Interspecific larvae competence and mandible shape disparity in cutworm pest complex (Lepidoptera: Noctuidae)

Journal Pre-proof Interspecific larvae competence and mandible shape disparity in cutworm pest complex (Lepidoptera: Noctuidae) Selene Niveyro, Hugo A. Benítez PII:

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DOI:

https://doi.org/10.1016/j.jcz.2019.10.004

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JCZ 25678

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Zoologischer Anzeiger

Received Date: 10 July 2019 Revised Date:

28 September 2019

Accepted Date: 14 October 2019

Please cite this article as: Niveyro, S., Benítez, H.A., Interspecific larvae competence and mandible shape disparity in cutworm pest complex (Lepidoptera: Noctuidae), Zoologischer Anzeiger, https:// doi.org/10.1016/j.jcz.2019.10.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier GmbH.

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Interspecific larvae competence and mandible shape disparity in cutworm pest

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complex (Lepidoptera: Noctuidae).

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Selene Niveyro1 & Hugo A. Benítez2

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1

7

Pampa, Ruta 35 Km 334, Santa Rosa 6300, La Pampa, Argentina.

8

2

9

Velásquez #1775 Arica, Chile.

Cátedra de Zoología Agrícola, Facultad de Agronomía, Universidad Nacional de La

Departamento de Biología, Facultad de Ciencias, Universidad de Tarapacá, General

10 11

ABSTRACT

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Cutworm pest complex (Lepidoptera: Noctuidae) varies globally in terms of regional

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dominance, crops attacked and feeding behaviour. In the Pampean region, extensive

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extensions of farming lands are affected by at least five cutworm species belonging to

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the genus Agrotis, Feltia and Peridroma. Due to the competitive environment that

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exposes these insects to stressors, certain individual features such as morphology could

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provide useful insights about functional diversity related to its dispersion and

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dominance. In this study, we analyze whether the shape of the mandibles is a

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contrasting trait between seven cutworm species, and we discuss whether this

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morphological trait can be an influential factor in the abundance and predominance of

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species within the complex between the southern and northern part of the Pampean

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region. Using geometric morphometrics tools, the results evidenced that cutworms

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complex differs in this functional trait. Closer phylogenetically species of Agrotis were

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widely different between them, while species phylogenetically distant belonging to

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Feltia and Peridroma were lightly similar. These results showed that functional traits

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have a fundamental importance to develop a predictive framework linking the herbivory

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damage with the herbivore functional diversity.

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Keywords: Noctuoidea; Agrotis; functional diversity; morphological trait; ecomorphology;

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cutworms.

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1. INTRODUCTION

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Noctuoidea is the largest and most diverse superfamily of Lepidoptera with more than

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40,000 recognized species (Wagner, 2001). Among them, numerous species are

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important pests, including the cutworm species. Cutworm is the common name given to

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the larvae of noctuid moth species feeds on roots, offspring of crop plants and grasses.

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Cutworms have great potential for spring damage in the Pampean region (Argentina),

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where they cut lucerne sprouts, seedlings of corn, sorghum, soybeans, and sunflowers

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(Aragón, 1985). In severe cases, the damage by the cutworms forces the reseed of the

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crop. Therefore, the economic thresholds for cutworm complex are very low, in summer

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crops it is estimated at 1 caterpillar per m2 (Aragón, 1985) and in lucerne 1-2 caterpillars

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per crown (Villata, 1993).

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Cutworm pest complex varies globally in terms of regional dominance, life cycle and

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feeding behaviour (Ayre & Lamb, 1990; Wang et al., 2015). In the Pampean region,

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vast extensions of farming lands are affected by at least five cutworm species: Agrotis

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ipsilon (Hufnagel), Agrotis robusta (Blanchard), Feltia deprivata (Walker), Feltia

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gypaetina (Guenée), Feltia lutescens (Blanchard), Feltia irritans (Köhler) and

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Peridroma saucia (Hubner) (Aragón, 1985). The composition of the complex and the

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dominance of species vary greatly in the region. Until the 90s, the damages by

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cutworms were mainly caused by Agrotis ipsilon (Aragón, 1985), while by the middle

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of the decade this situation has been modified by presenting prolonged and severe attack

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by the species Agrotis robusta and Feltia gypaetina (Aragón, 1995). In the north part of

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the region, Agrotis ipsilon is still the dominant species, while Agrotis robusta is

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registered as the main species in the southern and drier part of the region (Corró Molas

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et al., 2017). Samples taken in recent years (2014-2018) also indicated fluctuations

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among summer seasons in the proportion of the rest of the cutworm species that make

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up the complex e.g. Feltia lutescens and especially Feltia deprivata and Peridroma

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saucia (Niveyro data unpublished). However, the density of these species in the

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complex always remains low when compared to Agrotis genus. Regardless of the higher

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abundance of one or another species, the five species coexist sympatrically around the

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whole region and generally share, compete or exploit alternative resources to survive

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and reproduce (Corró Molas et al., 2017; Matley et al., 2017).

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It is a practice in agroecosystem to reduce the pest influence incorporating different

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resource pools or partitioning them to reduce herbivory, similar species can co-exist

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within distinct ecological niches (Chesson, 2000). For cutworms, the ecological niche is

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mainly driven by competition for resources and feeding interactions among organisms,

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although other factors such as morphological, functional and physiological constraints

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may contribute (Frankie & Ehler, 1978; Worner & Gervey, 2006). Since a competitive

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environment exposes these insects to stressors, certain organismal aspects such as

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morphology could provide useful insights about the expression of the phenotype-

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environment interaction (Benítez et al., 2014) and functional diversity related to

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dispersion and dominance (Mikac et al., 2016). Different rearing environments may

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produce dissimilar stress level in insects, which could be reflected on their multiples

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morphological diversity where the organism development modulates their own shape

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variation in response to changes in the environment (Chazot et al., 2016) There are

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several issues for which morphological analyses play an important role in

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agroecosystem. For instance, ecomorphological studies have revealed constraints and

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selective factors affecting the fitness response to certain environments of particular

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phenotypes (Bower & Piller, 2015, Sherratt et al., 2018). Besides, morphological

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diversity can reflect functional diversity in a community (Deraison et al., 2015). In

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cutworm complex, morphological variation, particularly in functional structures such as

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mandible, can influence the herbivory affecting crops, as well as, reflect an adaptive

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capacity of the species to expand and colonize other regions with the consequent

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economic damage. In all these cases, morphology reveals certain organismal aspects

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that relate an individual to its environment, hence its importance. Indeed, the

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relationship between morphology and ecology could provide useful insights about the

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expression of the phenotype-environment interaction and the related evolutionary

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history (Piersma & Drent, 2003).

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In recent years, the development of a morphological quantitative toolkit used in multiple

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studies in ecomorphology known as geometric morphometrics (GM) has made it

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possible to detect small shape variations that used to be difficult to distinguish with

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traditional linear morphometrics (Adams & Rohlf, 2000). GM is a coordinate-based

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method, which means that its primary data are cartesian coordinates of anatomically

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distinguishable landmarks (i.e. discrete anatomical points that are arguably homologous

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among all the individuals under analysis) (Adams et al., 2013).

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GM techniques have never been applied on the mandible of Lepidoptera`s larvae.

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Nevertheless, the only published approximation to this topic has been done in an

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agronomic system and it is a study on beetle larvae (Benítez et al., 2014).

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In this study, the mandibular shape belonging to the cutworm complex present in the

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Pampean region was analysed. Taking regional dominance of species into

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consideration, the hypotheses stated is that mandibular shape is a contrasting trait

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among the species within the cutworm complex.

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2. MATERIALS AND METHODS

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2.1. Laboratory mass rearing of cutworms

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Five gravid female moths of seven cutworm species: Agrotis ipsilon (Hufnagel), Agrotis

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robusta (Blanchard), Feltia deprivata (Walker), Feltia lutescens (Blanchard), F.

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gypaetina (Guenée), Feltia irritans (Köhler) and Peridroma saucia (Hubner), were

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collected using a light trap located in the campus of the Faculty of Agronomy,

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UNLPam, La Pampa, Argentina (36° 33'9 '' S; 64° 18'8'' W, 220 masl). Moths collected

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come from fields where larvae are exposed to natural competitive environments. Each

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moth collected was placed individually in plastic cups of 165 cc and fed a sugar solution

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of 10% honey and distilled water. Tissue paper was placed inside the plastic cup to

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facilitate the oviposition. Newly emerged larvae were separated in individual plastic

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cups of 120 cc to avoid cannibalism and reared with artificial diet based on bean meal,

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yeast extract, wheat-germ, sorbic acid, ascorbic acid and formaldehyde (following the

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methodology of Niveyro et al., 2015) until the fifth instar larvae was achieved. The

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temperature of the laboratory was 25 ± 2 ºC, with 65 ± 10% relative humidity and the

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photoperiod was 16:8 h Light–dark cycle. Five male specimens of each species were

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also collected in the light trap to confirm species identification by genitalia dissection

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(Aragón, 1985; Lafontaine, 2004). Left and right mandibles of each species (N = 194

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mandibles) were dissected and preserved in 70% ethyl alcohol: A. ipsilon (n = 14), A.

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robusta (n = 38), F. deprivata (n= 60), F. lutescens (n = 34), F. gypaetina (n = 12), F.

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irritans (n = 6) and P. saucia (n = 30). Larvae were not sexed for morphometrics

125

analysis.

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2.2. Shape analysis

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The concave surface of left and right mandibles of each species was photographed using

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a Leica digital camera on a binocular of Leica EZ4 stereo-microscope and saved in

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JPEG format using the Leica Application Suite version 1.7 (Leica Microsystems

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Limited, Switzerland). Photographs were digitized with tpsUtil version 1.58 and

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tpsDig2 version 2.17 (Rohlf, 2013). Twelve anatomical landmarks (LM) were digitized

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to capture the shape of the mandibles (Fig. 1, Table 1). The first tooth of the mandible,

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hereinafter named as lobe, was not considered in the analysis for being an inner lobe

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and impractical to locate in all samples. A generalized Procrustes analysis was applied

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to the landmark to remove the information of size, position and orientation from the

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shape variables (Rohlf & Slice, 1990; Dryden & Mardia, 1998). Left and right mandible

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were digitized twice and a Procrustes ANOVA was calculated to compare the influence

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of the digitizing error (Klingenberg & McIntyre, 1998) Both sides were analysed

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(matching symmetry) independently and the for graphical reasons the left side was used.

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Principal component analysis (PCA) based on the covariance matrix of individual

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mandible shape was performed in order to graphically visualize the mandible shape

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variation related to cutworm species in multidimensional space.

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A Canonical variate analysis (CVA) was performed in order to find the shape features

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that best distinguish among the groups based on the mandible shape changes and to

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quantify the morphological distance between different species. The results were

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reported as the p-values of Mahalanobis distances and Procrustes distances

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(Klingenberg & Monteiro, 2005). To evaluate the effect of size on the shape (allometry)

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a multivariate regression analysis was computed using the centroid size as an

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independent variable and the Procrustes coordinates as the dependent variable

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(Monteiro, 1999). All the morphometric analyses were performed using the software

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MorphoJ 1.06d (Klingenberg, 2011).

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3. RESULTS

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The Procrustes ANOVA to assess the measurement error showed that the mean square

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of digitalization error was much smaller than the Individual discarding measurement

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error of the landmarking process (Table 2). The first three components of the PCA

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accounted for 77.8% of the total variation (PC1: 38.52%, PC2: 25.06%, PC3: 14.24%).

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In general, the largest difference in mandible shape was explained by the distance

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between landmark 3 and 4, and from the landmarks in the apical end of the lobes (i.e.

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landmark 1, 7, 9, 11) to the landmark in the intersection (i.e. landmark 8, 10 and 12).

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In the genus Agrotis, differences in the mandible shape showed that A. ipsilon has a

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wider mandible with elongated and pointed incisor lobes than A. robusta. Conversely,

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A. robusta has the narrowest mandible among all the species tested and its incisor lobes

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have a broad shape with sharper crests (Fig. 2). Furthermore, regarding the genus

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Agrotis, the scatterplot of the PCA showed that A. robusta samples was more

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homogeneous group than A. ipsilon samples. In contrast, A. ipsilon samples shared the

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morphological space with all the remaining species (Fig. 3. a).

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In the genus Feltia, there were also differences, but they were not as noticeable as in the

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genus Agrotis. F. deprivata and F. lutescens have the widest mandible, F. irritans the

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narrowest one, and F. gypaetina presents intermediate width values. Moreover,

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differences in landmark 1, 7, 9 and 11 among species indicate that F. irritans has the 3rd

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and 4th incisor lobes less prominent than the rest of the samples. Comparisons among all

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genera and species showed that P. saucia shares characteristics with F. deprivata but it

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differs from A. robusta and A. ispilon by its wider mandible shape and less protuberant

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incisor and molar lobes.

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After maximizing the variance, the scatterplot of the CVA showed clusters between the

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cutworm species and the permutation between them was statistically significant (P

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<0.05) (Table 3). The CVA analysis showed that A. ipsilon is a completely separated

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group from other species and with the maximum variation in its mandible shape. Even

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though it was observed some samples of F. deprivata, F. lutescens and P. saucia share

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the morphospace, these species tended to be rather separated (Fig. 3.b).

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Multivariate regression analysis in Fig. 4 showed that there was a 10% of allometry

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influenced by the mandible shape on the centroid size, whereas the effect can be noticed

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on the specimens of A. robusta, which could have affected the morpho-space of the

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shape. After analysing a PCA from the residual of the regression (data without size

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influence) can be clearly evidence that the shape of A. robusta influenced by allometry

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(Supplementary Fig. 1).

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4. DISCUSSION

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The results of this study indicated a wide range of mandibular shape within the cutworm

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complex. Phylogenetically closest species of Agrotis were widely different, while other

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species phylogenetically distant such as Feltia and Peridroma were lightly similar.

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Mandibular shape was mainly defined by its width (distance between landmarks 3 and

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4) and the shape of their incisor lobes (landmarks 1, 7, 9, 11). In noctuid larvae, the

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incisor process or lobes in the mandible are generally considered to have been used for

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biting while the molar lobes are mostly for crushing (Das, 1937). The differences

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observed in the lobes of the mandible may generate a functional change on how the

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larvae can cut and crush or macerate the plant tissues. Considering this functional

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difference, it is likely that species with stronger molar and incisor lobes may have

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biological advantages in grassland areas where vegetation is dominated by grass plants

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with high lignin and fiber contents. Consequently, it was notorious in most of the

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samples tested here, a greater sign of wear in the molar lobes than in the incisor lobes of

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the mandible. This suggests that once the molar lobes are worn, the feeding of the larva

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may depend mostly on the incisor lobes. According to the CVA, the dominant species,

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A. robusta, differs from the rest of the cutworm species by its narrow mandible and the

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shape of its incisor lobes. In consequence, the argument stated in this study is that in A.

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robusta the lower depth in the notches of the incisors lobes generates a more compact

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structure and stronger than in the remaining species.

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It is well known that A. ipsilon is considered one of the biggest pests worldwide; these

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results showed the contrary for the semi-arid areas of the Pampean Region, being

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another species of Agrotis better represented and more competitive among them. The

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geometric morphometric analysis showed that A. robusta besides being noticeable

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different from all the examined species, it has the longest life cycle in comparison to all

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the species tested (insect-rearing data not shown), and the mandible shape variance it

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could allow this species to occupy all the different nutritional spaces around the

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southern and semi-arid areas of the Pampean region.

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Because of that and due to the higher competence pressures, lower population density is

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noticeable for A. ipsilon in the semi-arid areas of the Pampean region, compared to the

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different locations worldwide. Some tools in geometric morphometrics can be used to

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detect developmental instability between populations as a consequence of fitness

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competence (Klingenberg, 2015), being fluctuating asymmetry the most commonly

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used. Preliminary shape asymmetries were calculated (results not showed) finding that

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A. ipsilon has higher values of fluctuating asymmetry than A. robusta. Analyzing the

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level of developmental instability product of competence would be the next steps to

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follow, in order to detect the fitness behaviours related to the competition between

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larvae in the agricultural environment. Besides, it should be borne in mind the presence

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of biological control and climatic conditions could influence the abundance and

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predominance of cutworm dispersion. Also, new experimental designs are necessary to

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assess how the competitive environment drives the morphological diversity we report in

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this study.

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Considering that these species are agricultural pests, the results achieved here also have

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implications for pest management practice. In this sense, the simultaneous occurrences

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of cutworm species in the same ecological niche and the results obtained here raise

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questions about how changes in the proportion of the species within the complex may

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influence the herbivory levels on the crops. According to literature, herbivory level on a

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plant community increases as the functional diversity within the associated insect

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community increases (Shoener, 1974, Duffy et al., 2007). Hence the impact of the

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cutworm complex on crop plant biomass may not only depend on the number of species

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per se, but also on the particular traits of the species present (Deraison et al., 2015).

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Changes in functional diversity of mandibular traits within the cutworm complex could

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modify the cut of sprouts and seedlings of summer crops. The results of this study

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evidenced that cutworm’s complex differs in functional traits, mandibular shape, and

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this functional characteristic in species is of fundamental importance to determine the

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degree of damage that can be expected and to develop a predictive framework linking

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the herbivory damage with the herbivore functional diversity. Broadening knowledge of

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functional traits in insect pest can help to establish accurate values of economic damage

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thresholds in order to reduce the indiscriminate use of pesticide.

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ACKNOWLEDGMENT

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This work was supported in part by grant Pfort CyT-2017 Nº 140/2018 UNLPam and

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Research Project No I-144/17 FA-UNLPam. HB Thanks to the Universidad de

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Tarapacá, UTA Mayor 9719-17 and CONICYT Redes de Investigación REDI170182.

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We thank two anonymous reviewers for their valuable suggestions.

254 255

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FIGURE AND TABLE LEGENDS

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Table 1. Location of the 12 landmarks to capture the cutworm mandible shape.

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Landmarks

Location

1

Apical end of the 2nd incisor lobe

2

Beginning of the mandibular condyle

3

Middle of the mandibular condyle

4

Opposite to mandibular condyle

5

Apical end of the 2nd molar lobe

6

Intersection between the 1st and 2nd molar lobe

7

Apical end of the 1st molar lobe

8

Intersection between the 3rd incisor lobe and the 1st molar lobe

9

Apical end of the 4th incisor lobe

10

Intersection between the 3rd incisor and 4th incisor lobe

11

Apical end of the 3rd incisor lobe

12

Intersection between the 2nd and 3rd incisor lobe

345 346

Table 2. Procustes ANOVA results to assess the measurement error for shape change in

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the cutworm samples.

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Source of variation

SS

MS

31.386625

0.333900

94

75.42

<0.0001

Side

0.621406

0.621406

1

140.36

<0.0001

Individual*Side

0.416154

0.004427

94

1.54

0.0066

Error 1

0.547017

0.002879

190

Individual

df

F

P

349 350

Table 3. Procrustes distance between species (upper right triangle) and Mahalanobis

351

distance between the centroids of the groups (lower left triangle) derived from canonical

352

variation analysis. P-values for the pairwise are marked with asterisk (*) to indicate

353

significantly different (P < 0.05), double asterisk (**) when they are highly significant

354

(P< 0.001), and ns non-significant. AI: A. ipsilon, AR: A. robusta, FD: F. deprivata, FG: F.

355

gypaetina, FI: F. irritans, FL: F. lutescens and PS: P. saucia.

356

AI

AR

FD

FG

FI

FL

PS

AI

-

0.0643**

0.0465*

0.0493ns

0.0600 ns

0.0513 *

0.0561**

AR

4.1726*

-

0.0878**

0.0441*

0.0654*

0.0711**

0.0895**

FD

4.3152*

3.7874**

-

0.0544**

0.0633*

0.0313**

0.0238 ns

-

0.0404 ns

0.0326 ns

0.0525 ns

FG 4.6071** 2.4330** 2.5317**

357 358 359 360 361 362 363 364

FI

5.0036** 3.2899** 3.8140**

3.2692*

-

0.0424 ns

0.0667 ns

FL

5.2135** 3.3537** 2.0239**

1.9227*

3.6539**

-

0.0315 *

PS

4.9945** 4.0220** 1.8175** 2.8999**

4.3700**

1.8417**

-

365

Fig. 1. Ventral view of the concave surface of a cutworm mandible indicating landmark

366

locations. ams: anterior mandible setae, pms: posterior mandible setae,

367 368

Fig. 2. Wireframe representation of the average mandible shape variation and their

369

corresponding landmarks for the ventral view. AI: A. ipsilon (red), AR: A. robusta

370

(black), FD: F. deprivata (green), FG: F. gypaetina (yellow), FL: F. lutescens (orange),

371

FI: F. irritans (blue), and PS: P. saucia (pink).

372 373

Fig. 3. a) Principal component analysis (PCA) and b) Canonical variate analysis (CVA)

374

comparing the left mandible shape from seven cutworm species. The figures show the

375

first two PCA and CVA axes and the wireframe visualization of the average shape for

376

the species. Different colours and letters indicate the species. AI: A. ipsilon (red), AR:

377

A. robusta (black), FD: F. deprivata (green), FG: F. gypaetina (yellow), FI: F. irritans

378

(blue), FL: F. lutescens (orange) and PS: P. saucia (pink).

379 380

Fig. 4. Regression analysis of Score 1 and the centroid size. Different colours indicate

381

the species testes. AI: A. ipsilon (red), AR: A. robusta (black), FD: F. deprivata (green),

382

FG: F. gypaetina (yellow), FI: F. irritans (blue), FL: F. lutescens (orange) and PS: P.

383

saucia (pink).

384 385

Supplementary Figure 1: Principal component analysis of the average mandible shape

386

(left and right) from seven cutworm species a) using the covariance matrix of the

387

individual shape variation and b) using the covariance matrix of the residual of the

388

multivariate regression after an allometry correction. AI: A. ipsilon (red), AR: A.

389

robusta (black), FD: F. deprivata (green), FG: F. gypaetina (yellow), FI: F. irritans

390

(blue), FL: F. lutescens (orange) and PS: P. saucia (pink).

391

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Author declaration We wish to confirm that there are no known conflicts of interest associated with this publication. All the sources of funding for the work described in this publication are acknowledged in the manuscript.