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Vol. 62. Núm. 2.Abril - Junho 2018
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Vol. 62. Núm. 2.Abril - Junho 2018
Páginas 83-168
Systematics, Morphology and Biogeography
DOI: 10.1016/j.rbe.2017.12.004
Morphometric and molecular differences among Calvertius tuberosus (Coleoptera: Curculionidae) populations associated with Andean and coastal populations of Araucaria araucana in the La Araucanía Region, Chile
Luis Huala Jimeneza, Ramón Rebolledo Ranzb, Rubén Carrillo Lópezb, Mario Elguetac, Marco Paredes Honoratoa,
Autor para correspondência

Corresponding author.
a Universidad de La Frontera, Departamento de Ciencias básicas, Temuco, Chile
b Universidad de La Frontera, Departamento de Ciencias Agronomicas y Recursos Naturales, Temuco, Chile
c Museo de Historia Natural, Área de Entomología, Santiago, Chile
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Tabelas (1)
Table 1. Names and sequences of ISSR primers (Korpelainen et al., 2007).

Calvertius tuberosus (Curculionidae) lives exclusively on Araucaria araucana trees (commonly known as pehuen) in southern Chile. In this study, morphometric and molecular genetic analyses of Andean and coastal populations of C. tuberosus were performed to evaluate evolutionary divergence associated with the discontinuity of the Araucaria forest between the coastal and Andean regions. Specimens of C. tuberosus were collected in Nahuelbuta National Park, Villa Las Araucarias, and Malalcahuello National Reserve and were classified and stored at the Animal Biotechnology Researching Laboratory (LINBA), University of La Frontera, Temuco, Chile. Thirteen morphometric parameters and the expression patterns of ISSR (inter-simple sequence repeat) markers were analyzed. Morphometric data revealed high phenotypic similarity between coastal populations. The genetic analysis revealed a high similarity between coastal populations (genetic identity, 93%), which were differentiated from the Andean population (genetic identity, 84%). This study contributes new genotypic and phenotypic data for the C. tuberosus populations in forest ecosystems of A. araucana, and clarifies the associations between these characteristics and the geographic distributions of populations.

Araucaria forests
ISSR markers
Texto Completo

In Chile, there are over 4200 species of Coleoptera belonging to 97 families (Elgueta, 2008), representing 30% of all insects in Chile, with little diversification in most genera (Vergara et al., 2006) and high levels of endemism at the species level. Many of these genera are shared with those found in Australia and New Zealand, instead of South American tropical zones (Arias, 2000). Curculionidae is a diverse family, with almost 4600 genera and 51,000 species (Oberprieler et al., 2007).

Calvertius tuberosus (Fairmaire & Germain, 1860) (Coleoptera: Curculionidae) is found between the Biobío (36°46′22″S, 73°03′47″W) and La Araucanía (38°54′00″S, 72°42′00″W) regions in southern Chile (Arias, 2000), and is exclusively found on Araucaria araucana ((Mol) Koch, 1869) (Pinales: Araucariaceae) trees, with a frequency of almost 30% (Elgueta et al., 2008).

C. tuberosus is the largest among the 23 species of curculionids and other related families, on this host (Kuschel, 2000). Adults walk on the trunk or feed on leaves and soft shoots, and larvae are found in the subcortical zone in fallen or standing trees, where they feed on phloem, although they frequently become xylophages at the end of their development (Barriga et al., 1993; Morrone, 1997; Kuschel, 2000; Elgueta and Marvaldi, 2006). Morrone (1997) indicated that this beetle is a secondary invader that does not attack healthy trees, but enters branches previously affected by bark beetles (Scolytinae).

Araucaria araucana is an endemic species in South American temperate forests along the Andes mountains from 37°27′S to 40°03′S (Moreno et al., 2011). Approximately 97% of its populations form extensive pure forests, often on steep volcanic hills, and are associated with temperate rainforest species (Hechenleitner et al., 2005).

Some relatively small and disjunct populations occur in the Nahuelbuta mountains at the Nahuelbuta National Park (33°37′00″S, 79°02′00″W) and surrounding areas, including Villa Las Araucarias (38°00′17″S, 72°57′56″W). In this latter area, Araucaria are found in highly altered environments dominated by mixed forest of Nothofagus spp. (Fagales: Nothofagaceae) and exotic trees, such as Eucalyptus globulus (Labill) and Pinus radiata (D. Don).

The remaining Araucaria forests belong to private owners and are permanently subject to high levels of disturbance by the inadequate extraction of their edible fruit, fires, logging, and substitution with commercial forest plantations (Donoso et al., 2006). The ecosystems of both areas are characterized by a moist Mediterranean climate with differences related to altitude and exposure; their soils are composed of metamorphic materials and, in some locations, granite (Donoso et al., 2008). It has been proposed that geographical isolation between coastal and Andean A. araucana populations results in genetic population differentiation (Raffi and Dodd, 1998).

Morphometry was among the first methods used in biodiversity and phylogenetic studies and is still applied, despite the wide range of molecular techniques used currently (Wanek and Sturmbauer, 2015). Morphometric analyses are used in taxonomy, but are also used in coevolution and phylogenetic studies of diverse groups of insects, such as aphids, bees, grasshoppers, and beetles (Sánchez-Ruiz and San Martín, 2000). Morphometric measurements are widely used in approaches that integrate systematics with molecular data and can lead to taxonomic revisions, comparable to phylogenies created from DNA. When correctly selected, morphometric parameters can be used to establish phylogenetic relationships, especially for species that are not easy to distinguish owing to a lack of diagnostic characters (Przybycien and Waclawik, 2015).

At the genetic level, nucleotide sequence differences can be used to study evolutionary relationships among species. For instance, Woese and Fox (1977) classified prokaryotes based on ribosomal genes. Phylogenies created from DNA sequences can provide useful insight into the evolutionary history of genes and organisms (Yang and Rannala, 2012). During evolution, genetic material accumulates mutations that potentially result in phenotypic changes (Olsen and Woese, 1993).

Inter-simple sequence repeats (ISSRs) are a sensitive genetic marker for studies of polymorphism (Bornet and Branchard, 2004) within populations based on the absence or presence of a genomic element and the length of the amplified intermediary sequence (Zietkiewicz et al., 1994). ISSRs in the genomes of plants and animals are highly variable and therefore are commonly used in population genetic studies (Tikunov et al., 2003). ISSR analyses do not require high concentrations of DNA, and primer development does not require previous knowledge of the genome sequence of the organism under study (Joshi et al., 2000). The high degree of polymorphism and wide distribution of microsatellites enable the detection of low levels of differentiation (Yua, 2011).

Other molecular tools have been used to characterize A. araucana populations. For example, Marchelli et al. (2010) studied their possible pre-Pleistocene origin using chloroplast and mitochondrial DNA sequences. Based on nuclear and mitochondrial gene sequence analyses, Sequeira and Farrell (2001) investigated the phylogenetic relationships and the estimated divergence times of bark beetles associated with Araucaria in Australia and South America. The structure and genetic diversity of populations in South America have been studied based on the composition of foliar epicuticular wax alkanes (Rafii and Dodd, 1998), RAPD markers (Bekessy et al., 2002), nuclear microsatellites (Martín et al., 2014), and AFLPs (Marconi et al., 2011).

In this study, the morphological and genetic characteristics of C. tuberosus specimens from coastal populations (Nahuelbuta National Park, hereafter Nahuelbuta, and Villa Las Araucarias) and the Andean National Reserve (Malalcahuello National Reserve, hereafter Malalcahuello) of the La Araucanía region, Chile were evaluated with respect to differences among A. araucana populations.

Materials and methodsPopulations

C. tuberosus were collected from the bark of fallen and standing A. araucana threes in three areas in the la Araucanía region, i.e., Malalcahuello (38°24′21.56″S, 71°35′46.08″W), Villa Las Araucarias (38°29′12.65″S, 73°15′41.13″W), and the Nahuelbuta National Park (37°47′33.69″S, 72°59′53.36″W).

Morphometric measurements

Forty specimens from Villa Las Araucarias (27 male, 13 female), Nahuelbuta (30 male, 10 female), and Malalcahuello (32 male, 8 female) were included in the analyses. For each specimen, 13 parameters were measured under a Leica EZ4 stereoscopic magnifier (Wetzlar, Germany). The morphometric parameters were as follows: prothorax length, prothorax base width, prothorax maximum width, elytra length, elytra base width, elytra maximum width, elytra minimum width, elytra apex width, pedicel length, flagellum length, rostrum length, rostrum apex width, and rostrum base width (Fig. 1). Additionally, coloration was recorded for specimens in each population. These morphometric measurements were used to create a dendrogram using Nei's (1972) model implemented in PAST 3.14 (Hammer et al., 2001).

Fig. 1.

Morphology of a generalized Curculionidae, with the morphometrics measurements used (adapted from Marvaldi and Lanteri, 2005).

Total DNA extraction from C. tuberosus

Individuals were stored at −80°C at the Animal Biotechnology Research Laboratory (LINBA), University of La Frontera, Temuco, Chile, after grinding each specimen in a China mortar following treatment for 10min with UV light, yielding 1–2mg of homogenized tissue from each insect. Samples were processed using the AxyPrep Multisource Genomic DNA Miniprep Extraction Kit (Axygen Biosciences, Tewksbury, MA, USA).

Design of ISSR markers

Seventeen ISSR primers (Table 1) were selected based on the methods of Korpelainen et al. (2007). Seven primers (AC-T, CA-G, GA-C, AG-C, AC-C, CA-A, CAG) were prepared at the Animal Biotechnology Researching Laboratory (LINBA) for PCR amplification with purified DNA of C. tuberosus, following the methods of Pérez de la Torre (2012), with modifications. The amplification conditions were as follows: 10min of denaturation at 95°C, 40s at 90°C (45 cycles), 45s of reheating at 50°C (45 cycles), 90s of initial extension at 72°C (45 cycles), 10min of final extension at 72°C. The reaction mixture contained the following: 10μL of Maxima SYBR Green qPCR Master Mix (2×), 1μL of DNA (200ng), 1μL of 17 ISSR primers, and 8μL of H2O ultra-pure (20-μL final volume). The amplification reaction was performed using a MultiGene Gradient Thermocycler (Labnet International Inc., Edison, NJ, USA).

Table 1.

Names and sequences of ISSR primers (Korpelainen et al., 2007).

Names  Sequences  Names  Sequences 
AC-T  [AC]8CAA  [CAA]5 
CA-G  [CA]9CA-A  [CA]9
GA-T  [GA]9CAG  [CAG]6 
GA-C  [GA]8ATG  [ATG]5 
GA-A  [GA]9GA-C  [GA]9
AG-T  [AG]8AC-G  [AC]9
AG-C  [AG]8CA-T  [CA]9
AG-G  [AG]9GA-C  [GAC]5
AC-C  [AC]8   
PCR amplification of ISSR markers

Of the 17 primers evaluated according to their patterns of polymorphism, five were selected ([AC]8-T, [GA]9-T, [GA]8-C, [GA]9-A, and [CA]9-G). The PCR conditions were as follows: 10μL of Maxima SYBR Green qPCR Master Mix (2×), 1μL of DNA, 1μL of each ISSR primer, and 8μL of ultra-pure H2O (final volume, 20μL). The DNA amplification procedure was performed using a MultiGene Gradient Thermocycler (Fig. 2).

Fig. 2.

ISSR patterns of expression for C. tuberosus from Malalcahuello (1-1, 1-2, 1-3), Villa Las Araucarias (2-1, 2-2, 2-3) and Nahuelbuta (3-1, 3-2, 3-3).

In the analysis of ISSR expression patterns, only polymorphic and reproducible bands were considered, and values of 1 or 0 were assigned to indicate presence or absence. Data were classified in a binary matrix and used to estimate genetic distances based on Nei, 1972 model and these distances were used to build a dendrogram by the UPGMA method (Sneath and Sokal, 1975) with POPGEN 1.32 (Yeh et al., 1999).


A dendrogram was generated based on ISSR-derived genetic distances, reflecting the morphometric relationships among specimens obtained from three populations of C. tuberosus. There was greater morphological similarity between the specimens obtained from Villa Las Araucarias and Nahuelbuta (both coastal mountain locations) than between coastal and Andean populations. Moreover, the specimens obtained from Malalcahuello exhibited similar morphological features to those of the specimens obtained from Villa Las Araucarias (Fig. 3).

Fig. 3.

Neighbor-joining dendrogram of morphometrics measurements; 1. Villa Las Araucarias, 2. Nahuelbuta, 3. Malalcahuello.

The specimens obtained from the Villa Las Araucarias and Nahuelbuta populations presented a marked black or dark brown color, while the Malalcahuello specimens exhibited a deep red color.

In the ISSR analysis, 45 bands were detected, of which 43 (96%) were polymorphic, with a size of 250–1100bp (Fig. 2). The coastal populations (Nahuelbuta and Villa Las Araucarias) exhibited a genetic identity of 93%. The Malalcahuello population (Andes Mountains) had genetic identities of 84% and 87% in comparisons with the Villa Las Araucarias and Nahuelbuta populations, respectively (Fig. 4).

Fig. 4.

ISSR analysis. (A): Dendrogram for the populations studied (control group A. viridans).

A genetic distance value of less than 0.07 was observed between the coastal populations. The genetic distances between the Andean population (Malalcahuello) and the Villa Las Araucarias and Nahuelbuta populations were 0.17 and 0.14, respectively (Fig. 4).


Differences in body size between males and females are common in insects; typically, females are larger than males (Posadas et al., 2007). In Curculionidae, sexual dimorphism is common in the rostrum, i.e., the female rostrum is generally larger and flatter than that of the male (Soto and Reyes, 2014). Nevertheless, in C. tuberosus, rostrum size did not differ substantially between females and males; the observed morphometric variation could be the result of geographic isolation among C. tuberosus populations.

The morphometric analysis indicated the presence of geographic variation; in particular, the coastal populations (Villa Las Araucarias and Nahuelbuta) exhibited similar morphological patterns, which differed from those of the Andean population (Malalcahuello) (Fig. 3). The morphological and genetic similarity of coastal populations of C. tuberosus could be attributed to a more recent biogeographic separation of these populations.

The morphological and genetic characteristics of the coastal and Andean populations of C. tuberosus could be a consequence of the geographical separation their host, A. araucana, which has been affected by large-scale environmental changes. For example, since Pleistocene glaciation events, the tree persisted in small populations during the Last Glacial Maximum (LGM) in the coastal range of Chile and some areas of the Andes Mountains (Villagrán, 2001; Martín et al., 2014), with a discontinuous distribution. The substantial distance between populations suggests the possibility of population genetic changes, such as inbreeding, gene drift, and altered gene flow (Marchelli et al., 2010).

More recent catastrophic events, such as the Villarrica, Llaima, Lonquimay, and Copahue volcanic eruptions (Heusser et al., 1988) and serious deforestation over the last century, have resulted in the degradation or replacement of about 120,000ha of native forest (Herrmann, 2006), leading to further differences between the Andean and coastal populations by a lack of a vegetative continuum, limiting insect dispersal.

Despite a close relationship between the populations of Villa Las Araucarias and Nahuelbuta, there were clear genetic and morphological differences between the specimens of C. tuberosus in the populations. Current data indicate that anthropic intervention at Cordillera de Nahuelbuta (the mountain range that connects both populations) has resulted in over 70% loss of native vegetation (Wolodarsky-Franke and Díaz, 2011) and produced habitat fragmentation and more substantial population isolation.

Differentiation in morphometric parameters between geographically separated populations explains, to some extent, evolutionary processes and is considered the first stage in allopatric speciation (Olivero et al., 2012). In C. tuberosus, the interruption of gene flow between populations in diverse historical periods could have resulted in reproductive isolation, which would eventually lead to genetic differentiation and evolutionary divergence.

The genetic differences and similarities among populations of C. tuberosus (Fig. 4) can be compared with genetic variation in its obligate host, A. araucana. These comparative analyses allow us to infer that changes in the geographic distribution of Araucaria influence the observed variation in both species, thereby explaining genetic variability in coastal and Andean populations (Bekessy et al., 2002).

The ISSR markers revealed a more distant relationship (based on distance and genetic identity) between the coastal populations (Nahuelbuta and Villa Las Araucarias) and the Andean population (Malalcahuello) than between the two coastal populations (Fig. 4). These results are consistent with previous analyses of A. araucana Andean and coastal populations indicating significant genetic differences using RAPDs (Bekessy et al., 2002) and microsatellites (Martín et al., 2014). Similar differences between Araucaria populations have been described by Rafii and Dodd (1997) based on the proportional composition of foliar epicuticular wax alkanes.

It is not possible to understand the natural world without extensive knowledge of the morphological characteristics of organisms, which play central roles in life cycles, geographical distributions, identification, conservation, evolution, development, and delimitation of species (Wortley and Scotland, 2006). The inclusion of morphological data substantially improves the results of molecular phylogenetic analyses, proving to be useful not only for the resolution of taxonomic problems, but also for coevolution studies and phylogenetic inferences (Przybycien and Waclawik, 2015). The linkage of morphometric and molecular analyses is powerful for studies of population structure and the adaptive significance of trait divergence (Lee and Lin, 2012), to identify biotypes of the same species on different hosts (Fekrat et al., 2014), and for the geographic delimitation of species (Schwarzfeld and Sperling, 2014). Therefore, integrative analyses including morphometric measurements and DNA sequences are needed for a comprehensive understanding of the autoecology of species.


This study contributes new genotypic and phenotypic data for the C. tuberosus populations in forest ecosystems of A. araucana and clarifies the relationships between these characteristics and the geographic distributions of the populations; accordingly, these findings extend our knowledge of C. tuberosus populations in Chile.

Conflicts of interest

The authors declare no conflicts of interest


This research was funded by FONDEF D10I1038 (Biodiversity information network to guide the properties of scientific research in support of public environmental policies) and DIUFRO DI14-0111 [Chemical and biological characterization of Maiten (Maytenus boaria) as a food attractant of A. superciliosus (Coleoptera: Curculionidae)]. DIUFRO DI13-TD01 supported the doctoral thesis.

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