Latest Cretaceous (Maastrichtian) cf. Agathis leaves from the Lefipán Formation are preserved with hole feeding, margin feeding, surface feeding, piercing and sucking, mining, galling, and oviposition damage. External foliage feeding (Fig. 1A–C) includes circular holes measuring 0.4–1.0 mm in diameter (DT1; Fig. 1A). The holes are surrounded by a 0.1–0.2 mm wide reaction rim, and the margins of the holes are not influenced by leaf venation. A shallow, semicircular excision into the leaf margin (DT12; Fig. 1B) measures 1.1 mm wide by 0.4 mm deep with a 0.3 mm wide reaction rim. Circular to polylobate patches of surface feeding (DT29; Fig. 1C) measure 1.4–3.9 mm in diameter.

Figure 1. 

Insect damage on cf. Agathis leaves from the Lefipán Formation A circular hole with dark reaction rim (DT1; MPEF-Pb 9835) B small excision into the leaf margin (DT12) C patches of surface feeding (DT29) D cluster of circular piercing and sucking marks (DT46; MPEF-Pb 9837) E dark circular galls (DT32; MPEF-Pb 9829) F elliptical oviposition mark (DT101; MPEF-Pb 9841) G elliptical oviposition mark (DT101) H linear serpentine mines following leaf venation (DT139; MPEF-Pb 9836) I close-up of mines in (H) J detail of frass trail in (H) K oblong blotch mine (DT88; MPEF-Pb 9839).

A cluster of black marks probably represents piercing-and-sucking damage (DT46; Fig. 1D). The marks, or punctures, measure 0.1–0.2 mm in diameter. Some of these punctures are composed of a black rim that surrounds lighter tissue, interpreted as a reaction rim surrounding a pinpoint feeding site.

An oblong blotch mine lacking frass, occurring along the central axis of a leaf, was previously described by Donovan et al. 2020 (Fig. 1K). A serpentine mining association, characterized by a linear path largely confined by parallel venation (DT139; Fig. 1H–J), is also associated with a cf. Agathis leaf. The mine varies in width between 0.5–0.7 mm with no obvious directional width increase. The origin and overall trajectory of the mine, or possibly multiple mines, is difficult to discern because the entire leaf was not recovered, and some detail is obscured by poor preservation (Fig. 1I). Linear mine paths lie between adjacent parallel veins (Fig. 1J), although the mine sporadically crosscuts or straddles the veins. The central frass trail measures 0.3–0.4 mm wide (35–80% of the mine width) and is densely packed. The moderately to tightly sinusoidal frass trail is intermittent and may consist of spheroidal pellets. The lateral margins of the mine are smooth and parallel-sided (Fig. 1J).

Carbonized, oval galls measure 1.0–1.1 mm in maximum diameter by 0.8–0.9 mm in minimum diameter (DT32; Fig. 1E). The long axes of the galls are parallel to the leaf veins. The galls have a slight positive relief relative to the leaf surface.

Oviposition lesions are composed of inner elliptical, disturbed tissues surrounded by scar tissue, such as callus (Fig. 1F, G). The inner disturbed tissue areas measure 0.6 mm long by 0.2 mm wide, and the reaction tissue is 0.1–0.4 mm wide. The oviposition lesions are oriented parallel to major venation.

Insect feeding on early Paleocene (Danian) Agathis immortalis at PL2 in the Salamanca Formation includes hole feeding, margin feeding, surface feeding, piercing and sucking, mining, and galling. External foliage feeding (Fig. 2A–F) includes circular holes measuring 0.3–3.4 mm in diameter (DT1, DT2; Fig. 2A, B) surrounded by 0.2 mm wide reaction rims. Shallow, approximately circular excisions into the leaf margin (DT12; Fig. 2C, D) measure 5.2–33.0 mm long by 1.0–1.8 mm deep into the leaf blade. Reaction tissue surrounding the excisions measures 0.1–0.3 mm wide. Circular to oblong surface feeding zones lack reaction rims (DT29; Fig. 2E, F) and measure 5.0–8.5 mm long by 1.3–2.7 mm wide. Leaf veins within the surface feeding zones are faintly visible or locally not visible.

Figure 2. 

External foliage feeding, piercing and sucking, and leaf mining on Agathis immortalis from Palacio de los Loros 2 A small, circular hole (DT1; MPEF-Pb 6010) B circular and elliptical holes (DT2; MPEF-Pb 6042) C semicircular excision into the leaf margin (DT12; MPEF-Pb 6091) D elongate excision into the leaf margin (DT12; MPEF-Pb 9768) E zones of surface feeding (DT29; MPEF-Pb 9774) F patch of surface feeding (DT29; MPEF-Pb 6030) G cluster of piercing and sucking marks (DT46; MPEF-Pb 9766) H detail of piercing and sucking marks in (G) with central depressions I detail of piercing and sucking marks in (G) (DT46; MPEF-Pb 5959) J detail of piercing and sucking marks with central depressions in (K) (DT46; MPEF-Pb 9779) K clusters of piercing and sucking marks (DT46; MPEF-Pb 5959) L Frondicuniculum flexuosum blotch mine (DT421; paratype MPEF-Pb 6001) M F. flexuosum blotch mine (DT421; holotype MPEF-Pb 5970).

Circular piercing-and-sucking marks (DT46; Fig. 2G–K) measure 0.1–0.3 mm in diameter. Some punctures have a depression at their centers (Fig. 2H–J). The punctures are typically clustered on a leaf.

Agathis immortalis is associated with Frondicuniculum flexuosum, an elongate-ellipsoidal blotch mine with undulatory margins with a wrinkled appearance (DT421; Donovan et al. 2020). The mines are positioned along leaf margins with their long-axes parallel to leaf veins. Frass is deposited as distinct pellets or more amorphous packets, either along one margin of the mine or throughout (Fig. 2L, M). We assign F. flexuosum to DT421 (Suppl. material 1) for use in future versions of the “Guide to Insect (and Other) Damage Types on Compressed Fossil Plants” (Labandeira et al. 2007), and detailed descriptions and an ichnotaxonomic treatment of F. flexuosum were provided by Donovan et al. 2020.

Agathis immortalis is associated with three gall DTs. Distinctive ellipsoidal to near-circular galls with thickened walls (DT115; Fig. 3A–J) measure (1.0) 2.0–5.0 (9.9) mm long by (0.9) 1.5–3.0 (3.9) mm wide. The galls are oriented with their long axes parallel to the leaf veins. The outer walls of the galls are composed of a thickened layer of woody tissue (Fig. 3I, J) preserved as carbonized material and measuring 0.2–0.9 mm wide. The hardened outer wall surrounds unthickened tissue where the internal chamber was located. The galls are situated on epidermal tissue, and epidermal cells are visible in the center of some galls (Fig. 3B–D). However, leaf veins are not visible within the galls. For those galls that preserve epidermal tissue, a small, black splotch, possibly representing the position of the larval chamber or an exit hole, is located near the center (Fig. 3B–D). The thickened outer rims of the galls may have extended over the entire surfaces of these structures (Fig. 3E). Circular galls with an outer rim of thickened tissue surrounding a zone of unthickened tissue (DT11; Fig. 3K) measure 0.7–1.6 mm in diameter. The thickened rim, 0.2–0.4 mm wide, is striated and carbonized. Although common at PL2, this gall type is not found on Agathis at any other locality.

Figure 3. 

Galls on Agathis immortalis from Palacio de los Loros 2 (A–K) A elliptical galls with thickened outer walls surrounding epidermal tissue (DT115; MPEF-Pb 9767) B detail of oval gall with thickened walls in (A) C detail of gall in (B) showing files of epidermal cells and a dot representing the larval chamber or exit hole D detail of cluster of elliptical and circular galls in (A) E elliptical gall covered in carbonized, thickened tissue (DT115; MPEF-Pb 5977) F galls with thickened outer walls and central larval chamber (DT115; MPEF-Pb 6029) G galls with thickened outer walls (DT115; MPEF-Pb 6027) H five galls with carbonized, thickened walls (DT115; MPEF-Pb 9773) I detail of elliptical gall with carbonized, thickened walls in (H) J detail of elliptical gall with carbonized, thickened walls in (H) K circular galls with thickened outer tissue surrounding unthickened inner area (DT11; MPEF-Pb 5983).

The third gall DT associated with Agathis immortalis is defined as a columnar gall protruding above the leaf surface (DT116; Fig. 4A–L). The galls measure 1.1–1.8 mm in diameter and are ornamented with rounded or pointed bumps (Fig. 4E–K), which appear to be arranged concentrically. Some galls have a substantial oval depression near their center (Fig. 4I–L), which may be a feature of the standard morphology of the galls, analogous to extant galls made by Neuroterus numismalis (Hymenoptera, Cynipidae) on oak leaves (Jankiewicz et al. 2017), an exit hole, or a fungal ostiole. Each gall is surrounded by a thin rim of tissue (Fig. 4F–H), which wraps around the top of the gall on some specimens. The tissue rims measure 0.02–0.12 mm wide and approximately 0.3–0.4 mm tall, marked by horizontal and vertical striations (Fig. 4F–H). These columnar galls appear to be deeply set in the leaf tissue and result in a concave pit when removed (Fig. 4A, D). Most specimens are replaced by or filled in with amber derived from ambient leaf resins.

Figure 4. 

Galls (DT116) on Agathis immortalis from Palacio de los Loros 2 A columnar galls and pits where galls were not preserved (MPEF-Pb 6023) B detail of gall in (A) C columnar gall with striated side (MPEF-Pb 6088) D detail of pit in (A) E columnar scale with striated side overlapping the top (MPEF-Pb 5995) F side view of gall in (E) showing horizontal and vertical striations on the ventral cover G side view of gall in (E) H detail of gall in (E) under epifluorescence showing vertical and horizontal striations I columnar gall (MPEF-Pb 5960) J columnar gall preserved as amber (MPEF-Pb 9750) K columnar gall preserved as amber (MPEF-Pb 9750) L SEM image of gall in (J) showing texture (MPEF-Pb 9750).

Enigmatic structures possibly representing armored scale-insect covers (Diaspididae) are associated with leaves (Fig. 5A–G, I–O) and a cone scale (Fig. 5H). The structures, which we refer to as “covers” in the description, may be preserved as amber casts of the dorsal cover and/or ventral cover or “collar” (Fig. 5A–D, F, G, H); impressions of the dorsal covers (Fig. 5E, F, I–M); or impressions where the ventral covers were once located. The dorsal covers are approximately circular to oval, more or less flattened, marked with concentric growth rings, and are surrounded by an attached ventral cover (DT86). Dorsal covers measure 0.87–1.65 mm in maximum diameter by 0.77–1.5 mm in minimum diameter. Concentric growth rings on dorsal covers (Fig. 5A, C, D, K–N) are spaced 0.02–0.08 mm apart. The concentric rings may represent instar growth increments, although the boundaries between the first and second instar and adult phases of the dorsal covers are unclear. An ovoidal bump or pustule (Fig. 5K–N) near the center of the covers in the area presumably made by the first instar nymph may be present, possibly marking the location of the first instar exuviae or the position of stylets from the piercing-and-sucking insects into the subjacent targeted tissues. The ovoidal bumps measure 0.17–0.20 by 0.13–0.16 mm in dimension. On one specimen, rod-like structures radiate from the center of the cover (Fig. 5N, O). The ventral covers that surround the dorsal covers measure 0.06–0.20 mm wide and typically have greater relief than the surrounding unaffected area. The covers share similarities to the galls illustrated in Fig. 4, including the ventral collar and concentric rings, and ovoidal bump or hole on the dorsal side, suggesting that these structures may be related.

Figure 5. 

Enigmatic structures, possibly armored scale insect (Diaspididae) covers, (DT86) on Agathis leaves (A–G, I–M) and a cone scale (H) from Palacio de los Loros 2 A cover with concentric growth rings (MPEF-Pb 6096) B cover with concentric growth rings (MPEF-Pb 6096) C cover in (A) under epifluorescence D detail of (C) showing concentric growth rings E clusters of cover impressions (MPEF-Pb 6113) F embedded ventral cover (MPEF-Pb 6020) G embedded ventral cover (MPEF-Pb 5985) H cover preserved as amber (MPEF-Pb 5861) I impression of cover (MPEF-Pb 5996) J impressions of two covers (MPEF-Pb 5996) K detail of upper cover impression in (J) L detail of cover impression in (I) M detail of lower cover in (J) showing concentric growth rings N cover showing rod-like structures (MPEF-Pb 9750) O close-up of rod-like structures in (N).

Insect and pathogen damage on early Eocene A. zamunerae at LH in the Huitrera Formation includes hole feeding, margin feeding, surface feeding, piercing and sucking, mining, galling, and a rust fungus. External foliage feeding (Fig. 6A–G) includes circular to elliptical holes measuring 0.2–1.1 mm in length and 0.1–0.7 mm in width (DT1; Fig. 6A). The holes are surrounded by thin reaction rims, which measure <0.1 mm wide. A slot-feeding hole (DT8; Fig. 6B) measures 2.8 mm long and 0.3–0.6 mm wide with a 0.1 mm wide reaction rim. The long axis of the hole is parallel to the leaf veins. Shallow, approximately circular excisions into the leaf margins (DT12, Fig. 6C–F) measure 1.0–28.2 mm long and 0.2–2.8 mm deep. Flaps of unconsumed, apparently necrotic tissue measure 0.1–0.5 mm wide, and veinal stringers may be present (Fig. 6F). Polylobate surface feeding zones with reaction rims (DT30; Fig. 6G) measure 1.3–3.0 mm long by 0.2–1.3 mm wide. Reaction rims measure 0.2 mm wide.

Figure 6. 

External foliage feeding, leaf mining, and rust fungus damage on Agathis zamunerae from Laguna del Hunco A oval hole (DT1; MPEF-Pb 6328) B parallel-sided slot feeding (DT8; MPEF-Pb 6368) C excision into the leaf margin (DT12; MPEF-Pb 6329) D adjacent, shallow excisions into the leaf margin (DT12; MPEF-Pb 6311) E shallow excision into the leaf margin with veinal stringers (DT12; MPEF-Pb 6361) F detail of veinal stringers and reaction tissue in (E) G small zone of surface feeding (DT29; MPEF-Pb 6356) H circular to elliptical piercing and sucking marks (DT46; MPEF-Pb 6303) I Frondicuniculum lineacurvum blotch mine (holotype MPEF-Pb 6336) J probable blotch mine with breached epidermal tissue (DT251; MPEF-Pb 6361) K two probable blotch mines with breached epidermal tissue (DT251) and a rust fungus (MPEF-Pb 6303) L detail of blotch mine in (K) M dark, circular gall with slight relief (DT32; MPEF-Pb 6346) N concentric rings of aecia on a rust fungus spot (MPEF-Pb 6303) O detail of rust fungus in (N) P detail of two aecia in (N).

Circular to elliptical piercing-and-sucking punctures (DT46; Fig. 6H) are characterized by a black spot or a rim encircling an inner circle of lighter tissue where the puncture was located. These piercing-and-sucking punctures measure 0.11–0.18 mm in minimum and 0.14–0.22 in maximum diameter.

Agathis zamunerae is associated with the ichnotaxon Frondicuniculum lineacurvum, an oblong blotch mine with smooth, gently curving margins described previously (Donovan et al. 2020). The mines follow the leaf axis, and their long axes are parallel to leaf veins. Frass, including amorphous matter and spherical to hemispherical pellets locally replaced by amber, is deposited mostly along the mine margin when present (Fig. 6I). Putative linear blotch mines with smooth margins and breached epidermal tissue are also found on A. zamunerae (DT251; Fig. 6J–L). Epidermal tissue was breached on most specimens, leaving flaps of tissue along the internal margin of the mine, although epidermal tissue is still preserved across the width of some mines (Fig. 6L). Detailed descriptions of these mines, including the ichnotaxomic treatment of F. lineacurvum, were provided by Donovan et al. 2020.

A dark, oval gall measures 3.4 mm long by 2.8 mm wide (DT32; Fig. 6M). The long axis of the gall is parallel to the leaf venation. The gall has slight relief above the leaf surface and is characterized by a smooth, carbonized texture.

Enigmatic structures, possibly representing female diaspidid covers, are flattened, approximately circular to oval, and measure 0.90–1.63 mm in diameter (DT86; Figs 7–9). In our descriptions, we use the term “covers” to refer to these structures. The dorsal covers are marked by concentric rings (Figs 7B–D, H–K, 8H, I, 9F, G), which may represent cover growth increments during the first and second instar and adult phases. The first instar covers measure 0.31–0.44 mm minimum by 0.29–0.57 mm maximum diameter. The covers constructed by the first instars are marked by an ovoid depression along the edge of the cover, which may indicate the position of the remnant of the first instar exuviae or the stylet fascicle as it penetrated subjacent epidermal and deeper tissues (Figs 7H–K, 8G–I). The ovoid depressions measure 0.22–0.24 mm long by 0.14–0.18 mm wide, with the long axes typically parallel to the maximum diameter of the entire dorsal cover. Material added by second instars increases the diameters of the dorsal covers to 0.58–0.92 mm by 0.62–0.75 mm, and the fully formed adult dorsal covers measure 0.7–1.47 mm by 0.7–1.33 mm. Each dorsal cover is surrounded by a ventral cover (Figs 8J–L, 9C–E), which is deeply embedded in the leaf tissue and measures 0.04–0.15 mm wide. The tops of the ventral covers typically protrude above the leaf surface and the dorsal covers. Although the entire heights of the ventral covers are not usually visible, measured heights are 0.55–0.75 mm. Some scales are only represented by pits (Fig. 8E) or depressed rims where the ventral cover was originally positioned but subsequently detached (Figs 7E, 9I, J).

Figure 7. 

Enigmatic structures, possibly armored scale insect (Diaspididae) covers (DT86) on Agathis zamunerae branches (A–D) and leaves (E–K) from Laguna del Hunco A impressions of covers (MPEF-Pb 6307) B two covers on a branch in (A) C detail of cover with concentric growth rings in (A) D detail of cover with concentric growth rings in (A) E depression where ventral cover was positioned (MPEF-Pb 6349) F depression where cover was probably located (MPEF-Pb 6360) G possible scale insect covers (MPEF-Pb 6383) H detail of cover in (G) I cover in (H) under epifluorescence showing concentric growth rings J detail of cover in (G) K cover in (J) under epifluorescence showing concentric growth rings.

Figure 8. 

Enigmatic structures, possibly armored scale insect (Diaspididae) covers (DT86) on Agathis zamunerae from Laguna del Hunco (MPEF-Pb 6324) A covers on leaves and a branch B row of covers along the central axis of a leaf C four covers D dorsal and ventral covers E impression of cover F five covers G ventral cover and poorly preserved dorsal cover H impression of dorsal cover with concentric growth rings I dorsal cover with concentric growth rings J view of ventral cover under epifluorescence K protruding ventral cover with horizontal and vertical striations L ventral cover with horizontal and vertical striations under epifluorescence.

Figure 9. 

Enigmatic structures, possibly armored scale insect (Diaspididae) covers on Agathis zamunerae from Laguna del Hunco (MPEF-Pb 9842) A cover impressions and amber casts on leaves and a branch B raised ventral cover C dorsal cover surrounded by exposed, vertically-oriented ventral cover D detail of ventral cover showing horizontal and vertical striations E ventral cover in (D) under epifluorescence F dorsal cover with concentric growth rings G dorsal cover in (F) H counterpart to (A) I impression of ventral cover in (H) J impressions of ventral covers in (H).

A probable rust fungus (Fig. 6K, N–P) developed as a gall on which nearly concentric rings of circular to oval aecia (aeciospore-producing structures) are embedded. The aecia are 0.45–1.28 mm in diameter and have slight relief relative to the leaf surface (Fig. 6P). A depressed rim, 0.10–0.25 mm wide, surrounds each aecium, suggesting that the aecia were deep-set in the host tissue and/or that the aecia were cupulate. The circular gall in which the aecia are embedded is delimited by a depressed rim (0.3 mm wide), most clearly visible on the basal side of the gall as oriented in Fig. 6P. Leaf veins within the spot are distorted, possibly caused by thickened tissue growth. The gall measures 13.2 mm in the direction parallel to the leaf veins, and it spans the width of the leaf (15.6 mm).

Insect damage on early middle Eocene A. zamunerae at RP in La Huitrera Formation includes margin feeding, skeletonization, and mining. External foliage feeding (Fig. 10A, B) includes an excision into the leaf margin composed of two, adjacent, approximately circular feeding areas (DT12; Fig. 10A) that measure 13 mm across and are incised 1.8 and 3.7 mm, respectively. Ragged reaction tissue with visible vein stringers measures 0.6–0.8 mm wide. A patch of skeletonized tissue composed of multiple, closely spaced, holes with some leaf veins intact is surrounded by a dark reaction rim (DT17; Fig. 10B) measuring 0.3–0.5 mm wide.

Figure 10. 

External foliage feeding and leaf mines on Agathis zamunerae leaf from Río Pichileufú A arcuate excisions into the leaf margin (DT12; BAR 5002-20) B zone of skeletonized tissue with reaction rims (DT17; USNM 545229) C Frondicuniculum lineacurvum blotch mine (paratype USNM 545226) D probable blotch mines with breached epidermal tissue (DT251; USNM 545227) E detail of blotch mine in (D).

The ichnotaxon Frondicuniculum lineacurvum, an elongate-ellipsoidal blotch mine with smooth margins, occurs on A. zamunerae at both RP and LH, as previously reported (Donovan et al. 2020). The best-preserved example occupies approximately 85% of a leaf surface (Fig. 10C). Putative oblong blotch mines with flaps of unconsumed tissues along the rims of the mines are also found on A. zamunerae at RP (DT251; Fig. 10D, E). Detailed descriptions of these mines were provided by Donovan et al. 2020.

Enigmatic structures resembling female diaspidid covers (Figs 11, 12) are similar to those found at LH. Dorsal covers measure 0.70–1.08 mm in diameter and are marked by two concentric rings (0.40 by 0.38 mm and 0.53–0.55 by 0.55–0.57 mm wide, respectively), possibly representing instar growth increments (DT86; Fig. 11). The first instar covers are usually associated with an oval or semicircular depression (0.22–0.23 mm long by 0.15–0.16 mm wide; Fig. 11D–I) surrounded by a raised rim or flange (0.02–0.06 mm wide; Fig. 11H). Dorsal covers are marked by small circular to ellipsoidal shapes visible under epifluorescence (Fig. 11E, G, I), which correspond to bumps on the surface. The bumps measure 0.01–0.02 mm in diameter and decrease in size moving from the first instar through the adult portions of the scales. The first instar covers appear to be thicker than the material added by second instars and adults (Fig. 11E, G, I), probably attributable to the presence of exuviae. A deeply-set ventral cover (0.02–0.06 mm wide) surrounds each dorsal cover (Fig. 12A–C). Ovoid pits may represent where covers were originally positioned (Fig. 12D–G).

Figure 11. 

Enigmatic structures, possibly armored scale insect (Diaspididae) covers (DT86) on an Agathis zamunerae leaf from Río Pichileufú (USNM 545228) A leaf with covers B dorsal covers C dorsal covers D dorsal cover with concentric growth rings E cover in (E) under epifluorescence F dorsal cover with concentric growth rings G cover in (F) under epifluorescence H detail of rim surrounding semicircular impression in (G) I dorsal cover under epifluorescence showing concentric growth rings J dorsal cover with concentric growth rings K dorsal cover with concentric growth rings.

Figure 12. 

Enigmatic structures, possibly armored scale insect (Diaspididae) cover impressions (DT86) on Agathis zamunerae from Río Pichileufú A depressions where ventral covers were positioned (USNM 545223) B detail of lower depression in (A) C rim where ventral cover was positioned (USNM 545223) D pits where scale insects were possibly positioned (USNM 545226) E pit where scale insect was possibly located (USNM 545226) F pit surrounded by reaction tissue where scale insect was possibly positioned (USNM 545226) G Pit where scale insect was possibly located (USNM 545226).

Relatively few herbivorous insect associations with extant Agathis have been documented in the literature, as summarized by Donovan et al. (2020). Here, we provide brief descriptions of insect and fungal damage that we observed on Agathis herbarium specimens. Although not intended to be a comprehensive survey, the damage is representative of the diversity we found while looking for analogs to the fossil damage on thousands of herbarium sheets in several major herbaria. Much of this extant damage diversity that resembles the fossils is from unknown culprits, representing a major opportunity to discover living insect diversity and evolutionary history based on fossil analogs. Some of the leaf mines were previously illustrated by Donovan et al. (2020) but are re-illustrated and briefly mentioned in the text here for completeness. Extant Agathis species delimitations and range information follows (Farjon 2010). See the following section for comparisons of the fossil and extant damage.

On Agathis australis (Fig. 13; from the North Island of New Zealand), we observed external foliage feeding, including arcuate margin feeding (DT12; Fig. 13A) and removal of the leaf apex (DT13; Fig. 13B). A flap of dead tissue bordered by a black reaction rim forms at the sites of external foliage feeding damage (Fig. 13A, B). Galls are black, circular to ellipsoidal, with minor relief compared to the leaf surface (Fig. 13C–E). Most galls are single-chambered, but some are conjoined or are multi-chambered with exit holes. Galls were found on the adaxial leaf surfaces, and the long axes of the galls tend to be oriented parallel to leaf veins. Finally, Parectopa leucocyma (Lepidoptera, Gracillariidae) (Wise 1962) mines are common, typically characterized by an initial blotch phase transitioning into a serpentine trail. The mines follow the leaf margin and end in a gall near the petiole (Fig. 13F, G).

Figure 13. 

Insect damage on Agathis australis from New Zealand A excision into the leaf margin (Capt. Wilkes, U.S.N., 1838-42 (GH)) B excision through the leaf apex (Capt. Wilkes, U.S.N., 1838-42 (GH)) C blister galls with exit holes (K 0000230) D detail of blister galls in (C) E black, ellipsoidal galls (E.H. Wilson, February 2, 1921 (A)) F Parectopa leucocyma (Gracillariidae) moth mine (Capt. Wilkes, U.S.N., 1838-42 (GH)) G Parectopa leucocyma mines (K 000553313).

Agathis lanceolata (New Caledonia; Fig. 14A–E) is associated with external foliage feeding, including semicircular excisions into the leaf margin (DT12) and removal of the leaf apex (DT13). Galls on A. lanceolata include ellipsoidal blisters oriented parallel to leaf veins (Fig. 14A). Houard (1914, 1922) briefly described ellipsoidal blister galls on the upper surfaces of A. lanceolata leaves. The galls were not illustrated, but he may have been referring to galls similar to those in Fig. 14A. A dark, circular gall with a flattened top and a central indentation was found on a cultivated tree in New Caledonia (Fig. 14B). A curved serpentine mine with smooth margins is packed with frass (Fig. 14C). Two blotches of indeterminate origins, possibly induced by pathogens (fungi, viruses, or bacteria), were found on A. lanceolata. First, a teardrop shaped blotch with raised epidermal tissue that measures 15.1 mm long by 7.7–8.7 mm in width (Fig. 14D) has an acuminate upper margin and is surrounded by a wrinkled reaction rim 0.3–0.4 mm wide. The second blotch is oblong and is positioned along the leaf edge (Fig. 14E). The blotch is 14.4 mm long by 1.4–3.1 mm wide.

Figure 14. 

Insect and pathogen damage on Agathis lanceolata (A–E), A. montana (F), A. moorei (G–K), A. ovata (L–P) from New Caledonia A ellipsoidal blister galls with exit holes (K 000553125) B black columnar gall (E 00119757) C Serpentine mine (K 000553127) D teardrop-shaped blotch damage (K 0000305) E linear blotch damage (GH 2372 B) F elongate mine along the leaf margin (A 8571) G excision into the leaf margin surrounded by reaction tissue and exuding resin (E 00106192) H circular gall (K 0000357) I linear blotch mines with breached epidermal tissue on the right mine (K 000553352) J blotch mines (K 000553352) K blotch mine (K 000553352) L, M galls with thickened margins surrounding epidermal tissue with circular exit holes (E 00399687) N Chrysomphalus aonidum (Diaspididae) scale insects (E 00036880) O diaspidid scale insect covers (K 0000291) P diaspidid scale insect covers (K 00053124).

On Agathis montana (New Caledonia), we observed an elongate mine following the margin of a single leaf (Fig. 14F). The mine measures 59.7 mm long by 0.2–4.3 mm wide and is surrounded by a 0.1 mm wide reaction rim. The mine begins near the base of the leaf and follows the leaf margin, terminating at the leaf apex. The mine gradually widens as the miner consumed leaf tissue, except for two expanded protrusions, and spans the width of the leaf at the apex. The lateral margins of the mine are smooth, and linear to gently undulous frass appears to be composed of spheroidal pellets.

Agathis moorei (New Caledonia; Fig. 14G–K) is associated with shallow, arcuate margin-feeding excisions (DT12; Fig. 14G) flanked by lighter colored dead tissue and dark reaction rims. The wounded edges may exude resin. Galls are circular with minor relief and a raised black rim (Fig. 14H). Full-depth blotch mines are falcate or linear and smooth with parallel margins (Fig. 14I–K). Epidermal tissue was weathered away in some specimens (Fig. 14I, J).

Agathis ovata (New Caledonia; Fig. 14L–P) is associated with ellipsoidal to ovate galls with a dark, thickened rim (Fig. 14L, M). The tops of the galls are flat and consist of epidermal tissue with distinct files of cellular proliferations and a circular exit hole. Diaspidids, including Chrysomphalus aonidum (Fig. 14N) and unidentified species (Fig. 14O, P) also occur on this species.

Agathis macrophylla (Fiji, Vanuatu, and the Solomon Islands; Fig. 15A–O) is associated with semicircular excisions into the leaf margin (DT12; Fig. 15A) and feeding through the leaf apex (DT13) with flaps of dead tissue bordered by a rim of darkened tissue. Surface feeding consists of thin, fairly linear files of damaged tissue (Fig. 15B). Galls include hemispherical, black protrusions from the adaxial surface (Fig. 15C) of leaves and small, circular blister galls with central exit holes. Serpentine mines are curved and end in an elliptical terminal chamber (Fig. 15D) or travel in a zigzag pattern and terminate with a gall at the leaf base (Fig. 15E, F). The terminal gall is similar to mines made by Parectopa leucocyma on Agathis australis in New Zealand, although P. leucocyma mines have an initial blotch phase (Wise 1962) and do not mine in a zigzag pattern. Elongate, oblong to elliptical blotch mines are positioned along leaf margins with their long axes parallel to leaf veins (Fig. 15G–I). Diaspidid scale insects cause piercing-and-sucking damage, leaving a discolored orange mark where the scale cover was located (Fig. 15L, M). Pit-gall-inducing diaspidids are also associated with leaves of A. macrophylla (Fig. 15N, O). The only other gall-inducing diaspidid that has been documented on conifers is Leucaspis podocarpi, which makes leaf margin rolls on Prumnopitys taxifolia (Podocarpaceae) in New Zealand. Leucaspis podocarpi is also associated with Podocarpus cunninghamii Colenso and Podocarpus totara G.Benn ex D.Don (Podocarpaceae), but it does not induce galls on these species (Henderson and Martin 2006). A blotch, probably fungal-induced, occurs along the leaf margin (Fig. 15J). Kauri rust (Aecidium fragiforme) fungal galls are also common (Fig. 15K).

Figure 15. 

Insect and fungal damage on Agathis macrophylla (A–M) A semicircular excision into the leaf margin (K 0000265) B adjacent rows of surface feeding (Fiji, K B40) C round, black gall (New Caledonia, E 00131862) D serpentine mine with elliptical terminal chamber (Fiji, E 0000340) E serpentine mine terminating in a gall (Fiji, K 0000346) F serpentine mine terminating in a gall (Fiji, K 0000346) G possible blotch mine along leaf margin (Fiji, K 0000322) H elongate blotch mine (Fiji, K 0000327) I blotch mine along the leaf margin (Fiji, K 0000327) J fungal blotch along the leaf margin (Fiji, K 16421) K Kauri rust (Araucariomyces fragiformis) (Vanuatu, S.F. Kajewski 282 (K)) L armored scale insect (Diaspididae) (Fiji, K 350) M diaspidid scale insect (Fiji, E 00127892) N, O pit gall-inducing scale insects (Fiji, GH 01153259).

Agathis atropurpurea (Queensland, Australia; Fig. 16A–C) is associated with slot feeding (DT8; Fig. 16A) characterized by a thin rim of necrotic tissue along the inside of the hole. Ovoid to elliptical and polylobate blister galls are positioned on the adaxial sides of leaves and are marked by centrally located circular exit holes (Fig. 16B). An oblong blotch mine with smooth, gently curving margins is positioned near the leaf margin (Fig. 16C).

Figure 16. 

Insect damage on Agathis atropurpurea (A–C), A. microstachya (D–F), and A. robusta (Fig. I–R) A slot feeding (Australia, K 000553290) B blister galls with circular exit holes (Australia, K 000553290) C elongate blotch mine (Mount Bartle Frere leaf litter, Queensland, Australia) D ellipsoidal blotch mine (Australia, K 0000199) E blotch mine lacking epidermal tissue (Australia, K 0000199) F ellipsoidal blotch mine (Australia, E 00210640) G ellipsoidal blotch mines (Australia, K 111455) H blotch mine (Queensland, Australia; A.K. Irvine 00417 (A)) I ellipsoidal blister galls with exit holes (Queensland, Australia, K 000553277) J serpentine mine (Australia, CANB 590252) K serpentine mine with ellipsoidal terminal chamber (Australia, K 000553286) L ovate blotch, probable pathogen damage (Australia, NSW 381650) M ovate blotch, probable pathogen damage (Australia, NSW 381650) N elongate fungal blotch (Queensland, Australia, A 01153261) O ellipsoidal blister galls with exit holes (New Guinea, K 000553264) P circular blister galls with exit holes (New Guinea, E 00127882) Q globose, black gall (New Guinea, E 00127880) R coccid scale insect (New Guinea, K 000553265).

Agathis microstachya (Queensland, Australia; Fig. 16D–F) is associated with elongate-ellipsoidal blotch mines that have smooth margins positioned with their long axes parallel to the leaf veins. In one specimen (Fig. 16E), the epidermal tissue was removed due to weathering, revealing an abraded inner texture caused by insect feeding.

Agathis robusta occurs in both Queensland, Australia (Fig. 16G–K) and New Guinea (Fig. 16L–O). In the Australian material, we found semicircular excisions along the leaf margin (DT12) and apex feeding (DT13). Probable blotch mines with breached epidermal tissue have a slot-like appearance. Ellipsoidal blister galls are characterized by a central exit hole and are surrounded by black rims (Fig. 16I). Serpentine mines are tightly wound (Fig. 16J, K) and some end in an elliptical terminal chamber (Fig. 16K). Ovate blotches caused by pathogens are positioned near the central axes of leaves (Fig. 16L, M) and measure 17.7–20.5 mm long by 8.6–8.9 mm at their widest diameter. The margins of the blotches are minutely ragged and surrounded by 0.1–0.2 mm wide reaction rims. Elongate fungal blotches occur along the leaf margin (Fig. 16N). In New Guinea specimens, A. robusta is associated with small circular holes (DT2). Blister galls on A. robusta in New Guinea (Fig. 16O, P) are indistinguishable from those found on Australian members of the species (Fig. 16I). Globose, black galls (Fig. 16Q) and a single coccid scale insect (Fig. 16R) were also found on the leaf surface.

Agathis labillardieri (New Guinea; Fig. 17A–E) is associated with external foliage feeding, including small circular holes (DT2), slot feeding (DT8; Fig. 17A), semicircular excisions into the leaf margin (DT12), apex feeding (DT13), and linear traces of surface feeding that parallel the leaf veins. Galls include densely packed blisters with single or multiple chambers (Fig. 17B, D) and elliptical galls composed of brown, thickened tissue (Fig. 17C). Sinusoidal serpentine mines also occur on A. labillardieri (Fig. 17E).

Figure 17. 

Insect damage on Agathis labillardieri (A–E) and A. dammara (F–U) A slot feeding (New Guinea, K 0000171) B densely-packed blister galls (New Guinea, K 0000161) C elliptical galls composed of brown, thickened tissue (New Guinea, A 63040) D circular blister galls with exit holes (New Guinea, E 00036878) E sinusoidal serpentine mine (New Guinea, SING NIFS bb.30358) F feeding along the leaf apex (Maluku Islands, K 000553215) G polylobate blister galls with exit holes (Maluku Islands, A 106) H blister gall with circular exit hole (Maluku Islands, A 106) I serpentine mine with thin frass trail (Maluku Islands, A 121) J ellipsoidal blotch mine (Maluku Islands, A 121) K serpentine mine (Maluku Islands, K 0000041) L serpentine mine ending in blotch (Maluku Islands, K 0000078) M elongate rows of parallel surface feeding (Brunei, A 4330) N slot feeding (Sulawesi, Indonesia, K 0000067) O serpentine mine terminating in a blotch (Sulawesi, Indonesia, K 0000067) P serpentine mine with oval terminal chamber (Sulawesi, Indonesia, K 516) Q slot feeding (Philippines, K 0000137) R excision into the leaf margin that expands towards the center of a leaf (Philippines, K 3091) S linear serpentine mine (Philippines, K 3091) T globose woody gall (Philippines, K 000553230) U blister gall with central exit hole and darkened rim (Java, Indonesia, E 00420594).

On Agathis dammara (eastern Malesia; Fig. 17F–U), external foliage feeding includes circular (DT2) and polylobate (DT5) holes, slot feeding (DT8; Fig. 17N, Q), arcuate excisions (DT12), feeding through the leaf apex (DT13; Fig. 17F), and trenched excisions into the leaf margin that expand towards the center of the leaf (DT15; Fig. 17R). Elongate, squiggly rows of surface feeding run parallel to each other (Fig. 17M). Circular to polylobate blister galls with central circular exit holes (Fig. 17G, H, U) and a globose woody gall (Fig. 17T) are found on the adaxial surfaces of leaves. Various serpentine mine morphologies occur on A. dammara, including overlapping (Fig. 17I), forking (Fig. 17K), or linear trails (Fig. 17S) that end in an elongate blotch (Fig. 17L), polylobate terminal chamber (Fig. 17O), or a gall (Fig. 17P). Elongate-ellipsoidal blotch mines also occur (Fig. 18J).

Agathis borneensis (Borneo to Sumatra; Fig. 18A–L) is associated with external foliage feeding, including arcuate margin feeding (DT12; Fig. 18A) and apex feeding (DT13) with a thin band of thickened reaction tissue. Galls on the adaxial leaf surface include ellipsoidal to polylobate blister-like bumps with circular exit holes (Fig. 18B) and hardened, cylindrical protrusions with flat tops (Fig. 18C). A coccid scale insect or psyllid (Fig. 18D) was found associated with A. borneensis. Mines include winding or overlapping serpentine trails (Fig. 18E, F), serpentine mines transitioning into an oval terminal chamber (Fig. 18G), and oblong to elliptical blotch mines (Fig. 18H–L). The presence of silk in one of the blotch mines suggests that it was created by a lepidopteran caterpillar (Fig. 18I, J).

Figure 18. 

Insect damage on Agathis borneensis (A–L), A. lenticula (M–P), A. kinabaluensis (Q–S), A. flavescens (T–W) A arcuate margin feeding (Sarawak, Malaysia, K 17672) B ellipsoidal to polylobate blister galls with exit holes (Kalimantan, Indonesia, A B1468) C hardened, cylindrical galls with flat tops (Brunei, K 000553181) D coccid scale insect (Kalimantan, Indonesia, E 00032242) E serpentine mine (Penang Island, Malaysia, K 000553157) F sinusoidal serpentine mine (Penang Island, Malaysia, SING 90799) G serpentine mine with terminal chamber (Sulawesi, Indonesia, A 4109) H elongate blotch mine (Sarawak, Malaysia, K 000553188) I blotch mine with silk (Brunei, K 000553179) J detail of silk in mine in (I) K blotch mine containing frass (Brunei, SING 91231) L close-up of frass in blotch mine in (K) M arcuate margin feeding (Sabah, Malaysia, K 000553185) N adjacent rows of surface feeding (Sabah, Malaysia, K 000553187) O circular to polylobate galls with flat tops and exit holes (Sabah, Malaysia, K 000553185) P elongate blotch mine along the leaf margin (Sabah, Malaysia, K 000553187) Q circular to ellipsoidal galls with exit holes (Sabah, Malaysia, K 000553243) R circular, raised galls (Sabah, Malaysia, K 000553243) S tightly overlapping serpentine mine (Sabah, Malaysia, K P644) T shallow excisions into the leaf margin U blister galls surrounded by a thin black rim (Malaysia, A P544) V serpentine mines packed with frass (Malaysia, A P542) W overlapping serpentine mine (Malaysia, A P542).

On Agathis lenticula (Sabah, Malaysian Borneo; Fig. 18M–P), external foliage feeding includes arcuate excisions into the leaf margin (DT12; Fig. 18M) and curved, adjacent rows of surface feeding (Fig. 18N). Galls are circular to polylobate with flat tops, consist of one or two chambers, and have circular exit holes (Fig. 18O). A possible elongate, elliptical blotch mine or path of pathogen damage occurs along the leaf margin (Fig. 18P) measures 30.7 mm long by 1.0–4.7 mm wide. The miner targeted upper leaf tissue, leaving a thin layer of epidermal tissue intact. The mine has smooth margins, and a 0.1 mm wide enveloping reaction rim. The specimen was damaged, and much of the inner tissue was lost. No evidence of frass is present in the mine.

Agathis kinabaluensis (Mt. Kinabalu, Sabah, and Mt. Murud, Sarawak, Malaysian Borneo; Fig. 18Q–S) is associated with galls characterized by hemispherical eminences (Fig. 18Q, R), typically with their long axes oriented parallel to the leaf veins. Some galls are surrounded by a black rim and many have a central circular to elliptical exit hole. The galls appear to be single-chambered. Serpentine mines are characterized by an overlapping path filled with frass in some portions (Fig. 18S).

Agathis flavescens, restricted to two mountains in Peninsular Malaysia (Fig. 18T–W), is associated with shallow excisions into leaf margins with black reaction rims (DT12; Fig. 18T). Blister galls surrounded by a thin, black rim and associated with a central, circular exit hole (Fig. 18U) occur on the adaxial surfaces of leaves. Serpentine mines are characterized by densely packed frass, slightly increasing width, and either a curvilinear (Fig. 18V) or overlapping trajectory (Fig. 18W).

Overall, similar external-feeding damage is found at all four Cretaceous–middle Eocene fossil sites (Table 1). Small, circular holes (DT1) are associated with Agathis leaves during the Late Cretaceous (Lef; Fig. 1A), early Paleocene (PL2; Fig. 2A, B), and early Eocene (LH; Fig. 6A). Arcuate excisions into the leaf margins (DT12) occur at Lef (Fig. 1B), PL2 (Fig. 2C, D), LH (Fig. 6C–F), and middle Eocene RP (Fig. 10A); and zones of surface abrasions are found at Lef (Fig. 1C), PL2 (Fig. 2E, F), and LH (Fig. 6G). External foliage feeding damage is made by insects with mandibulate, chewing mouthparts across orders such as Lepidoptera, Coleoptera, and Orthoptera, many of which can produce comparable damage types (Carvalho et al. 2014). Although the presence of similar external foliage feeding damage on Agathis at multiple fossil localities does not necessarily suggest that related insects inflicted the damage, the diversity of damage at all fossil sites suggests that Agathis species hosted an array of externally feeding herbivores, similar to extant members of the genus (Donovan et al. 2020). Previously documented, extant, external foliage-feeding insects on Agathis include chrysomelid and curculionid beetles (Ecroyd 1982), geometrid and tortricid moths (Ecroyd 1982; Nuttall 1983), and phasmids (Plant-SyNZ database 2017) on Agathis australis in New Zealand. In addition, we found numerous examples of external foliage feeding on herbarium specimens throughout the range of the genus (Figs 13A, B, 14G, 15A, B, 16A, 17A, F, M, N, Q, R, 18A, M, N, T).

We found similar circular piercing and sucking marks (DT46) in fossil assemblages spanning the latest Cretaceous to early Eocene (Lef – Fig. 1D; PL2 – Fig. 2G–J; and LH – Fig. 6H), suggesting long-term associations of Agathis with hemipteran insects. Evidence for piercing and sucking is common on angiosperms from Lef, PL2 (Donovan et al. 2017), and LH (Wilf et al. 2005a), and on the conifer Retrophyllum (Podocarpaceae) from Lef and LH (Wilf et al. 2017). On extant Agathis, all known hemipteran associations are scale insects (Figs 14N–P, 15L–N, 16R; Cohic 1958; Williams 1985; Brun and Chazeau 1986; Cox 1987; Williams and Watson 1990; Ben-Dov 1994; Mille et al. 2016), although undocumented associations involving other hemipteran and thysanopteran groups are probable. In addition, kauri thrips, Oxythrips agathidis Morison (Thysanoptera, Thripidae), probe A. robusta leaves in Queensland, Australia, and can cause major defoliation (Morison 1941; Heather and Schaumberg 1966).