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Corresponding author: Alfonsina Arriaga-Jiménez ( ponchis.arriaga@gmail.com ) Academic editor: Andrey Frolov
© 2015 Alfonsina Arriaga-Jiménez, Lise Roy.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Arriaga-Jiménez A, Roy L (2015) Co1 DNA supports conspecificity of Geomyphilus pierai and G. barrerai (Coleoptera, Scarabaeidae, Aphodiinae) and is a good marker for their phylogeographic investigation in Mexican mountains. ZooKeys 512: 77-88. https://doi.org/10.3897/zookeys.512.9646
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Members of Geomyphilus are associated with rodent burrows, such as pocket gophers and prairie dogs. In Mexico, they are found in the mountains of the Mexican Volcanic Belt and in Sierra Madre Oriental. Our study aims to initiate the exploration of the dispersal modes of Geomyphilus pierai and G. barrerai from burrows of pocket gophers. In order to estimate the dispersal scale of the beetles, the utility of mitochondrial and nuclear molecular markers for studying the phylogeographic structure of this complex of species (G. pierai and G. barrerai) was tested from 49 beetle individuals. High intraspecific and intra-mountain nucleotidic diversity was captured from this sample using Co1 mitochondrial sequences, whilst the ITS2 nuclear ribosomal sequence did not allow observing informative variation. Mitochondrial phylogenetic analysis revealed that the specific delineation between the two species under study was doubtful. In this preliminary study, Co1 was shown to be a good marker for elucidating dispersal routes of the burrowing rodent-associated beetles.
Dung beetles, pocket gophers, phylogeography, Co1, ITS, Mexico
The subfamily Aphodiinae is one of the most diversified groups within the Scarabaeoidea (classification sensu
Members of Geomyphilus are endemic of the Nearctic region and are associated with rodent burrows. Common hosts include pocket gophers (Geomyidae) and prairie dogs (Sciuridae: Cynomis), but some data indicate that rarer Aphodiinae species may be found in association with kangaroo rats (Heteromyidae: Dipodomys), voles (Cricetidae: Microtus), or other rodents (
While the habits and distribution patterns of their associated rodents are rather well-known (
We undertook a preliminary molecular study to investigate Geomyphilus dispersal modes and clarify whether the preferred route is underground or aerial. Measuring the amount of gene flow between the populations in different mountains could help understanding the spatial scale of the Geomyphilus dispersal. Indeed, one may presume that if the species are mainly moving within the rodent galleries, the gene flow between the populations in different mountains should be less than if they fly outside.
To initiate the exploration of the dispersal routes of coprophagous scarabaeids narrowly associated to rodents, the development of molecular genotyping is needed, allowing to characterize the population genetic structure at different spatial scales within at least one species living in rodent burrows. Candidate molecular markers should provide genetic variation detectable at spatial scales appropriate to assess aerial dispersal. We assumed that the aerial dispersal might allow individuals to quickly fly from one mountain to another, whilst the underground dispersal most likely would limit the beetle circulation to a single mountain or at most to the interconnected mountains. Therefore, we decided to work on the individuals sampled from the nests located in the different mountains of MVB.
Moreover, several species of the genus Geomyphilus can be found together in the same gopher nest (
Here we present the results from a preliminary molecular investigation of G. pierai and G. barrerai aiming at (1) assessing interspecific molecular divergence and (2) testing molecular markers to allow phylogeographic studies to be performed in these species. We sequenced one mitochondrial and one nuclear DNA regions from individual beetles sampled from the burrows of pocket gophers (C. merriami and T. umbrinus) in four mountains of the MVB. The mountains under study were: La Malinche, Cofre de Perote, Pico de Orizaba and Sierra Negra (Fig.
To collect Geomyphilus beetles, the gopher nests were excavated (only those that seemed recently used). The galleries were followed up to find the nests and latrines where the dung beetles might be found. This sampling technique is very effort-consuming because the nests can be located very deep and they are not always found. Once the nests and latrines were reached, the organic matter was removed as well as the soil within 20 cm from it, and then all the soil was carefully inspected for scarabs. The depth of the nest and latrines varies depending on the type of soil, slope and surrounding vegetation. They were found from 30 cm to 1.5 m deep in different biotopes (scrublands and grasslands) in the studied mountains (Fig.
The DNA was extracted from ethanol-preserved specimens. Either of two different techniques were used, both designed to obtain molecular information while keeping voucher cuticle of each individual. For the first one, after complete evaporation of ethanol, the cuticle of each specimen was cut at two points on the prothoracic-mesothoracic joint soft integument in order to facilitate the lysis of internal tissues. This was done with a sterile syringe needle on a glass slide before insertion of individual in a vial for proteinase K digestion. For the second technique, one leg was removed from the beetle, then put in an Eppendorf vial and crushed with a pestle. For both techniques, the entire cut individual or the crushed leg were immersed in 100 ml Buffer ATL + 15 ml Proteinase K and incubated for 19–30 h at 70 °C, before proceeding to the DNA purification using Qiagen’s DNeasy Blood & Tissue Kit following the manufacturer’s protocol. The specimens’ cuticles (tech. 1) were conserved in glycerol and the body remaining (tech. 2) in ethanol as vouchers.
Checking congruence between phylogenetic topologies from genes with different transmission patterns such as mt DNA (maternal transmission) and nuclear DNA (biparental transmission) is useful to confirm cryptic species (see
Polymerase chain reaction (PCR) was performed using the Qiagen’s Taq PCR Core kit. The following reagent concentrations were used for a final volume of 25 µL per tube: 1×PCR buffer, 0.036U/µl Qiagen Taq Polymerase, 300 µM dNTPs and 0.6 µM of each Co1 primer or 0.43 µM of each ITS2 primer, and 2.5 mM MgCl2 for CO1 or 3.0 mM MgCl2 for ITS2. For the Co1, thermal cycling parameters comprised an initial denaturation step of 10 minutes at 94 °C, followed by 5 cycles of 94 °C for 40 s, 49 °C for 60 s and 62 °C for 60 s, and 35 cycles of 94 °C for 40 s, 52 °C for 60 s and 62 °C for 60 s, with a final elongation at 62 °C for 10 min. For the ITS region, thermal cycling parameters comprised an initial denaturation of 10 minutes at 94 °C, followed by 5 cycles of 94 °C for 40 s, 56 °C for 60 s and 72 °C for 60 s, and 35 cycles of 94 °C for 52 s, 52 °C for 60 s and 72 °C for 60 s, with a final elongation at 72 °C for 10 min. Purification and sequencing was realized by Genoscreen (France, Lille) using a 96-capillary sequencer ABI3730XL.
Obtained DNA sequences were aligned using Muscle (
In order to estimate the utility of the two DNA markers to assess the phylogeographic structure of this complex of species (G. pierai and G. barrerai; Fig.
Nucleotide diversity: (Pi, Dxy and Da) between G. pierai and G. barrerai.
Pi | ||||
---|---|---|---|---|
Cofre-Vigas | Pico Orizaba | Sierra Negra | Malinche | |
Cofre Perote | - | |||
Pico Orizaba | 0.02305 | - | ||
Sierra Negra | 0.01918 | 0.01622 | - | |
Malinche | 0.02143 | 0.02083 | 0.0193 | - |
Dxy | ||||
Cofre-Vigas | Pico Orizaba | Sierra Negra | Malinche | |
Cofre Perote | - | |||
Pico Orizaba | 0.02605 | - | ||
Sierra Negra | 0.025 | 0.01898 | - | |
Malinche | 0.02615 | 0.02286 | 0.034 | - |
Da | ||||
Cofre-Vigas | Pico Orizaba | Sierra Negra | Malinche | |
Cofre Perote | - | |||
Pico Orizaba | 0.00623 | - | ||
Sierra Negra | 0.01439 | 0.00725 | - | |
Malinche | 0.01206 | 0.00765 | 0.02799 | - |
Species boundaries between G. barrerai and G. pierai were not confirmed either by mitochondrial or nuclear data. When considering concordance between morphological taxonomic assignation and molecular information, not only none of the 46 Co1 polymorphic sites and none of the 4 ITS2 polymorphic sites did segregate accordingly, but also, the distribution of the two taxa across the Co1 topology was dispersed and intermingled, without any clade grouping together all individuals of any of them (Fig.
Gb | Gp | Gb vs Gp | Gp vs Np | Gb vs Np | |
---|---|---|---|---|---|
n | 8 | 20 | 28 (20 and 8) | 20/3 | 8/3 |
k | 13.8570 | 14.5630 | 15.0030 | 30.6800 | 43.6000 |
Pi | 0.0208 | 0.0219 | 0.0225 | 0.0461 | 0.0654 |
Dxy | 0.0236 | 0.1250 | 0.1256 | ||
Da | 0.0023 | 0.1141 | 0.1152 |
Subsequently considering the sample as monospecific, so much variation in the Co1 sequence dataset, obtained from the present locally-restricted sample is promising, providing basic tools for further analyses. Besides, several different mitochondrial lineages were found within the beetle sample, with no clear correspondence between mountains and genetic structure (Fig.
The size of the circles is proportional to the haplotype frequency. The length of links between haplotypes is proportional to the number of mutated positions. The median vectors that represent hypothetical intermediates or unsampled haplotypes are shown in small open dots. The haplotype circles’ color corresponds to the sampling origin (one color per mountain).
The authors thank Marco and Giovanni Dellacasa for the identification of all the material and M.A. Arnaud who assisted in the English proof-reading of the manuscript. J. L. Sánchez- Huerta and A. Aceves- Aparicio consent to include their pictures of G. barrerai and G. pierai as part of our paper. The fieldwork was funded by the Instituto de Ecología, the Societé d’Entomologie de France, with the Bourse Germain Cousin assigned to AAJ, and the doctoral school ED#60 of the University Paul-Valéry Montpellier. Fernando Escobar helped during the fieldwork. We thank the CEFE platform of molecular biology for the help and the facilities for the molecular work. We thank the reviewers and editor whose comments contributed to improve the final version of the manuscript.
EMBL accession numbers of beetle DNA sequences
Data type: DNA sequences
Explanation note: accession numbers (EMBL) of each Co1 and ITS DNA sequence obtained from beetle individuals and integrated into analyses in the present study.