondary veins, and it does not appear to be affected by
tertiary venation. The larva may have caused the blotched
appearance of the mine by producing an intestiniform feeding
path delimited by secondary venation and removing mesophyll
tissue between the veins. The margins of the mine are confluent
along secondary venation and interveinal tissue. Reaction tissue
along the margins of the mine is 0.14–0.18 mm wide.
cf. Ternstroemites aureavallis
One gall was observed on cf. Ternstroemites aureavallis
(Fig. 14I), positioned at the intersection between the primary vein
and a secondary vein (DT33), and crossing the secondary vein. It is
circular, with a diameter of 3.24 mm, a darker outer rim
measuring 0.75 mm wide, and an inner, circular chamber
measuring 0.34 mm in diameter.
Unknown dicot leaf morphotypes and leaf fragments
Insect damage for Dicot morphotypes 1–4 is illustrated in
Figure 15. The associations on Dicot leaf morphotype 1 (Fig. 15A–
D) include hole feeding, margin feeding, and two mine associa-
tions. Hole feeding is circular to elliptical, ranging in diameter
from 0.16 to 3.52 mm (DT2, DT4) with reaction rims ranging in
width from 0.06 to 0.15 mm (Fig. 15A). Smooth, semicircular
margin feeding (DT12) measures 1.69 cm across and 0.92 cm
deep, with a probable secondary feeding incision towards the
midvein measuring 1.47 mm wide and 6.04 mm deep (Fig. 15D).
The smooth, large, primary incision resembles leaf-harvesting
damage made by extant Megachilidae, the leaf cutting bees [50].
A single blotch mine on Dicot leaf morphotype 1 (Fig. 15A) is
bounded by secondary veins and was assigned to DT36 [19]. The
left side of the mine is bound by a tertiary vein, and the right side
of the mine consists of two polylobate extensions, with the
uppermost extension reaching the midvein. There is a dark
reaction tissue surrounding the mine, which has a similar
appearance in color and shape to the reaction tissue surrounding
the hole feeding on the same specimen. The mine is filled with
particulate frass. The specimen was not available for further study,
and finer details of the frass were not resolvable from the
photograph.
Another mining association on Dicot leaf morphotype 1,
characterized by a serpentine/linear path, was assigned to DT91
[19] (Fig. 15B). The oviposition site is circular (Fig. 15C) and is
followed by an initial threadlike, serpentine trail completely filled
with frass and measuring 0.19 mm in width. The second portion
of the mine is marked by a moderate width increase to 0.25 mm
and a linear path following the leaf margin. The third portion
continues to follow the margin, but there is a width increase to
0.52 mm. Frass fills the width of the mine in the first two sections.
The center 55% of the last section is filled with frass, and the last
2.81 mm of the mine is free of frass. Because of the poor
preservation of the mine, the nature of the frass is indeterminate.
Dicot leaf morphotype 2 (Fig. 15E) has a circular hole (DT2;
15E, lower arrow) measuring 4.40 mm in diameter and semicir-
cular margin feeding (DT12; 15E, upper arrow) measuring
0.70 cm wide at the margin and 0.33 cm deep. Dicot leaf
morphotype 3 (Fig. 15F) is associated with oval holes (DT2;
Fig. 15F, arrow) that measure 0.12–0.24 cm in diameter. Dicot
leaf morphotype 4 (Fig. 15G) features margin feeding assignable to
DT14. The removed portion is 1 cm wide at the margin and
tapers to 3 mm at the deepest part, where it meets the midvein.
Oviposition scars are found on three unidentifiable leaf
fragments (Fig. 16A–B). The scars are ovoid or elliptical and
measure between 0.45–1.70 mm in length and 0.40–0.70 mm in
width. The thickened reaction tissues surrounding the entry slits
measure 0.15–0.30 mm wide. Many oviposition scars appear on
each damaged leaf but are not arranged in any discernable
pattern.
Leaves and fruits without insect damage
Undamaged leaves and fruits are illustrated in Figure 16C–E.
Insect damage was not found on Paleonelumbo macroloba
(Fig. 16C) and Paranymphaea crassifolia (Fig. 16E) leaves nor
on Polyptera manningii fruits (Fig. 16D) at Mexican Hat. The lack
of damage can probably be explained by the small sample size
examined for each species, namely two specimens each of
Paleonelumbo macroloba and Paranymphaea crassifolia, and one
specimen of Polyptera manningii. Leafy branches of the only
conifer, Glyptostrobus europaeus (Cupressaceae) were very abun-
dant; although they were not censused, damage on this species was
noted as extremely rare (PW and CCL pers. obs. 2004).
Results and Discussion
Our results indicate both that leaf-mining and presumed leaf-
miner diversity at Mexican Hat is even higher than previously
recognized, and, equally importantly, that none of the Mexican
Hat mines can be linked back to the local Cretaceous mining
fauna. Thus, there is no evidence for survivorship of any of the
diverse Cretaceous leaf miners over the K-Pg boundary, even on
well-sampled, surviving plant families (Cercidiphyllaceae and
Platanaceae). These results show that the high insect damage
Figure 13. Insect damage on ‘‘
Populus
’’
nebrascensis
.A: Skeletonized tissue with reaction rim (DT17) bounded by primary veins and possible
piercing and sucking marks (DT46). Arrow indicates area expanded in the inset, showing detail of the piercing and sucking marks (USNM 560165). B:
Circular holes (DT1) through interveinal tissue and oval holes at the intersection of primary and secondary veins (DT57; USNM 560166). C: Ovoidal
hole at the intersection of primary and secondary veins with well-developed reaction rim (DT57; USNM 560167). D: Gall with thickened outer rim and
unthickened inner area (DT11) on interveinal tissue (USNM 560167). E. Circular gall positioned on a secondary vein (DT34) with darkened outer rim
(USNM 560168). F: Linear mine following the primary vein to a secondary vein, characterized by a gradual width increase and frass-packed trail with
spheroidal pellets (DT91). Arrow indicates area expanded in the inset, showing detail of the initial frass trail (USNM 498158). G: Detail of linear path in
(F). Closeup of the frass trail and spheroidal pellets in inset. Arrow indicates area expanded in the inset.
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Figure 14. Insect damage on
Browniea serrata
(A–E), ‘‘
Ficus
’’
artocarpoides
(F–H), and cf.
Ternstroemites aureavallis
(I). A: Circular (DT1,
DT2), polylobate (DT3), and elongate slot-feeding (DT8) holes on Browniea serrata. Arrow indicates area expanded in the inset, showing detail of the
slot-feeding hole (USNM 560169). B: Circular (DT1) and polylobate (DT3) holes on Browniea serrata. (USNM 560170). C: Circular galls on interveinal
tissue (DT32) and secondary veins (DT34) on Browniea serrata (USNM 560171). D: Piercing and sucking marks with central depressions (DT46) on
Browniea serrata. Arrow indicates area expanded in the inset, showing detail of a piercing and sucking mark (USNM 560172). E. Serpentine mine
packed with spheroidal frass pellets (DT91; DMNH 35832) F: Blotch mine with internal frass trail (DT36) and polylobate holes (DT3) on ‘‘Ficus’’
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diversity at Mexican Hat represents an influx of novel insect
herbivores during the early Paleocene and not a refugium for
Cretaceous leaf miners.
Affinities of Mexican Hat mines
Lepidopterans are the most diverse leaf-mining insects [51].
The leaf-mining habit is practiced by basal lepidopteran groups
and may have only evolved once [52,53]. Numerous probable
lepidopteran mines have been described in the fossil record [54],
dating back to the Early Cretaceous [55] or possibly latest Jurassic
[56,57], although the lepidopteran leaf-mining habit may extend
considerably earlier, to the mid Jurassic [58]. The abundance of
fossil lepidopteran mines, compared to other orders, may be
attributable to the early origin of mining behavior within
Lepidoptera or to their overall preference for woody over
herbaceous plants [53,59], the latter of which are much less likely
to be fossilized.
Lepidopteran miners make serpentine and blotch mines, and
mine paths rarely overlap with earlier traces. Most produce solid
frass deposited in pellets. The frass trails are usually deposited in a
medial, linear path, or dispersed throughout the mine width [41].
Exit holes are small and circular [41], semicircular [42], or in slits
[60]. These characteristics, especially mine shape and frass,
suggest that lepidopteran larvae made most of the mines at the
examined localities. Mexican Hat mines included in DT37, DT41,
DT91, and DT92, all exhibit features associated with lepidopteran
mines, and they share many similarities with those of extant
Nepticulidae and Gracillariidae micromoths. Nepticulidae larvae
create serpentine or linear mines with a continuous central frass
trail, often composed of ellipsoidal or spheroidal frass pellets. The
widths of the mines increase with each instar [61]. Gracillariidae
larvae typically make significantly larger blotch and serpentine
mines. Their serpentine mines, often produced by species of
Phyllocnistis, are characterized by a median frass trail, which
typically extends unbroken through the mine, except for the last
instar immediately before creation of the terminal chamber. The
final instar does not feed, and it makes a cocoon in the mine
terminus [60]. The mines on J. glabra that lack evidence of frass
(DT42 and DT105; Fig. 6A, 4I), unless this is taphonomic, were
probably also made by lepidopteran larvae because almost all
miners known to remove frass from their mines are Lepidoptera
[41]. Alternatively, these mines could have been made by a sap-
feeding species.
Leaf-mining Hymenoptera occur within one of the basal sawfly
superfamilies, Tenthredinoidea [59,62], particularly in the families
Tenthredinidae, Argidae, and Pergidae [59]. Most hymenopteran
leaf miners are found in Tenthredinidae subfamilies Heterarthri-
nae and Nematinae, which evolved mining behavior separately
from each other [63]. The fossil record of Tenthredinoidea is fairly
rich and extends back to the Jurassic [64]. The earliest putative
tenthredinid is from the earliest Cretaceous, but this record may
represent a stem group [65,66]. Many fossil sawflies from the
Eocene have been assigned to Tenthredinidae [67–72].
Tenthredinidae generally produce blotch mines with pelletized
frass that is dispersed irregularly through the mine or near the
margins. The mine may begin with a short serpentine phase or
start as a blotch. Exit holes are typically large and circular [41].
This Tenthredinidae mine morphology matches the DT36 mines
on P. raynoldsii (Fig. 3A–D). There is one known species of
Tenthredinidae associated with extant Platanus (Platanaceae),
Bidigitus platani, which mines Platanus racemosa at least in the
Californian part of its range [73–75]. The mine morphology of
this modern association differs from DT36 on P. raynoldsii
because the B. platani mine has a short, initial serpentine phase.
The presence of Tenthredinidae mines at Mexican Hat on
Platanus extends the Tenthredinidae-Platanus mining association
to the early Paleocene. Other possible fossil Tenthredinidae leaf
mines have been described on host plants other than Platanus,
including from the Miocene of Romania [76], and others have
noted morphological similarities between extant Tenthredinidae
mines and leaf mine fossils from the Albian and Turonian [77,78]
and the Eocene [79].
Dipteran leaf miners are found in several basal and derived fly
families, but the most speciose is Agromyzidae [80]. Agromyzids
produce serpentine and blotch mines, often with irregular paths
and overlapping sections. They do not feed on cell walls, are
limited to sap-feeding by the mouthhook design of their
mouthparts, and produce fluidized frass, which forms small
clumps. Because the larvae (maggots) feed on their sides relative
to the leaf surface and often alternate between borders of the mine,
their frass may be deposited in alternating rows. A thorough
description of dipteran mine characteristics, particularly of
Agromyzidae, was given by Winkler et al. [26]. The agromyzid
mine on P. raynoldsii at Mexican Hat, Phytomyzites biliapchaensis,
represents an extinct association because no modern agromyzids
feed on Platanus. This contrasts with the proposed Tenthredini-
dae mine (DT36) on P. raynoldsii, which appears to represent a
long-term surviving relationship between Tenthredinidae and
Platanus.
Coleoptera may have been the first order of leaf miners, possibly
responsible for the earliest known mines, from the Triassic (i.e.,
[81,82]). Coleopteran mines can resemble lepidopteran mines,
although coleopteran excision areas are usually larger and
typically form robust, full-depth mines with particulate frass
[41]. Leaf-mining coleopterans are much less diverse today than
leaf-mining lepidopterans. No clear evidence of coleopteran mines
has been found at Mexican Hat.
Mining diversity and host specificity at Mexican Hat
Leaf mine diversity at Mexican Hat is much higher than at any
other Paleocene locality examined in this study, or at any other
Paleocene site from the United States known to the authors, who
have collectively excavated at least 200 Paleocene plant localities
from the Rocky Mountains and Great Plains. The full suite
includes 9 mine DTs on 8 host plants (18 insect-host plant pairs
total; Table 3), increasing from an earlier count of 7 mines on 6
host plants (14 DT-host plant pairs total) [19]. However, as
detailed below, host-specific variation strongly suggests that an
even higher number of actual mining species was present at
Mexican Hat. For comparison, Pyramid Butte, which lies just
20 cm above the K-Pg boundary 15 km north of Marmarth,
North Dakota, had the next highest number of mine DTs (2) out of
the early Paleocene sites. Mines at Pyramid Butte were found on 2
host plants (2 insect-host pairs total) from a collection of 549
specimens. The Battleship locality, located 3.6 m below the K-Pg
boundary 5 km northeast of Marmarth, had the highest number of
mines at a Cretaceous site, 5 mine DTs on 10 hosts (10 insect-host
plant pairs total), although somewhat less than half the number of
artocarpoides (YPM 65820). G: Detail of frass trail in (F) showing spheroidal frass pellets. H: Polylobate hole (DT5) on ‘‘Ficus’’ artocarpoides (USNM
560173). I. cf. Ternstroemites aureavallis with a gall at the intersection of primary and secondary veins (DT33). Inset shows details of the gall, including
a darkened outer rim and center (USNM 560174).
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Figure 15. Unknown dicot leaf morphotypes with insect damage. A: Blotch mine delimited by secondary veins with central frass pellets
(DT36, arrow), and circular and oval holes (DT2, DT4) on Dicot leaf morphotype 1 (USNM 560178). B: Poorly-preserved serpentine mine on Dicot leaf
morphotype 1. Arrow indicates circular oviposition damage (DT91; arrow expanded in C; USNM 560179). C. Detail of (B) showing circular oviposition
damage. D: Smooth, semicircular margin feeding (DT12), and probable secondary feeding incision towards the midvein (DT15) on Dicot leaf
morphotype 1 (USNM 560180). E: Oval hole feeding (DT2; indicated by lower arrow) and semicircular margin feeding (DT12; indicated by upper
arrow) on Dicot leaf morphotype 2 (USNM 560181). F: Oval hole feeding (DT2, arrow) on Dicot leaf morphotype 3 (USNM 560182). G: Deep margin
feeding incision reaching the midvein (DT14, arrow) on Dicot leaf morphotype 4 (USNM 560183).
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specimens were sampled (459) than at Mexican Hat (1073
specimens). When compared by resampling at equal sample size
(400 leaves) [19], Battleship had ca. 5 mine DTs and Mexican Hat
ca. 3 mine DTs.
Leaf-mining larvae produce characteristic damage that can
provide taxonomic clues [41], but many species produce very
similar morphologies. Four mine DTs at Mexican Hat appear on
multiple host plants, but their placement within a single DT in no
way implies that all the damage was made by the same insect
species. Rather, because most leaf miners are specialized [53],
mine characters unique within one host plant may suggest one-to-
one host specificity of the leaf-mining insect species. Mine DTs
occurring on multiple host plants include DT36 (P. raynoldsii,
Dicot leaf morphotype 1), DT41 (C. genetrix,Z. flabella), DT91
(P. raynoldsii,Z. flabella,J. glabra,C. genetrix,‘‘P.’’ nebrascensis,
B. serrata, Dicot morphotype 1), and DT282 (P.raynoldsii and J.
glabra). The following analysis of fine differences within DTs,
among host plants, suggests a higher number of leaf-mining species
at Mexican Hat than implied by the number of mine DTs.
Blotch mines assigned to DT36 are associated with five P.
raynoldsii specimens (Fig. 3A–D) and one Dicot morphotype 1
specimen (Fig. 15A). Damage type 36 mines on both hosts exhibit
some similarity, including lack of a central chamber, frass pellets
dispersed throughout the mine, and channeling of the mine path
by secondary venation. However, host-specific differences also are
present. The mines on P. raynoldsii contain initially small and
closely packed frass. Larger pellets, produced by later instars, are
spread farther apart (Fig. 3D). The differently-sized frass pellets do
not usually overlap, allowing for the inference of larval instar shifts
and feeding trajectory. In contrast, frass in the DT36 mine on
Dicot morphotype 1 is packed closely together near the center of
the mine, with no clear spatial differentiation in pellet sizes
(Fig. 15A; a more detailed look at the frass was not possible
because the specimen was unavailable). Although the mine on
Figure 16. Oviposition (A–B), rare angiosperm leaves (C, E), and one fruit (D) from Mexican Hat. A: Oviposition scars on unidentifiable
leaf fragment. Arrow indicates area expanded in the inset, showing detail of oviposition scars (USNM 560184). B: Oviposition scars on unidentifiable
leaf fragment. Arrow indicates area expanded in the inset, showing detail of oviposition scar (USNM 560185). C. Paleonelumbo macroloba leaf, without
insect damage (rotated to fit layout; USNM 560175). D: Polyptera manningii fruit, which is correlated with Juglandiphyllites glabra, without insect
damage (USNM 560176). E: Paranymphaea crassifolia without insect damage (USNM 560177).
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Dicot morphotype 1 does not cross secondary veins for an
extensive range, parts of the secondary vein were removed where
the larva crossed the vein. Removal of vein tissue also is observed
along the primary vein (Fig. 15A). Vein tissue removal is not
evident on any of the DT36 mines on P. raynoldsii. The DT36
mine on Dicot leaf morphotype 1 has a thick reaction rim
surrounding the mine margin (Fig. 15A), but reaction tissue is not
apparent on the P. raynoldsii mines. The overall sizes of the DT36
mines on P. raynoldsii are larger. In summary, it is very likely that
these occurrences of DT36 on two different host plants represent
separate leaf-mining taxa.
Mines of the DT41 type are associated with one specimen each
of Z. flabella (Fig. 8F–H) and C. genetrix (Fig. 10E–G). The mines
on both specimens exhibit serpentine paths with expanding widths
and smooth margins, and although affected by secondary
venation, other host-specific differences also are apparent. The
DT41 mine on Z. flabella begins with an extended threadlike
phase measuring around 0.10 mm wide (Fig. 8H), compared to a
0.27 mm wide initial phase on C. genetrix (Fig. 10G). Frass fills the
width of the mine on Z. flabella (Fig. 8H), but it fills only 32–65%
of the center of the mine path on C. genetrix (Fig. 10F) The mine
on Z. flabella also features a dramatic widening associated with
loosening of the otherwise tightly packed frass trail (Fig. 8G),
whereas no evidence of a change in frass trail density is noticeable
on C. genetrix. The overall length of the mine on Z. flabella is also
much greater, including a longer threadlike phase, compared to
the mine on C. genetrix, although the latter specimen is not fully
preserved. In summary, it is very likely that these two instances of
DT41 on different host plants represent separate leaf-mining taxa.
Mines assigned to DT91 are associated with 20 specimens of P.
raynoldsii (Fig. 2A–H), 6 specimens of Z. flabella (Fig. 8A–E), 4
specimens of J. glabra (Fig. 5A–D), 1 specimen of C. genetrix
(Fig. 10A–D), 1 specimen of ‘‘P.’’ nebrascensis (Fig. 13F–G), 1
specimen of B.serrata (Fig. 14E), and 1 specimen of Dicot
morphotype 1 (Fig. 15B–C). Similarities among all DT91 mines at
Mexican Hat include a serpentine mine path, ellipsoidal frass
pellets, pronounced width expansion, and control of mine margins
by tertiary venation.
Despite these similarities, several host-specific differences exist
among DT91 mines (Table 4). One distinct feature of DT91 mines
on P. raynoldsii is a dramatic increase in width at the terminal
portion (Fig. 2A, D, E, G). Secondary veins typically delimit the
mine path, leading to a winding pattern (Fig. 2A, E). On other
specimens, the mine continues on a similar path, but the width of
the mine greatly increases, and frass pellets are deposited in
clusters near the margin (Fig. 2D, G). The larvae that produced
DT91 mines on P. raynoldsii fed through tertiary venation during
the terminal phase of the mine, a syndrome not observed on any
other host plant (Fig. 2A, E). The DT91 mines on Z. flabella
originate near a secondary vein and loop around to the adjacent
secondary vein, following it until termination (Fig. 8A, D, E). The
single DT91 mine on C. genetrix follows a similar trajectory to that
on Z. flabella, but the frass trail ends 22 mm before the terminus.
A frass-free terminus is common in DT91 mines, but the length of
the frass-free phase on C. genetrix is much longer than on any
other observed specimen (Fig. 10B). The frass trail on C. genetrix is
also unique for DT91 at Mexican Hat, with spheroidal frass pellets
embedded in a non-particulate frass matrix (Fig. 10C–D). The
frass trail of DT91 mines on J. glabra also differs from mines on
other host plants. Frass is spheroidal and spread out in a loose trail
or completely absent in some areas (Fig. 5A–D). The distance
between frass pellets is greater than for similar mines on other host
plants. The DT91 mine on ‘‘P.’’ nebrascensis follows a linear path
along primary and secondary veins through its entire course,
which has not been observed on any other DT91 mine (Fig. 13F–
G). The DT91 mine on Dicot morphotype 1 follows a linear path
along the margin initially, but then turns along a secondary vein
and widens (Fig. 15B). A circular oviposition site is clearly visible at
the origination of the mine (Fig. 15C) but is not observed on any
other host plants. Individual frass pellets are ambiguous, but this
Table 3. Mine damage types by host plant at Mexican Hat.
Host Plant Leaves Mine DTs Leaves with Mines
Platanus raynoldsii 1207 DT36 3
DT91 20
DT104 63
DT282 1
Juglandiphyllites glabra 396 DT42 1
DT91 4
DT92 2
DT105 1
DT282 1
Zizyphoides flabella 231 DT41 1
DT91 6
Cercidiphyllum genetrix 214 DT41 1
DT91 1
‘‘Populus’’ nebrascensis 85 DT91 1
Browniea serrata 12 DT91 1
‘‘Ficus’’ artocarpoides 4 DT37 1
Dicot morphotype 1 12 DT36 1
DT91 1
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may be an artifact of preservation. The DT91 mine on B. serrata
follows a serpentine path across secondary veins. The frass trail,
possibly made up of spheroidal frass pellets, appears to fill the
entire mine width, although preservation is poor, and the
specimen is fragmented.
Differences in DT91 mines among host plants strongly suggest
that more than one species made them. Evidence for multiple leaf-
mining species is clearly seen, for example, by comparing the
dramatic width increase and winding path on P. raynoldsii, the
loosely packed frass trail with spheroidal pellets on J. glabra, the
sparse frass pellets surrounded by a possibly fluidized frass matrix
on C. genetrix, and the oviposition site damage on Dicot leaf
morphotype 1. These differences allow for a conservative estimate
of five leaf-mining species, although seven or more are possible.
Epidermal mines assigned to DT282 occur on P.raynoldsii
(Fig. 3E) and J.glabra (Fig. 4K) at Mexican Hat. A serpentine
path with dark frass or reaction tissue along the margins and a
frass-free central chamber characterize this DT. Mines on both
host plants feature similar morphologies, but both specimens are
poorly preserved and lack proximal and terminal portions. The
lack of preserved features prevents comparisons of the full range of
mine characteristics. Based on shared distinctive morphological
traits, however, DT282 mines on P.raynoldsii and J.glabra were
probably made by closely related species.
Despite the fine variation within mine DTs among host plants
detailed above, there are potential issues with precisely inferring
species-level mining diversity based on minor host-specific
differences among leaf mines. Leaf architecture varies among
host plants and can affect mine paths. Mining larvae may avoid or
follow leaf veins because of tissue-specific variations in nutritional
quality. Vein arrangement may be one of the most important
factors controlling feeding path and mine shape [83]. Because leaf
anatomical characters differ among plant species and affect mine
path, care must be taken to avoid interpreting slight differences in
path morphology driven by venation differences as representing
different mining species. Seasonal changes can affect leaf
nutritional quality, and in turn affect the amount of time spent
feeding within a leaf and overall mine length [41]. Nutritional
differences among host plant species may also have an effect on
mine length for polyphagous leaf miners. However, despite
possible issues with interpreting slight host-specific variations
among mines, we have listed above many host-consistent
differences that are not obviously related to leaf venation or other
factors.
In conclusion, the species-level diversity of mining insects at
Mexican Hat must have been significantly higher than the number
of DTs originally assigned to those mines [18]. Six mine DTs were
recorded in [19], and two additional mine DTs were added in this
study, but host-specific morphological differences among mine
DTs, detailed above, suggest higher mining diversity than
previously recognized. In summary, these results suggest that at
least 15 insect species created the leaf mines at Mexican Hat, but
the presence of 18 or perhaps even more mining species is possible.
No survivors? Comparison with Cretaceous leaf mines
We found no conclusive evidence for the survival of any of the
diverse Cretaceous leaf-mining fauna over the K-Pg boundary
(Table 5). Earlier studies [11,19] found four mine DTs that crossed
the boundary in the Western Interior, USA: DT41, DT42, DT43,
and DT59. Two of these (DT41 and DT42) occur at Mexican
Hat. In the case of DT43 (not illustrated), the differences between
Hell Creek and Fort Union samples were so great on re-
examination that we did not consider this an example of a possible
surviving leaf mine association. Damage type 59 has not been
Table 4. Comparison of DT91 leaf mine morphology by host plant at Mexican Hat.
Plant host Mine width (mm) Margins
Frass trail
percentage Pellet shape
Pellet length x
width (mm) Distinctive features
#
P. raynoldsii 0.32–7.89 wavy 41–77% spheroidal/ellipsoidal 15–130610–75 dramatic widening during terminal phase,
removal of tertiary veins
20
J. glabra 0.38–2.33 wavy 25–70% spheroidal 50650 distinctive spheroidal frass, sparse/spread out
frass trail
4
Z. flabella 0.45–1.9 wavy 30–50% ellipsoidal 45–90645–82 close association with secondary veins, most
densely packed frass trails of DT91 mines
6
C. genetrix 0.28–0.90 initially smooth, then wavy 62–43% spheroidal with fluidized frass matrix 33633 frass trail composed of spheroidal pellets in a
fluidized frass matrix
1
‘‘P.’’ nebrascensis 0.33–0.85 wavy 100–80% spheroidal/ellipsoidal 42–77 linear path along primary to secondary vein 1
B. serrata 0.10–0.88 smooth 100% probably spheroidal N/A N/A – poor preservation and incomplete fossil 1
Dicot morphotype 1 0.19–0.52 smooth 100–55% unknown N/A oviposition damage mark at proximal trail 1
Frass trail percentage range from earliest trail to latest trail.
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found at Mexican Hat, but it has been found in the supplemental
local Paleocene collections and therefore is discussed here. As
detailed below, although there are some morphological similarities
within the DTs that cross the boundary, there is no convincing
evidence to suggest that the damage was made by the same leaf-
mining taxon. The potential boundary-crossing DTs are also not
found on related host plants.
Damage type 41 (Fig. 17) is one of the most common mine DTs
from the Cretaceous North Dakota sites, with 22 specimens
censused in [11] and 6 additional specimens examined in this
study. The earliest local (Williston Basin of North Dakota)
appearance of this mine DT is 103 meters below the K-Pg
boundary, and the latest known local Cretaceous appearance is
3.6 m below the K-Pg boundary at the Battleship locality [10,11].
Paleocene DT41 mines are found at Mexican Hat and at several
late Paleocene sites in Wyoming. Although similar morphological
characteristics, such as a serpentine path with smooth margins, are
observed consistently in DT41 mines from both the Cretaceous
and Paleocene, there are many host-specific morphological
differences that suggest that DT41 mines in the Cretaceous were
made by numerous different leaf-mining species, whereas the
Paleocene DT41 mines were made by only a few species.
A consistent feature of all Paleocene DT41 mines found in this
study is the position at the base of a multi-veined leaf, occurring on
Z. flabella (Fig. 8F) and C. genetrix specimens (Fig. 10E–K). No
examined Cretaceous DT41 mine is situated in a similar position
(Fig. 17). The consistently small size (length and width) of some
Cretaceous leaf mines precludes them from being related to any
known Paleocene DT41. For example, a delicate mine associated
with leaf morphotype HC81 (Fig. 17A) is initially around 0.13 mm
wide and filled entirely with frass. The mine widens to 0.45 mm by
its terminus, and only the middle 35% of the mine is filled with
frass. The widest portion of the mine has less than half the greatest
width of any Paleocene specimens. Another Cretaceous mine, on
‘‘Artocarpus’’ lessigiana (Fig. 17B), increases minimally in width
throughout its path, from 0.25–0.30. The mine path also makes
angular turns, which differs from observed Paleocene mines.
Other Cretaceous mines assigned to DT41 differ in path
morphology. Examples include a tightly sinusoidal and looping
path on leaf morphotye HC34 (Fig. 17C), a coiled and intestini-
form path (Fig. 17D), a tightly sinusoidal path with many angular
turns on an unidentified leaf (Fig.17E), and a mostly linear mine
lacking any major width increase on ‘‘Dryophyllum’’ subfalcatum
(Fig. 17H). Differences in frass trail morphologies also occur in
Cretaceous DT41 mines compared to examined Paleocene
specimens. One example occurs on an unidentified leaf (Fig. 17F)
and on the leaf morphotype HC266 (DMNH 20015). The mines
are initially threadlike but widen throughout their course. The full
widths of the mines are filled with spheroidal frass pellets that
increase in size and spread farther apart as the mine widens
(Fig. 17G). Aborted mines (Fig. 17J) have also been included in
DT41. These mines are typically around 0.1 mm in width with no
width increase, and 5 mm or less in length. Other Cretaceous
DT41 mines have a frass-free terminal portion, measuring ca.
2.8 mm in length (Fig. 17I). All examined Paleocene DT41 mines
are filled with frass until termination.
The consistent positioning of mines at the bases of multi-veined
leaves and association with basal primary veins, as found on
Paleocene DT41 on C. genetrix, has not been found on any
examined Cretaceous specimens. These characteristics, plus the
extended threadlike phase followed by an expansion into a
loosened frass trail observed on Z. flabella at Mexican Hat, further
suggest that leaf miners responsible for DT41 mines at Mexican
Hat and in the local Paleocene differed from those of the
Cretaceous.
Table 5. Comparison of leaf mine damage types from the Cretaceous Hell Creek Formation, Williston Basin, North Dakota; local
early Paleocene localities in the Fort Union Formation, Powder River and Williston Basins, of North Dakota and Montana; and
Mexican Hat, Powder River Basin, Montana.
Mine DT Hell Creek Fm. Mexican Hat Local Early Paleocene
DT35 x
DT36 x
DT37 x
DT40 x
DT41 x x
DT42 x x
DT43 x x
DT45 x
DT59 x x
DT60 x
DT65 x
DT90 x
DT91 x x
DT92 x
DT104 x
DT105 x
DT109 x
DT282 x x
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Another potential K-Pg boundary crosser, DT42, (Fig. 6), was
found in the latest Cretaceous of North Dakota (five specimens,
Fig. 6B–C) [11] and Mexican Hat (one specimen, Fig. 6A). Mines
assigned to DT42 are defined by a linear, widening path, which
lacks frass, and ragged margins. All Cretaceous DT42 specimens
differ from the Mexican Hat specimen (Fig. 6A). A typical
Cretaceous DT42 (Fig. 6B) features a linear skeletonized portion
that follows a secondary vein before a dramatic expansion to a
small blotch with thickened reaction tissue. Another Cretaceous
mine (Fig. 6C) follows primary and secondary veins. The mine
expands dramatically towards the end of its path, starting at
0.61 mm and widening to 1.04 mm, although it is possible that
this is an incomplete mine. The 41% width expansion is about half
of the expansion of the Mexican Hat specimen (81%). Tissue at
the end of the Cretaceous mine was completely removed. The
Mexican Hat DT42 (Fig. 6A) expands in width throughout the
preserved portion and is strongly influenced by primary and
secondary venation. Part of the margin is wavy because it is
delimited by tertiary venation. The gradual expansion differs from
most Cretaceous specimens, which exhibit a dramatic widening
Figure 17. Mines assigned to DT41 from the latest Cretaceous of North Dakota and Montana, USA. A: Thin, serpentine mine,
characterized by a gradual width increase and central frass trail (HC81; Somebody’s Garden – DMNH loc. 9824; DMNH 19619). B: Frass-packed
serpentine mine with minimal width increase and angular turns on ‘‘Artocarpus’’ lessigiana (Triceps – DMNH loc. 1855; DMNH 20005). C: Threadlike
mine with looping path (HC34; Allison’s Attachment – DMNH loc. 568; DMNH 20503). D: Leaf mine with tightly coiled, intestiniform path, packed with
frass (Mud Butte, Dean Street – DMNH loc. 428; DMNH 7483). E: Serpentine leaf mine packed with frass (Unidentifiable; Triceps – DMNH loc. 1855;
DMNH 7511). F: Partially preserved serpentine leaf mine with gradual width increase and dispersed, spheroidal frass pellets (arrow expands to G;
Unidentifiable; Triceps – DMNH loc. 1855; DMNH 20000). G: Detail of frass trail in (F) showing dispersed, spheroidal frass pellets. H: Smooth-margined,
linear mine with parallel sides on ‘‘Dryophyllum’’ subfalcatum (Dragonfly – DMNH loc. 565; DMNH 7908). I: Serpentine mine with thin, central frass trail
starting near leaf apex (Skunk Hunt – DMNH loc. 4301; DMNH 35834). J: Aborted mine (HC199 (Laurales); Mud Butte, Dean Street – DMNH loc. 428;
DMNH 7544).
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near termination (Fig. 6B, C). In summary, it is unlikely that the
Cretaceous and Paleocene DT42s represent the same taxa.
Damage type 59 mines are characterized by a linear path
adjoining and following secondary and primary veins. The mine
path increases minimally in width, but it ends with an expanded
ovoidal or ellipsoidal terminal chamber (Fig. 18A). The two
Cretaceous DT59 mines are much smaller than both mines on the
Paleocene specimen (which is from the site Paleocene Leaf,
DMNH loc. 563, and not Mexican Hat; Fig. 18A). The
Cretaceous DT59 in Fig. 18B only follows a secondary vein
before expanding into an ovoidal terminal chamber measuring
5.0767.14 mm. The mine path increases from 0.39 mm wide at
the oviposition site to 0.81 mm wide surrounding the terminal
chamber. The Paleocene DT59 path is 0.56 mm wide and
expands to 1.98 mm wide surrounding the terminal chamber,
which measures 7.12611.83 mm (Fig. 18A). The overall sizes of
the termini of the Paleocene DT59 specimens are much larger
than on the Cretaceous specimens, and the association with both
secondary and primary veins is not observed in either Cretaceous
specimen.
The variety of morphologies within Cretaceous DT41 mines
and other boundary crossing DTs, and the number and identities
of hosts that they are found on, suggest that the diversity of leaf-
mining Lepidoptera was significantly greater during the latest
Cretaceous than previously recognized. Similarly, the lack of
evidence for the survival of any leaf-mining forms over the K-Pg
boundary suggests a near total extinction of leaf miners [11]. In
contrast, the analysis of Paleocene DT41 mines in this study did
not suggest greater diversity than recognized in past studies [19].
With similar collection sizes, DT41 mines were found on over 20
Cretaceous host plants and only 3 host plants in the Paleocene,
further suggesting a major extinction of the local microlepidop-
teran mining fauna.
Comparison of Mexican Hat and other Paleocene insect
damage
Three Mexican Hat leaf mine DTs (DT41, DT91, and DT282)
are found at other local or regional Paleocene sites. Damage type
41 is associated with Z. flabella (Fig. 8F–H) and C. genetrix at
Mexican Hat (Fig. 10E–G) and with C. genetrix at the early late
Paleocene (ca. 59 Ma) sites Haz-Mat, Washakie Basin of
southwestern Wyoming (Fig. 10H–I), and Skeleton Coast, Bighorn
Basin of northwestern Wyoming (Fig. 10J–K). All DT41 mines on
Paleocene C. genetrix arise at the base of the multi-veined leaf and
are usually associated with primary veins. A serpentine path,
packed with frass and increasing in width throughout, defines the
mines. Mine width is initially around 0.1 mm and widens up to
0.95 mm, and margins are typically smooth.
The presence of the C. genetrix-DT41 association at Mexican
Hat, Haz-Mat, and Skeleton Coast shows that this mining
association persisted for at least 6 million years, thus providing a
striking but singular example of a long-lasting leaf-mining
association from Mexican Hat. Earlier studies have also provided
evidence for the persistence of insect-plant relationships over
geologic time. Opler [44] suggested a minimum Miocene age for
eleven leaf mine associations based on the indistinguishable
morphology of extant and fossil mines on related oaks. Hispine
beetle surface-feeding damage has been found on Zingiberales in
the latest Cretaceous (Hell Creek Formation) and regional Eocene,
and the association still occurs today in the Neotropics [84].
Biogeographic evidence has also been used to suggest long-term
insect-plant relationships and to explain modern distributional
patterns [85,86]. Fossil evidence for long-term associations
between plants and insects has usually been found at the family
or genus level, but here we present a one to one insect-plant
association spanning 6 million years that may have involved a
single, or perhaps a few closely related mining species.
Probable mines, assigned to DT282 and associated with P.
raynoldsii (Fig. 3E) and J. glabra (Fig. 4K) at Mexican Hat, are
also found on three ‘‘P.’’ nebrascensis specimens (Fig. 18C–D) at
Pyramid Butte, ND. The morphology is very similar among mines
on all three hosts, although the proximal and terminal portions are
not preserved on any of the specimens, suggesting a taphonomic
bias against preservation of these features. Major veins do not
affect the mine paths, suggesting that the mines traveled through
epidermal tissue. The mines are all poorly preserved, which
prevents detailed comparison of the frass. Despite the poor
preservation, the mines are morphologically similar overall, which
suggests that they were made by a few species of closely related
epidermal miners.
Damage type 91 mines are associated with P.raynoldsii,J.
glabra,Z.flabella, C.genetrix,‘‘P.’’ nebrascensis,B.serrata, and
Dicot leaf morphotype 1 at Mexican Hat, and they are also found
at three other early Paleocene localities (‘‘P.’’ nebrascensis at YPM
loc. 87150, ND; Z. flabella at YPM loc. 8403, ND; unidentifiable
leaf fragment at Pyramid Butte, ND). The mine on ‘‘P.’’
nebrascensis at YPM loc. 87150 (Fig. 18E) is initially serpentine,
with a densely-packed, sinusoidal frass trail composed of spheroi-
dal-ellipsoidal pellets. The terminal portion widens dramatically
into a blotch-like mine filled with ellipsoidal frass pellets. This
morphology is most similar to DT91 mines on P.raynoldsii at
Mexican Hat (Fig. 2), which typically increase dramatically before
termination. The mine on the unidentifiable leaf fragment from
Pyramid Butte, ND (Fig. 18F–G) is incomplete and lacks a
terminal chamber. The preserved portion includes a loosely
packed frass trail, which was deposited in a meniscate pattern
(Fig. 18G). This frass pattern is not present on any Mexican Hat
specimen, and the unique larval behavior necessary to produce the
meniscate shape suggests that a different leaf-mining species
created the mine. The mine on Z. flabella from YPM loc. 8403
(Fig. 18H) is very poorly preserved, but it shares some features
associated with DT91 at Mexican Hat, in particular a continuous,
medial frass trail composed of ellipsoidal-spheroidal pellets. The
poor preservational quality prevents a full comparison of mine
features, but it is probably related to the DT91 mines on Z.
flabella at Mexican Hat.
Although members of the Platanaceae and Cercidiphyllaceae
have been sampled extensively from both the local Cretaceous and
Paleocene [6–8,14,39], we found no evidence that these plant
families provided a refuge for Cretaceous leaf-mining insects
(Table 6, 7). As described above, no mines found on Cretaceous
specimens of either family were found on any confamilial or other
Paleocene species.
Possible causes of leaf miner extinction
Two, intimately related scenarios for the extinction of special-
ized insects at the K-Pg boundary have been suggested: a direct
extinction caused by catastrophic environmental conditions in the
wake of the asteroid impact, and extinction caused by the loss of
host plants [11]. Insect herbivores may track plant chemicals
[87,88] and switch hosts to plants with similar secondary
chemistry, often including closely related plants. Although there
were multiple Platanaceae and Cercidiphyllaceae species during
the latest Cretaceous, and host-switching can occur on ecological
time scales [89,90], the sudden plant extinction and small
surviving host-species pool may not have left enough time or
options for host-switching to occur.
Novel Insect Leaf-Mining after the End-Cretaceous Extinction
PLOS ONE | www.plosone.org 30 July 2014 | Volume 9 | Issue 7 | e103542

Because leaf-mining larvae are overwhelmingly host specialized
[53], a lack of suitable hosts, even for a short time period, could
cause extinction. Low light levels after the Chicxulub impact
caused a decrease in photosynthesis for a period of months to years
[91–93]. Angiosperms that crossed the K-Pg boundary may have
survived in seed banks, germinating when conditions were
suitable. Insect extinction during the extreme conditions directly
after the impact, when plants were not growing, may well explain
the low diversity of mines, even on boundary crossing plants.
Figure 18. Leaf mines assigned to DT59 (A–B), DT282 (C–D), and DT91 (E–H). A: Two linear leaf mines with widened terminal chambers
along the midvein of Paranymphaea crassifolia (DT59; Paleocene; Paleocene Leaf –DMNH loc. 563; DMNH 20055). B: Leaf mine with widened terminal
chamber along secondary vein on leaf morphotype HC265 (DT59; Cretaceous; Battleship – DMNH loc. 900; DMNH 7286). C, D: Probable mines on ‘‘P.’’
nebrascensis from Pyramid Butte, ND (DT282; YPM locality 86107; YPM 9796, YPM 9762). E. Initially serpentine mine with blotch-like terminal chamber
on ‘‘P.’’ nebrascensis (DT91; YPM loc. 87150; YPM 9636) F. Serpentine mine with loosely packed frass trail on an unidentifiable leaf fragment (DT91;
arrow expanded in G; Pyramid Butte - YPM loc. 86107; YPM 168194) G. Detail of frass trail in (F) showing meniscate pattern. H. Serpentine mine with
medial frass trail composed of spheroidal-ellipsoidal pellets on Z.flabella (DT91). Arrow indicates area expanded in the inset, showing detail of the
frass trail (YPM loc. 8403; YPM 9526).
doi:10.1371/journal.pone.0103542.g018
Novel Insect Leaf-Mining after the End-Cretaceous Extinction
PLOS ONE | www.plosone.org 31 July 2014 | Volume 9 | Issue 7 | e103542

Why Mexican Hat? Possible causes of high damage
diversity
What is different about Mexican Hat? The site captures an
otherwise unknown ‘‘decoupled’’ [19] plant-insect ecosystem of
high insect richness on depauperate Paleocene plants, but its flora
and depositional environment were in no way unique for the
region or time period. Its dominant plants were very widespread,
and like Mexican Hat, the sediments of most regional Paleocene
plant localities were deposited in floodplain and swamp environ-
ments [6]. In modern rainforests, plant genera with wide ranges
can support insect faunas with low beta diversity [94], so low
diversity Paleocene floras dominated by widespread species might
be expected to support similar insect faunas across fossil localities.
We did not observe any Paleocene sites with insect damage
diversity comparable to Mexican Hat, even at Lebo Member
localities deposited nearby and at an approximately similar time
(Signal Butte, MT section [14,30,39], ca. 32 km from Mexican
Hat). The high diversity of leaf-mining damage at Mexican Hat,
and the lack of evidence linking the mines to local Cretaceous or
Paleocene localities, instead suggest an influx of novel herbivores
into the area, possibly within a very small spatial or temporal
window.
Differences in preservational quality can contribute to variation
in observed damage diversity among sites, but we do not find this
to be explanatory. Leaf fossils at Mexican Hat are certainly well-
preserved, and fourth or even fifth order venation is typically
visible. There is a noticeable color contrast between the fossil leaf
tissue and rock matrix that may increase leaf-mine visibility. Fossil
preservation varied among localities in this study, ranging from
poorly preserved (sandy sediments or faint fossil preservation) to
preservation comparable to Mexican Hat. Most of the sites that
are closest in age to Mexican Hat from the Williston Basin in
North Dakota and the Powder River Basin in Montana have
poorer preservation, but not nearly so poor, based on our
collective experience, that leaf mines would falsely appear to be
nearly absent. Despite differences in sediment size and preserva-
tion, Cretaceous leaf fossils in coarse-grained sandstones, such as
at Mud Buttes and Battleship, preserved diverse leaf mine DTs on
many host plants. The high preservational quality of the Mexican
Hat flora probably somewhat affected observed insect damage
diversity, but the presence of diverse leaf mine DTs even at the
more poorly preserved Cretaceous sites suggests that causes other
than taphonomic bias led to the patterns observed in this study.
Mexican Hat could represent a rare insect outbreak captured in
the fossil record [95]. There are many causes of outbreaks,
including changes in plant nutritional quality [96], changes in
climate (increases in temperature, drought, etc.; [96,97]), and
reduced top-down control from predators or parasites [98].
However, modern insect outbreaks are typically associated with
a sudden increase in population densities of a single insect
herbivore species, whereas Mexican Hat documents high diversity
at ordinal level, including agromyzid mines, rare hymenopteran
Table 6. Comparison of leaf mine damage types on Cercidiphyllaceae leaves from the Cretaceous Hell Creek Formation, Williston
Basin, North Dakota; local early Paleocene localities from the Fort Union Formation, Powder River and Williston Basins, of North
Dakota and Montana; and Mexican Hat, Powder River Basin, Montana.
Mine DT Hell Creek Fm. Mexican Hat Local Early Paleocene
DT35 X
DT41 X
DT42 X
DT45 X
DT65 X
DT91 X
doi:10.1371/journal.pone.0103542.t006
Table 7. Comparison of mine damage types on Platanaceae leaves from the Cretaceous Hell Creek Formation, Williston Basin,
North Dakota; local early Paleocene localities from the Fort Union Formation, Powder River and Williston Basins, of North Dakota
and Montana; and Mexican Hat, Powder River Basin, Montana.
Mine DT Hell Creek Fm. Mexican Hat Local Early Paleocene
DT35 X
DT36 X
DT41 X
DT42 X
DT43 X
DT45 X
DT65 X
DT91 X
DT104 X
DT282 X X
doi:10.1371/journal.pone.0103542.t007
Novel Insect Leaf-Mining after the End-Cretaceous Extinction
PLOS ONE | www.plosone.org 32 July 2014 | Volume 9 | Issue 7 | e103542

mines on P. raynoldsii, and diverse Lepidopteran mines on seven
host plants, along with a great variety of other damage. Also, fine-
grained leaf-fossil deposits such as Mexican Hat are estimated to
represent 500–1000 years of deposition [99], and insect-damaged
leaves occur abundantly throughout the fossiliferous section, but
insect outbreaks occur on much shorter time scales.
The increase in mining diversity more closely, and plausibly,
resembles rapid increases in insect damage diversity associated with
warming, suggesting that the Mexican Hat assemblage lived during a
transient warming event that brought in a diverse immigrant fauna
of minute flying insects that fed on the depauperate, less mobile plant
assemblage and thus established the decoupled plant and insect
diversity. Insect herbivory, including both damage diversity and
intensity, is highest in the tropics [100,101], implying a correlation
between diversity and temperature. Increased insect herbivory in
response to rising temperatures has been observed in modern
agricultural and natural ecosystems [102,103]. Most importantly
here, several different warming events during the Paleocene-Eocene
transition in Wyoming, including the Paleocene-Eocene Thermal
Maximum (PETM), all led to significant increases in insect damage
diversity, including that of mines and galls, on geologically short time
scales [16,18,34]. High insect damage diversity has also been
observed during the early Eocene climatic optimum in Patagonia
[104] and in the middle Eocene of Germany [105], and thermophilic
formiciine ants migrated across the Arctic into North America from
Europe during hyperthermal conditions between the latest Paleo-
cene and early middle Eocene [106]. Damage type diversity declined
as temperatures decreased after the PETM in the western USA,
suggesting that the prior increase in DT diversity was driven by
climate change, and not a coincidental evolutionary radiation
[16,18]. Lower DT diversity has also been observed during cooler
climates in the Oligocene of Ethiopia [107] and Germany [108].
Short hyperthermal events have been documented in early
Paleocene marine sections, including during the early Danian
(Dan-C2 event [109,110], Top Chron 27 [111] or latest Danian
event [112], and the Danian-Selandian transition event
[113,114]). The Dan-C2 event has been detected in marine
carbonate d
13
C and d
18
O records in the Atlantic Ocean [109] and
western Tethys [110], but not in the Pacific Ocean [115]. A
terrestrial carbon isotope excursion, possibly concurrent with the
Dan-C2 event, has also been observed at the Boltysh crater in
Ukraine [116]. Pollen analysis there revealed an increase and
dominance of thermophilic Normapolles pollen during the carbon
isotope excursion, suggesting a shift to a warmer and drier climate
[116]. The age resolution of the Mexican Hat strata is currently
insufficient to correlate directly to these specific hyperthermal
events, but more broadly, the existence of a number of short-lived
marine hyperthermal events at this time suggests that an
undocumented warming event during Mexican Hat deposition is
a plausible scenario. More precise age constraints for Mexican Hat
and the discovery and sampling of new localities of the same age
are necessary to test the warming hypothesis. Paleogene
hyperthermals were geologically brief, so it is very unlikely that
any other local sites so far known were deposited during the same
short time span. We hypothesize that if localities of precisely the
same age as Mexican Hat are found, they will be associated with
similar levels of damage.
We note that recovery of insect damage diversity after the K-Pg
in the Western Interior USA finally began during late Paleocene
warming, before the recovery of plant diversity [117], and this
could be a general pattern given the potentially faster migration
potential of some insect vs. plant taxa, especially winged insects
with very small body sizes that are typical for the adult phases of
leaf miners. Mexican Hat may represent a ‘‘failed’’ version of this
pattern, wherein the proposed, short-lived increase in temperature
led to an increase in local insect damage diversity via immigration.
Current evidence suggests that impact effects on insect herbivores
decreased with increasing distance from the Chicxulub crater
[118–120], and thus pools of insect species would have existed
elsewhere that could have sourced the influx observed at Mexican
Hat. Insects can be rapidly transported long distances by wind as
aerial plankton, and microlepidopterans, hymenopterans, and
dipterans, including agromyzid flies, have been captured over
oceanic waters 460 km from the nearest land [121]. In contrast, a
short warming interval may not have left enough time for plant
ranges to shift effectively. The low diversity, homogenous plant
species diversity pool across a large geographic area during the
early Paleocene [6–8,14,19] may have also impeded any similarly
rapid increase in plant diversity. Modern ecological studies on
much shorter time scales show a comparable pattern in response to
temperature increase, with a disproportionate increase in insect
biomass compared to plant biomass [122].
Conclusions
Mexican Hat is the only known early Paleocene locality with high
insect damage diversity on a typical, low-diversity flora. Our results
suggest that the diversity of mining insects represented at Mexican
Hat is greater than previously recognized, but there is no evidence
linking any Cretaceous mines with those found at Mexican Hat.
There is also no convincing evidence for the survival of any
Cretaceous leaf miners over the K-Pg boundary regionally, even on
the well-sampled, surviving plant families (Platanaceae and
Cercidiphyllaceae). Cretaceous leaf mines are also much more
morphologically diverse than those of the Paleocene. All these results
suggest a severe regional extinction of leaf-mining insects rather than
the survivorship of Cretaceous insect taxa. Our results strongly
suggest that the high insect damage diversity on the low diversity
Mexican Hat flora is linked to an influx of novel insect herbivores
during the Paleocene, most plausibly caused by an undocumented,
transient warming event that relocated tiny flying insects.
Supporting Information
Table S1 Rescored damage types on Mexican Hat
census collection curated at USNM.
(XLSX)
Table S2 Mexican Hat damage types by host plant.
(XLSX)
Table S3 Leaf mines from local and regional Creta-
ceous and Paleocene localities.
(XLSX)
Appendix S1 Descriptions of new damage types.
(DOCX)
Acknowledgments
This research partly fulfills requirements for a Masters degree in
Geosciences by MPD, 2013. Thanks to E. Belt, T. Bralower, D. Davis,
D. Hughes, and M. Patzkowsky for valuable discussions and feedback;
Harding Land and Cattle Company for previous land access at Mexican
Hat; D. Danehy, T. Menotti, E. Currano, and R. Wilf for field assistance
on the 2004 dig; R. Wilf for commenting on figures; and the staff of the
Yale University Peabody Museum, Denver Museum of Nature and
Science, and Smithsonian National Museum of Natural History for help
with their collections, especially L. Ivy, D. Kline, C. Lucking, A.
Maccracken, I. Miller, and L. Petrie from the DMNS, S. Hu, P. Crane,
and the late L. Hickey from the YPM, and D. Greenwalt, R. Labandeira,
F. Marsh, S. Schachat, and J. Wingerath from the USNM.
Novel Insect Leaf-Mining after the End-Cretaceous Extinction
PLOS ONE | www.plosone.org 33 July 2014 | Volume 9 | Issue 7 | e103542

Author Contributions
Conceived and designed the experiments: MPD PW. Performed the
experiments: MPD. Analyzed the data: MPD. Wrote the paper: MPD.
Contributed substantially to data interpretation: MPD PW CCL KRJ DJP.
Commented on and substantially contributed to the manuscript: PW CCL
KRJ DJP.
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Novel Insect Leaf-Mining after the End-Cretaceous Extinction
PLOS ONE | www.plosone.org 35 July 2014 | Volume 9 | Issue 7 | e103542