Geometric Morphometrics to Interpret the Endophytic Egg-Laying Behavior of Odonata (Insecta) from the Eocene of Patagonia, Argentina

Geometric morphometrics to interpret the endophytic egg-laying behavior of Odonata (Insecta) from the Eocene of Patagonia, Argentina

Eugenia Romero-Lebrón,1,2 Raquel M. Gleiser,1,2,3 and Julián F. Petrulevičius1,4

1CONICET (Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas) 2Universidad Nacional de Córdoba - CONICET. IMBIV. Centro de Relevamiento y Evaluación de Recursos Agrícolas y Naturales (CREAN), Av. Valparaíso s/n, C.C. 509, (5016) Córdoba, Argentina , 3Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Cátedra de Ecología. Av. Vélez Sársﬁeld 299. C.C. 5000, (5016) Córdoba, Argentina 4División Paleozoología Invertebrados, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, (1900) La Plata, Argentina

Abstract.—Although the order Odonata has a rich fossil record, many questions about its reproductive biology remain unanswered. There are two strategies of egg laying among odonates, exophytic and endophytic, the latter being one of the most revealing vestiges of plant–insect association in the fossil record. We assessed whether geometric morphometrics based on elliptical series of Fourier allow expression of variability of shape in traces of Odonata eggs within a leaf of Eucalyptus chubutensis (Berry) González (in part), González, 2009 (Myrtaceae) from Laguna del Hunco (Chubut, Argentina) (early Eocene) and whether this variability is consistent with the ichnotaxonomy of this material. We found that the largest variation corresponds to the compression of the shape while the remaining two components reflect variations in the apex position and its curvature, which changed according to the relative position of the traces in the leaf. There was no evidence that the hardness of the leaf would affect the shape of the egg trace. We postulate that these traces could have been produced by one single female: Variations in the pattern observable in the fossil of an originally three- dimensional structure are consistent with differences in the position of the eggs inserted by a single female who has flexed her abdomen to insert the eggs as she approaches the apex of the leaf (behavior observed also in extant dragonflies). For the first time, endophytic egg traces are analyzed with geometrical morphometrics, and this allows us to make inferences on the oviposition behavior of a female that lived around 52 million years ago.

Introduction Odonata lay eggs by exophytic behavior, in which the females lack a well-developed ovipositor and release the eggs in the Odonatoptera is one of the oldest groups of winged insects, with water or deposit them on the surface of periaquatic objects. a very diverse record that includes several orders, four of them The females that have an exophytic behavior produce appearing in the lower Carboniferous (325 Ma, Petrulevičius ovoid-rounded eggs while the eggs of the endophytic females and Gutiérrez, 2016). Odonata is the only order of Odonatoptera are elliptical-elongated. that persists today and represents one of the most charismatic After mating, females with an endophytic behavior, some- groups of insects. It has an estimated 6,042 living species and times in tandem with the male, select a plant substrate to place some 608 extinct species (Zhang, 2013). their eggs. For this, the female contacts the substrate with its ovi- The odonatans are insects linked to the aquatic environ- positor, located at the end of the abdomen. The ovipositor has ment, where their immature stages develop. At the time of repro- cutting shells to lacerate the plant tissue, generating a cavity duction, sexually active Odonata gather in or around water where the egg is inserted. Between each egg deposition, the bodies to mate and lay their eggs. There are two oviposition strat- female can rotate the abdomen and relocate the ovipositor to a egies in this group: endophytic and exophytic (Corbet, 1980). new site, repeating the process. In addition, females may walk The endophytic behavior occurs in species with well-developed a few steps on the substrate to reach different sectors and repeat ovipositors with which they insert their eggs inside the plant tis- the process systematically until the egg deposition is ﬁnished sues. This strategy occurs in all extant families of Zygoptera, in (Matushkina and Lambret, 2011). At the time of piercing, the Epiophlebioptera, Epiophlebiidae, and in some families of Anis- female can bend the abdomen so that the tip of the ovipositor optera such as Aeshnidae (Lucas, 1900; Asahina, 1934; Corbet, rests between its middle and hind legs (Matushkina and Gorb, 1962). It also occurs among other modern insect groups such as 2000). Endophytic oviposition may take place in live or dead Orthoptera, Hemiptera, Coleoptera, Lepidoptera, and Hymenop- plant substrates (Martens, 2001; Petrulevičius et al., 2011). Fos- tera (Wesenberg-Lund, 1913, 1943; Hinton, 1981; Zeh et al., sil leaves that show tissue reactions associated with the lesions 1989; Zherikhin, 2002; Krassilov et al., 2007). Among the are interpreted to be alive at the time of oviposition (Pott extinct odonatopterans with this strategy, we can cite Argenti- et al., 2008; Sarzetti et al., 2009; Petrulevičius, 2013). The ﬁnd- noptera (Petrulevičius and Gutiérrez, 2016). The rest of the ings of fossil endophytic eggs since the Cenozoic are relatively 1126 Downloaded from https://www.cambridge.org/core. University of Athens, on 26 Sep 2021 at 15:26:51, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/jpa.2019.30 Romero-Lebrón et al.—Geometric morphometrics of the egg-laying behavior of Odonata 1127

frequent, being the most revealing vestiges of plant–insect asso- The more terms (harmonics) are added to the function, the better ciations in the paleontological record (Petrulevičius et al., 2011). is the adjustment of the curve. Then the Fourier analysis coeffi- Evidence of oviposition in fossil leaves is known from the cients become descriptors of the shape of said curve that can be Paleozoic (e.g., Adami-Rodrigues et al., 2004; Béthoux et al., compared with the corresponding coefficients of other curves by 2004; Laaß and Hoff, 2015) to the Cenozoic. Fossil traces of means of several multivariate statistics techniques. There are dif- egg insertion in leaf tissues are externally manifested by the ferent methods to analyze the variation in the shape of the con- presence of scars generated in response to the injury produced tours using the Fourier technique (Rohlf and Archie, 1984; by the ovipositor apparatus (e.g., Peñalver and Delclòs, 2004; Foote, 1989; Rohlf, 1990; Temple, 1992), but among them, Vasilenko, 2005; Sarzetti et al., 2009; Petrulevičius et al., the most used are the Fourier analysis expressed in polar coordi- 2011; Moisan et al., 2012), and fossilized eggs are only rarely nates and the elliptical Fourier analysis. preserved (Pott et al., 2008). These scars are generally oval The Fourier shape analysis has been used in studies where it (Labandeira et al., 2002; Zherikhin, 2002; Béthoux et al., was applied to several groups of extant and fossil plant and ani- 2004; Moisan et al., 2012; Petrulevičius, 2013) and are com- mal organisms (e.g., Christopher and Waters, 1974; Rohlf and monly arranged on the leaf following a certain pattern. Archie, 1984; White et al., 1988; Renaud et al., 1996). While Vasilenko (2005) proposed an ichnotaxonomic record to in paleontology numerous works have contributed data on include traces of oviposition, establishing the ichnogenus traces, to the best of our knowledge no previous studies have Paleoovoidus. The Paleoovoidus ichnogenus is characterized used the method of Fourier analysis to describe egg fossil traces. by having medium-sized, elongated, narrow, ovoid or lens- In most cases, traces reports have been qualitative descriptions shaped structures, with a regular arrangement in the leaf lamina. based on linear dimensions (length and width) or using geomet- These structures, defined by dark, surrounding reaction tissue, ric morphometrics with landmarks (e.g., coleopteran fossil are narrow at one end, and each often bears a dark spot (defin- pupation chambers; Guerrero-Arenas et al., 2018). ition proposed by Vasilenko, 2005 and redefined by Sarzetti Sarzetti et al. (2009) described several traces of Odonata’s et al., 2009). This ichnogenus currently has more than a dozen endophytic eggs present in an angiosperm leaf from Laguna del species (Gnaedinger et al., 2014). Depending on the oviposition Hunco (early Eocene). The authors indicated that in a leaf of pattern followed by the traces, they are classified among others Eucalyptus chubutensis (Berry) González (in part), González, in Paleoovoidus rectus Vasilenko, 2005 (if the traces follow a 2009 two ichnospecies are present; the first set of traces found linear pattern) and Paleoovoidus arcuatus (Krassilov, 2008) (if in the apex of the leaf were classified as Paleoovoidus rectus they follow a curved or zigzag pattern). and the successive traces as Paleoovoidus arcuatus. In this con- The quantification of the shape is an important aspect in text, an opportunity was presented to evaluate whether geomet- many of the paleontological studies. The first approaches to ric morphometrics allows expressing variability of shape in the study of forms were developed from indices derived from Odonata’s egg traces, to evaluate whether this variability is simple linear measurements as length and width of structures, consistent with the ichnotaxonomic attribution, and to investi- which give a relationship between linear dimensions, providing gate whether the methodology may provide additional informa- a reference of the proportions although not of the shape. Trad- tion or insight on the behavior of the female at the time of itional morphometrics relied on measures of linear distances oviposition. such as length and width, and sometimes ratios and angles, and patterns of shape variation can be described by using multi- Materials and methods variate statistical tools. However, one of the main disadvantages of this approach is that different shapes can produce equal results We worked with a fossil leaf of Eucalyptus chubutensis (Myrta- because no information is included on the relative locations of ceae), which shows 25 traces of Odonata’s endophytic eggs the measurements. By contrast, geometric morphometrics cap- located along the entire leaf surface (Fig. 1.1). Only those traces tures the geometry of the organism (Adams et al., 2004). of eggs that were well defined and had complete contours were Geometric morphometrics analyzes changes in shape as a considered. The leaf comes from the locality of Laguna del function of the relative positions of a series of homologous mor- Hunco, Chubut, Argentina (Fig. 1.4) (LH13-1269, morphotype phometrics points (landmarks). There are different approaches, TY21; Wilf et al., 2003), belonging to the early Eocene (51.91 ± which can be generally grouped as based on landmarks or 0.22 Ma.). The material was observed with a Nikon Magnifier based on boundaries. In cases where it is difficult to establish SMZ1000 with a built-in Nikon DS-Fi1-L2 camera. The fossil homologous points (as would be the case with egg traces), the leaf was placed on a support in a horizontal position perpendicu- shape of a structure can be captured by means of the coordinates lar to the charge-coupled device (CCD) of the camera. The dis- of the sequence of points that define its contour. There are differ- tance used between the objective lens of the camera and the ent methods to adjust a mathematical function to the sequence of sample to be photographed was constant to avoid deformation points that define an outline and then to use the parameters of the of the microphotographs. These photographs were obtained at adjustment function as descriptive variables in multivariate ana- a size of 2,560 × 1,920 pixels in TIF format. The images were lysis. Although there are numerous techniques for describing edited so that all the partial photos completed the leaf in its entir- closed contours (for a review of them, see Rohlf, 1990; Cramp- ety. We digitally placed a superimposed inserter in which the ton, 1995; Swiderski et al., 2002), usually some of the variants contours of each trace and midrib were marked in detail of Fourier analysis are used. In general, this technique consists of (Fig. 1.2). Classical and geometric morphometrics were per- analyzing the contribution of the coefficients of a trigonometric formed using that inserter. Drawing on the definition proposed function that reproduces a certain curve as accurately as possible. by Vasilenko (2005) and redefined by Sarzetti et al. (2009)

Downloaded from https://www.cambridge.org/core. University of Athens, on 26 Sep 2021 at 15:26:51, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/jpa.2019.30 1128 Journal of Paleontology 93(6):1126–1136

Figure 1. (1) Image of the leaf of Eucalyptus chubutensis (Myrtaceae) with traces of endophytic egg insertion of Odonata located along the entire leaf surface (MPEF-Pb-2216 - MPEF-IC 1376 / MPEF-IC 1392); scale bar = 1 cm. (2) Layer overlay to the main photograph of the leaf in which the contours of each trace and rib were marked in detail; the dashed line represents distance measured between a point at the base of the leaf to the apex of each of the traces of the eggs. (3) Detail of an egg trace with the scale (square 1 x 1 mm) in the lower margin. (4) Map of Patagonia, Argentina showing the locality of Laguna del Hunco, Chubut.

that considers the tissue reaction, the present work includes the Geometric morphometrics.—The separation of shape and size is dark reaction mark of the leaf. a critical problem in morphometrics studies because both variables are commonly related and the independent measurement Classical morphometrics.—In each trace of an egg, the of one of them is difficult due to its covariation with the other. following measurements were made: length (the longest part However, when trying to describe the morphological variation of the trace), width (the widest part of the trace), distance to of an object, the metric variation between the different units of the closest trace (from apex to apex), distance from the apex size needs to be standardized to reduce the effects of the variance of the trace to the leaf rib, angle of the trace with respect to in the analysis. the rib, area, and perimeter. The program ImageJ 1.51n was The Fourier shape analysis allows us to explore small dif- used for such measurements. In addition, the distance ferences in shapes (Kuhl and Giardina, 1982; Ferson et al., between a fixed point at the base of the leaf to the apex of 1985; Hammer and Harper, 2006), defined from the character- each of the traces of the eggs was measured (Fig. 1.2). The ization of a contour registered by a certain number of points distance obtained was transformed to logarithmic scale. (Williams, 1981; Hammer and Harper, 2006). From the defin- Spearman correlation analyses were performed between that ition of this contour, the Fourier analysis is based on the reduc- distance and the trace length or width to observe whether tion or transformation of a series of bidimensional coordinates these metrics of the trace vary with respect to its location on (x, y) to shape parameters (Hammer and Harper, 2006) that the leaf. will be expressed as harmonics and analyzed by multivariate

analysis (Williams, 1981; Rohlf and Archie, 1984; Lestrel, the leaf at the time of egg insertion. The same procedure was fol- 1997). lowed with PC 2 and PC 3. There are two types of Fourier analysis: polar analysis (or radial) and the elliptic functions analysis. The fundamental dif- Interaction of ichnotaxonomy with classical and geometric ferences are established in the type of coordinates (Hammer and morphometrics.—A comparison was made by classical Harper, 2006). The elliptical Fourier analysis represents a for- morphometrics between the groups interpreted from geometric mulation where the coordinates x and y are established in the morphometrics (apical, middle, and basal) and between the plane, point by point, around the contour as a third periodic ichnotaxonomic classification proposed by Sarzetti et al. function (Kuhl and Giardina, 1982). As a result, four coeffi- (2009; P. rectus and P. arcuatus) to observe whether classical cients are generated (cosine [cos] and sine [sin] of the x-axis morphometrics is sufficient to differentiate egg traces between [cosx-sinx] and y-axis [cosy-siny]) for each harmonic, unlike these classifications. The parameters to be compared were the two (cos-sin) generated from polar-type coordinates (Rohlf length, width, distance between successive traces, distance to and Archie, 1984; Ferrario et al., 1999; Navarro et al., 2004; the rib, angle to the rib, and area and perimeter of the trace by Sheets et al., 2006). The elliptical method allows definition of means of ANOVA followed by Tukey tests, where significant more complex shapes with better detail than polar analysis differences (p < .05) were found. Principal component scores (Hammer and Harper, 2006); for this reason, in the present of the three groups, based on geometric morphometrics, were work the contour analyses were performed by elliptical Fourier also compared using ANOVA followed by Tukey tests. analysis. The Fourier method involves the digitalization or delimita- Repository and institutional abbreviation.—The specimen tion of a contour from a number of points, generally using a examined in this study is deposited in the Egidio Feruglio photographic support (Hammer and Harper, 2006). In general, Paleontological Museum (M.E.F.), Trelew, Province of Chubut, as many parameters as necessary for a correct representation Argentina, with two collection numbers, one paleobotany of the shape must be defined. The greater the number of para- (MPEF-Pb-2216) and another ichnological (MPEF-IC 1376 and meters used, the more accurately the contour is captured. The MPEF-IC 1392). functions adjusted to the contours are called harmonics (Rohlf, 1990; Monteiro and Reis, 1999), curves that are added in des- Results cending order to describe the contour, so that the first harmonics describe the contour in a general way, while the last ones Classical morphometrics.—The mean length of the traces was represent small-scale variation, which, when the number of har- 0.83 mm, the mean width 0.26 mm, the mean distance to the monics is excessive, tends to pick up noises (Rohlf, 1990; closest trace 2.94 mm, the mean distance from the apex of the Haines and Crampton, 2000; Ubukata, 2004). It has been trace to the rib 1.07 mm, the mean angle of the trace with described that around 20–30 harmonics are usually sufficient respect to the rib 13.08°, the mean area 0.13 mm2, and the to define complex shape, although this varies with the shape, mean perimeter 1.78 mm (Table 1). No significant correlations and sometimes a smaller number is adequate (Williams, 1981; were found between the length (p = 0.34, r = −0.20) or the Lestrel, 1997; Hammer and Harper, 2006). width (p = 0.18, r = 0.28) of the trace and the location of the For geometric morphometrics, we worked with individual trace on the leaf (Fig. 2). images of the contour of each of the traces (Fig. 1.3). For the calculation of the Fourier coefficients, the free distribution Geometric morphometrics.—The analysis of the main statistical package SHAPE 1.3 (Iwata and Ukai, 2002)was components of the Fourier coefficients reduced the variability to used. The coefficients of the Fourier transformations were calcu- three main components (PC) (Figs. 3, 4) that comprise 92.92% lated through the normalization method based on the first har- of the variance observed in the shape. PC 1 explains 58.40% of monic. Twenty harmonics were taken (Hammer and Harper, the total variation, PC 2 explains 20.64% of the total variation, 2006) (we tested with a lower number of harmonics; 20 harmo- and PC 3 explains 13.87% of the total variation (Table 2). nics were better adjusted to the reference shape). With the As seen in Figure 4, the largest variation in the sample cor- numerous variables produced (four coefficients for each har- responds to the compression of the shape, while the remaining monic), a principal component analysis (PCA) was performed two components show localized variations in the position of using the covariance matrices, which reduced the dimensional- the apex of the trace and the curvature of the trace, respectively. ity, and new derived variables were created. They order the The superposition on the shape (Fig. 4, first column) shows the traces according to their shapes in a canonical space. For this, variation in the components as well as the location of the shape the PAST 3.15 free distribution statistical package (Hammer differences between the contours (data on two egg traces appear et al., 2001) was used. Finally, the PrinPrint (SHAPE 1.3 to be outliers: egg traces 3 and 6; however, results remained con- subprogram) was used to visualize the variation of shape sistent after removing those traces from the PCA, with the first represented by each main component. three components explaining 89.4% of the total variation). The base of the leaf hardens more quickly than the tip of the Figure 5 shows the trace shapes disposition along the leaf, leaf (Choong, 1996; Teaford et al., 2006), so a regression ana- based on the first two main components. For the positive values lysis was performed between the variables PC 1 and distance of PC 1 and positive values of PC 2, the PCA gathers traces of of each of the traces to a fixed point located at the base of the spherical eggs with the vertex downward (Fig. 5.1 ‘circle,’ leaf (Fig. 1.2) to observe whether the changes in the shape of mainly in the apical area of the leaf). For the negative values the trace would be linearly related to the hardness changes of of PC 1 and negative values of PC 2, it gathers traces of eggs

compressed with the vertex upward (Fig. 5.1 ‘star,’ mainly in the a a a a a cant

ﬁ central zone of the leaf). For the negative values of PC 1 and positive values of PC 2, it gathers traces of eggs compressed with the vertex downward (Fig. 5.1 ‘square,’ mainly in the 1.31 to 2.21 (1.75 ± 0.11) 1.11 to 2.62 (1.80 ± 0.10) 1.31 to 2.21 (1.75 ± 0.11) 1.11 to 2.27 (1.78 ± 0.16) 1.40 to 2.62 (1.81 ± 0.14) 1.11 to 2.62 (1.78 ± 0.08) basal area of the leaf). Spherical eggs with the vertex upward are rare (positive values of PC 1 and negative values of PC 2) and located mainly in the middle zone and the apical zone of ) Perimeter (mm) a a a a a 2 the leaf (Fig. 5.1 ‘triangle’). No linear relationship was found between the distance from each of the traces to a point located at the basal of the leaf and the compression of the trace (PC 1) (R2 = 0.047, p = 0.298), the position of the apex (PC 2) (R2 = 0.07 to 0.23 (0.15 ± 0.02) 0.06 to 0.22 (0.12 ± 0.01) 0.07 to 0.23 (0.15 ± 0.02) 0.06 to 0.17 (0.11 ± 0.02) 0.09 to 0.22 (0.13 ± 0.02) 0.06 to 0.23 (0.13 ± 0.01) 0.005, p = 0.732), or the curvature of the trace (PC 3) (R2 = 0.367, p = 0.001) (Fig. 2).

a a a Drawing on the ﬁrst two main components, the leaf may be a a qualitatively divided into three zones according to the shape of

(°) Area (mm the eggs: apical, middle, and basal (Fig. 5.2, 5.4). The apical cript letters within a column indicate statistically signi zone presents traces of spherical eggs, most frequently with Angle to the rib 1.10 to 25.80 (9.43 ± 3.21) 1.83 to 32.96 (15.13 ± 2.57) 1.10 to 25.80 (9.43 ± 3.21) 7.00 to 32.96 (17.13 ± 3.65) 1.83 to 27.81 (13.13 ± 3.73) 1.10 to 32.96 (13.08 ± 2.05) the apex downward. The middle zone presents traces of compressed eggs, with the apex upward. The basal zone of the leaf presents, in addition to the previous combination, traces of com- a b a b a pressed eggs, with the apex facing downward. Note the rare presence of spherical eggs with the apex upward. Consistently, there ﬁ rib (mm) were statistically signi cant differences between the scores of Distance to the 0.01 to 0.70 (0.50 ± 0.08) 0.21 to 4.60 (1.38 ± 0.30) 0.01 to 0.70 (0.50 ± 0.08) 0.59 to 4.60 (2.06 ± 0.05) 0.21 to 1.44 (0.71 ± 0.14) 0.01 to 4.60 (1.07 ± 0.21) PC 1 of the apical and middle zones, and between PC 3 scores cation based on geometric morphometrics from the analysis of main components (apical,

ﬁ of the basal compared to apical and middle zones (Table 3).

a a a a a Interaction of ichnotaxonomy with classical and geometric morphometrics.—The parameters measured according to (mm)

) and the classi classical morphometrics (length, width, distance between

closest trace successive traces, distance to the rib, angle to the rib, area Distance to the 1.00 to 5.33 (2.57 ± 0.42) 1.32 to 6.89 (3.16 ± 0.45) 1.00 to 5.33 (2.57 ± 0.42) 1.67 to 6.89 (3.50 ± 0.67) 1.32 to 5.61 (2.78 ± 0.59) 1.00 to 6.89 (2.94 ± 0.32) and perimeter of the traces) were compared between the three zones interpreted from the analysis of geometric P. arcuatus morphometrics (apical, middle, and basal) and between the a b a b b and two ichnospecies proposed by Sarzetti et al. (2009; P. rectus and P. arcuatus; Table 1). The only signiﬁcant differences

P. rectus were found in the widths of the traces of the apical zone 0.21 to 0.41 (0.31 ± 0.02) 0.17 to 0.30 (0.23 ± 0.01) 0.21 to 0.41 (0.31 ± 0.02) 0.17 to 0.24 (0.21 ± 0.01) 0.21 to 0.30 (0.25 ± 0.01) 0.17 to 0.41 (0.26 ± 0.01) (which coincides with P. rectus), which were wider compared to the other traces (p < 0.01), and in the distance of the egg trace to the rib, which was shorter on average in the apical and

a a a a a basal zones compared to the middle zone (p < 0.05).

Discussion (0.80 ± 0.05) (0.86 ± 0.08) (0.83 ± 0.07) (0.84 ± 0.05) (0.80 ± 0.05) (0.83 ± 0.04)

Classical morphometrics.—The descriptions of the fossil shapes N Length (mm) Width (mm) 9 0.64 to 1.01 16 0.53 to 1.21 have historically been made through linear measurements. Sarzetti et al. (2009) provide linear measurements of P. rectus (length ranging from 1.0 to 1.2 mm, width from 0.4 to 0.5 mm, and distance between successive traces 0.7 to 1.4 mm); these dimensions are different from those taken in the present P. rectus P. arcuatus Middle zoneBasal zone 8 8 0.53 to 1.10 0.64 to 1.21 Apical zone 9 0.64 to 1.01 research (lengths were 0.64 to 1.01 mm, widths 0.21 to 0.41 mm, distances between successive traces 1 to 5.33 mm). These differences could be partly because they considered P. rectus in an area that would contain 12 traces, while we identiﬁed 13, but four of them were not included in the analyses because they were incomplete (three of them are on the tip of the leaf, and if Comparative values by classical morphometrics between the ichnospecies (

cation we consider the spatial disposition, it is observed that these ﬁ eggs are placed in a curved pattern [similar to P. arcuatus]; Ichnospecies differences (p < 0.05). middle, and basal zones). Minimum to maximum range is shown, and mean ± standard error is included in parentheses. For each grouping, different supers Table 1. Geometric morphometrics classi AllFig. 5.5). They do 25 not 0.53provide to 1.21 data on the measurements of

Figure 2. Correlation graph between: (1) length of the trace and log of the distance to a point at the base of the leaf; (2) width of the trace and log of distance to a point at the base of the leaf. Regression graph between: (3) log of the distance to a point at the base of the leaf and PC 1; (4) log of the distance to a point at the base of the leaf and PC 2; (5) log of the distance to a point at the base of the leaf and PC 3.

P. arcuatus belonging to that leaf. The existence of variation work with linear measurements it is a frequent problem that produced by differences of criteria in the measurement is an could be reduced by using the geometric morphometrics important aspect to be considered because for those who technique.

Figure 4. Variation in the shape of the traces of the eggs present in the leaf of Eucalyptus chubutensis (Myrtaceae). The variation in the shape is represented in units of standard deviation from a mean shape, which is the average of the Fourier coefﬁcients for all the shapes analyzed. Principal components PC 4 and PC 5, which capture a very small fraction of the variation, are shown for illustrative purposes.

contains recent endophytic eggs of the family Aeshnidae. This material has eggs inserted by one female and arranged in two different patterns. The first area of the stem shows eggs arranged in a linear pattern, and the basal area shows eggs arranged in a zigzag pattern, an arrangement comparable to the traces of fossil eggs observed in the leaf of Eucalyptus chubutensis, showing that one female can oviposit in both patterns. This is also sup- ported by personal observations on living specimens in the field (Romero-Lebrón). Fossil materials with an arrangement like that can be found in work by Hellmund and Hellmund (1998) and Petrulevičius et al. (2011). Hellmund and Hellmund (1998) show a leaf of Daphnogene lanceolata Unger, 1850 (Lauraceae, specimen SMMGD Hw 253, fig. 5) from the lower Oligocene of Germany. This specimen is not complete, but a curved pattern can be observed at the base and a straight pattern at the apex. Petrulevičius et al. (2011) show a leaf of Sideroxylon salicites (C.O. Weber) Weyland, 1937 (Sapotaceae, specimen HW Ro 2.8, figs. 11, 12) from the upper Oligocene Figure 3. Graphs of the principal components analysis performed with the from Rott (also Germany) that has both ichnospecies (P. rectus covariance matrices on the Fourier coefficients. (1) PC 1 versus PC 2. (2)PC 1 versus PC 3. (3) PC 2 versus PC 3. Table 2. Eigenvalue and contributions of the main components calculated in the traces of the eggs. The marked similarity between the morphometrics of the Proportion Accumulated egg traces of the ichnospecies proposed by Sarzetti et al. Component Eigenvalue (%) (%) Indicator (2009), combined with egg insertion patterns of extant Odonata 1 0.008 58.40 58.40 Compression of the form (Matushkina, 2007), suggests that they could have been placed 2 0.003 20.65 79.05 Apex position by the same female. Matushkina (2007) photographed a stem 3 0.002 13.87 92.92 Curvature of Myriophyllum spicatum Linnaeus, 1753 (Haloragaceae) that Total variance 0.013

Figure 5. Trace shapes disposition along the leaf, based on the ﬁrst two principal components (PC 1 and PC 2) of a principal components analysis. (1) Plot of PC 1 versus PC 2. (2) According to the analysis of PC 1 versus PC 2, the leaf may be divided into three zones: apical zone, middle zone, and basal zone. The apical zone presents spherical eggs with their apex downward. The middle zone presents mainly compressed eggs with their apex upward. The basal zone of the leaf presents, in addition to the previous combinations, compressed eggs and with their apex downward. (3) Geometric morphometrics show an overlap of the range of shapes occurring between P. rectus and P. arcuatus.(4) Leaf divided into three zones: apical, middle, and basal zones (dotted line). (5) Possible pattern of the female placing its eggs. Scale bar = 1 cm.

at the apex of the leaf and P. arcuatus at the base). They state that the Lestidae family probably produced P. rectus, while the Coe- the Rott specimen exhibits only minor differences from the nagrionidae family were responsible for P. arcuatus. Based on Argentine material described by Sarzetti et al. (2009), and the preceding observations, our hypothesis is that it is more the authors comment that they find it curious that in their leaf probable that the same female has placed her eggs in both pat- the same association of P. rectus and P. arcuatus is repeated terns in an Eocene leaf of Laguna del Hunco from Argentina and in the same position within the leaf. Sarzetti et al. (2009) and leaves of Oligocene from Germany than that two different suggest that the oviposition traces are similar to the oviposition families of zygopteran have coincided in the same leaves numer- of Zygoptera (Odonata). Specifically, they say that members of ous times to generate the same disposition. The values obtained by classical morphometrics of the variable length of the trace agree with those reported by Moisan et al. (2012) for the length of present Odonata’s eggs of the family Table 3. Comparison of principal components (PC) scores for apical, middle, and basal zones along the leaf (mean ± standard error). For each PC, different Coenagrionidae, while the distances between successive traces superscript letters within a column indicate statistically significant differences agree with Matushkina and Gorb (2000) for the variable distance (p < 0.05). between successive eggs of current Odonata of the family Les- Class PC 1 PC 2 PC 3 tidae. We also have to consider that in Laguna del Hunco extinct Apical zone 0.07 ± 0.02a 0.01 ± 0.02a −0.02 ± 0.01a families of Odonata are recorded that could be the producers of Middle zone −0.06 ± 0.03b −0.03 ± 0.02a −0.01 ± 0.01a ̌ ab a b the traces (Petrulevicius, 2013): the Austroperilestidae (Zygop- Basal zone 0.02 ± 0.03 0.02 ± 0.02 0.04 ± 0.01 tera) and the Frenguelliidae, controversially considered either

Figure 6. Female of Coenagrionidae (Zygoptera) placing its eggs endophytically under water in a stem of Hydrocotyle bonariensis (Araliaceae). (1) Extended abdomen. (2) Abdomen at the height of the ﬁrst pair of legs. (3) Abdomen beyond the head. Dragonﬂy is 3.1 cm long (from the tip of the head to the tip of the abdomen).

basal Epiproctophora or Zygoptera (Petrulevičius and Nel, their abdomen ventrally to different degrees to lay their eggs 2003, 2009, 2013; Petrulevičius, 2017). Therefore, the informa- (Corbet, 1962; Fig. 6). Jödicke (1997) formulated the hypothesis tion obtained so far is not enough to assign the ichnospecies to a that the degree of curvature of the abdomen correlates with the family of Odonata. rigidity of the substrate and, therefore, with the force that a No significant correlations were found between the length female can exert during the penetration of the tissues of the of the trace or their width and the distance of each of the traces plant when inserting her eggs. to a point located at the base of the leaf, which means that nei- For birds, Denis Ávila and Olavarrieta (2011) and Denis ther the length of the trace nor their width would be modified by Ávila (2014) applied the method of Fourier analysis for studying the relative location of the trace on the leaf. These results would the shape of eggs. While in paleontology numerous works have support the hypothesis that these eggs were placed by a single contributed data on traces, to the best of our knowledge, no pre- female. In current species, differences are observed between vious studies have used the method of Fourier analysis to the length of eggs laid by different species (Moisan et al., describe insect egg fossil traces. The present work is the first 2012). contribution to the knowledge of the variation of the shape through the analysis of Fourier contour in traces, providing Geometric morphometrics.—The present study is based on the insights on the behavior of the female of Odonata during fact that the method of elliptical Fourier descriptors uses an oviposition. approach that is independent of the dimensions; therefore it is postulated that it is capable of detecting subtle differences in Interaction of ichnotaxonomy with classical and geometric the shape of the traces. The results obtained support that morphometrics.—According to the parameters calculated in the Fourier contour analysis allows exploring in detail the the traces of the eggs, the results support the assertion that variability in the shape and morphospatial representation of the performing only classical morphometrics analysis does not traces of Odonata’s endophytic eggs present in a leaf assigned provide sufficiently robust data to differentiate between the to the myrtle family (Myrtaceae) from the Laguna del Hunco. ichnospecies P. rectus and P. arcuatus (Table 1). Geometric Choong (1996) and Teaford et al. (2006) postulated that morphometrics also show an overlap of the range of shapes dicotyledonous leaves are harder at the base than at the tip. By occurring between P. rectus (apical zone) and P. arcuatus means of the linear regression analysis, we can infer that the (middle and basal zones; Fig. 5.3; Table 3).Thus, these two shape of the traces (PC 1, PC 2, and PC 3) would not be influ- trace fossil structures (which consider not only the egg traces enced by the hardness of the leaf at the time of egg inserting. measured but also their spatial pattern in the substrate) may Because of the tissue reaction of the leaf, we postulated that at have been made by one individual tracemaker. An ichnotaxon the time of oviposition, the leaf was alive. is a name assigned to a morphologically recurrent structure The contour analysis of the traces also provides information resulting from the life activity of an individual organism (or suggestive of the way in which the insertion of eggs would have homotypic organisms) modifying the substrate (Bertling et al., occurred. It has been observed that the females lay their eggs 2006). The results presented here suggest that both ichnotaxa with the pointed ends directed outward (Sawchyn and Gillott, were made by one individual and contribute to reconstructing 1974 [Lestidae]; personal observation by Romero-Lebrón [Coe- the reproductive behavior of the tracemaker. Although giving nagrionidae]). We can infer that the eggs were placed by a single different ichnotaxonomical names to different parts of a female, who initially began to insert her eggs at the base of the trackway because of changes in behavior should be generally leaf and then, with oscillatory movements, moved forward to avoided (Bertling, 2007), if the possibility that the two the apex of the leaf. Once she reached two-thirds of the length ichnotaxa were made by the same female is accepted, it does of the leaf, she stopped moving and bent her abdomen onward not necessarily invalidate the ichnotaxa assigned to each of the to complete the insertion of the eggs in this position, thus a pre- two components of the superstructure. Nevertheless, the dominance of eggs with their apex oriented downward (toward approach proposed in this work could be a useful starting point the base of the leaf; behavior observed by Romero-Lebrón in for further investigations in defining new ichnotaxonomical current Coenagrionidae). The females of Odonata can bend categories or species.

Conclusions Christopher, R.A., and Waters, J.A., 1974, Fourier series as a quantitative descriptor of miospore shape: Journal of Paleontology, v. 48, p. 697–709. Corbet, P.S., 1962 (facsimile 1983), A Biology of Dragonflies: Oxford, Classey. Fossil traces are more than just the mere modification of the sub- Corbet, P.S., 1980, Biology of Odonata: Annual Review of Entomology, v. 25, strate by the life activity of an organism. They are also important p. 189–217. Crampton, J.S., 1995, Elliptic Fourier shape analysis of fossil bivalves: Some sources of information about the behavior of the animals that practical considerations: Lethaia, v. 28, p. 179–186. made the traces. The results support that the methodology Denis Ávila, D., 2014, Application of the elliptic Fourier functions to the descrip- used allows detecting differences in the shape of the traces. Var- tion of avian egg shape: Revista de Biología Tropical, v. 62, p. 1469–1480. fl Denis Ávila, D., and Olavarrieta, U., 2011, ¿Existe la isomorfía en los huevos de iations in the shape of the trace re ect the two-dimensional pat- la familia Ardeidae (Aves, Ciconiiformes)?: Animal Biodiversity and Con- tern observable in the fossils of an originally three-dimensional servation, v. 34, p. 35–45. structure. Neither the length nor the width of the trace would be Ferrario, V.F., Sforza, C., Tartaglia, G.M., Colombo, A., and Serrao, G., 1999, Size and shape of the human first permanent molar: A Fourier analysis of the affected by the location on the leaf, which would support the occlusal and equatorial outlines: American Journal of Physical Anthropol- hypothesis that the eggs were placed by a single female. In add- ogy, v. 108, p. 281–294. Ferson, S., Rohlf, F.J., and Koehn, R.K., 1985, Measuring shape variation of ition, it is inferred that the shape of the trace would not be two-dimensional outlines: Systematic Biology, v. 34, p. 59–68. affected by the hardness that the leaf possessed at the time of Foote, M., 1989, Perimeter-based Fourier analysis: A new morphometric method the insertion of the eggs. Performing exclusively studies of clas- applied to the trilobite cranidium: Journal of Paleontology, v. 63, p. 880–885. Gnaedinger, S.C., Adami-Rodrigues, K., and Gallego, O.F., 2014, Endophytic sical morphometrics of the egg traces per se does not provide ovipositions on leaves from the Late Triassic, northern Chile: Ichnotaxo- enough data to differentiate between ichnospecies, while the nomic, palaeobiogeographical and palaeoenvironmetal considerations: geometric morphometrics allow making inferences in the ovi- Geobios, v. 47, p. 221–236. González, C.C., 2009, Revisión taxonómica y biogeográfica de las familias de position behavior of a female that lived 52 million years ago. angiospermas dominantes de la “Flora del Hunco” (Eoceno temprano), Chu- The variations in the shape of the trace are consistent with the but, Argentina [Ph.D. dissertation]: Buenos Aires, Universidad de Buenos positional differences of the eggs that would have been inserted Aires, 174 p. Guerrero-Arenas, R., Zúñiga-Marroquin, T., and Jiménez-Hidalgo, E., 2018, by a single female on a live leaf. The female would begin at the How much variation is in the shape of fossil pupation chambers? An base of the leaf and then flex its abdomen to place the eggs as it exploratory geometric morphometric analysis of Fictovichnus gobiensis approaches the apex of the leaf (behavior also exhibited by from the late Eocene of Oaxaca, southern Mexico: Boletín de la Sociedad Geológica Mexicana, v. 70, p. 361–368. extant species of Odonata). 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Tambussi for helpful discussions that led to writing this Hammerunterwiesenthal (Freistaat Sachsen): Abhandlungen des manuscript. We also thank comments from the editor and Staatlichen Museums für Mineralogie und Geologie zu Dresden, v. 43, three anonymous reviewers that helped improve the quality of p. 281–292. fi Hinton, H.E.,1981, Biology of insect eggs: Oxford, Pergammon Press. the manuscript. Funding support for the eld trip and laboratory Iwata, H., and Ukai, Y., 2002, SHAPE: A computer program package for quan- studies came from grants: DEB-1556666 from the National Sci- titative evaluation of biological shapes based on elliptic Fourier descriptors: ence Foundation of USA (NSF); PIP 0834 from the National Journal of Heredity, v. 93, p. 384–385. 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