Research Article |
Corresponding author: Ángel I. Ortíz-Ceballos ( angortiz@uv.mx ) Academic editor: Samuel James
© 2020 Diana Ortíz-Gamino, Josefat Gregorio, Luis Cunha, Esperanza Martínez-Romero, Carlos Fragoso, Ángel I. Ortíz-Ceballos.
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:
Ortíz-Gamino D, Gregorio J, Cunha L, Martínez-Romero E, Fragoso C, Ortíz-Ceballos ÁI (2020) Population genetics and diversity structure of an invasive earthworm in tropical and temperate pastures from Veracruz, Mexico. ZooKeys 941: 49-69. https://doi.org/10.3897/zookeys.941.49319
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Pontoscolex corethrurus (Müller, 1857) is an invasive tropical earthworm, globally distributed. It reproduces through parthenogenesis, which theoretically results in low genetic diversity. The analysis of the population structure of P. corethrurus using molecular markers may significantly contribute to understanding the ecology and reproductive system of this earthworm species. This work assessed the genetic diversity and population structure of P. corethrurus with 34 polymorphic inter simple sequence repeat markers, covering four populations in tropical and temperate pastures from Veracruz State. Nuclear markers distinguished two genetic clusters, probably corresponding to two distinct genetic lineages. The number of clones detected in the AC population was lower than expected for a parthenogenetic species. Also, the apparent lack of differences in population structures related to the geographic region among the populations studied may indicate that human-mediated transference is prevalent in these areas. Still, most individuals apparently belong to lineage A, and only a few individuals seem to belong to the lineage B. Thus, the admixture signatures found among the four populations of P. corethrurus may have facilitated a successful invasion by directly increasing fitness. In summary, addressing the genetic variation of P. corethrurus with ISSR markers was a suitable approach, as it evidenced the genetic diversity and relationships in the populations evaluated.
Agroecosystems, asexual reproduction, exotic earthworm, peregrine species, Rhinodrilidae
Earthworms are not only ubiquitous ecological engineers of soil that create biogenic structures; they also sustain the functioning of the ecosystem through their fundamental actions (
Pontoscolex corethrurus, formerly included in Glossoscolecidae but is now placed in Rhinodrilidae (
Parthenogenesis is common in earthworms, usually associated with dispersal, where a single propagule is usually sufficient to stablish a new population (
Around the world, P. corethrurus is distributed from 0 to 2000 m a.s.l., with an average altitude of 463 m (Fragoso et al. 1999). In Mexico, specifically in Veracruz State, its distribution ranges from 0 to around 1600 m a.s.l., living at an average temperature of 17 °C (
Sampling points were established according to the different attributes of the sites studied in Veracruz State, Mexico (
Attributes of earthworms sampling of four pastures in central Veracruz State, Mexico.
Sampling Site | Altitude (m a.s.l.) | Climate | Grass species | Soil texture (%) | ||
---|---|---|---|---|---|---|
Clay | Silt | Sand | ||||
Laguna Verde (LV) | 24 | Aw1(w)g | Paspalum conjugatum, Cynodon nlemfuensis | 26.6 | 28.1 | 45.3 |
Actopan (AC) | 480 | Aw0(w)gw” | Saccharum officinarum L. | 12.8 | 32.3 | 54.9 |
La Concepción (LC) | 973–1036 | (A)Ca(f)gw” | Paspalum conjugatum, Cynodon nlemfuensis | 26.6 | 28.2 | 45.3 |
Naolinco (NA) | 1566–1667 | Cb(fm)gw” | Paspalum conjugatum, Cynodon nlemfuensis, Pennisetum clandestinum | 12.8 | 32.3 | 54.9 |
Tail-wall tissue was used for extraction of genomic DNA. Total DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Mainz, Germany) following the manufacturer’s instructions. DNA was checked for quality by gel electrophoresis and quantified using a spectrophotometer (ND-2000, Nanodrop Technologies, Wilmington, DE).
Forty specimens of P. corethrurus (N = 10 per site) were used for ISSR screening. ISSR screening was based on five primers (Table
Primers used for PCR amplification of Pontoscolex corethrurus genomic DNA.
Primer | Sequence | Ta (°C) | Maximum number of bands | Estimated size (bp) |
---|---|---|---|---|
840 | GAGAGAGAGAGAGAGAYT | 59.5 | 8 | 2000-200 |
834 | AGAGAGAGAGAGAGAGYT | 61 | 7 | 2000-300 |
866 | CTCCTCCTCCTCCTCCTC | 70 | 6 | 2000-400 |
810 | GAGAGAGAGAGAGAGAT | 52.4 | 6 | 2000-400 |
807 | AGAGAGAGAGAGAGAGT | 54.4 | 7 | 2000-300 |
The amplified DNA fragments were transformed into a binary matrix (1 = presence, 0 = absence), as reported previously (
The genetic variance for all MLGs was estimated through an analysis of molecular variance (AMOVA) using the GenAlEx v.6.5 software (
Population structure was explored using the Bayesian clustering method implemented in STRUCTURE v.2.3.4 (
The hybridization status of individuals according to the Bayesian genetic clusters defined in Structure (defined as putative Lineage A and Lineage B) was further investigated using NEWHYBRIDS v1.1 (
Beyond the ecological relevance of P. corethrurus, information on genetic variability is relevant to determine the selective forces that act on the reproductive system of this species. In that sense, the survey carried out in this study was aimed to reveal the population genetic structure of P. corethrurus in natural landscapes covering tropical and temperate pastures. For this, a set of ISSR markers were used to assess the genetic diversity and population structure of P. corethrurus genotypes from four locations in Veracruz State, Mexico.
Following a PCR-based approach, ISSR primers produced bands on agarose gel that were suitable for assessing the genetic diversity and genetic relationship between and across populations of P. corethrurus. The PCR products ranged between ~200 and ~2000 bp from genomic DNAs of P. corethrurus. The total number of bands and polymorphism rates are shown in Table
Parameters of genetic variation in four Pontoscolex corethrurus populations living in central Veracruz State, Mexico.
Sampling Site | N | MLG | eMLG | Pb | Tb | H | G | E 5 | Hexp | Ia | rbarD |
---|---|---|---|---|---|---|---|---|---|---|---|
LV | 9 | 9 | 9 | 20 | 34 | 2.20 | 9 | 1 | 0.30 | 1.98* | 0.10* |
AC | 10 | 8 | 8 | 22 | 34 | 2.03 | 7.14 | 0.93 | 0.30 | 3.23* | 0.15* |
LC | 6 | 6 | 6 | 27 | 34 | 1.79 | 6 | 1 | 0.39 | 1.16* | 0.04* |
NA | 10 | 10 | 10 | 24 | 34 | 2.30 | 10 | 1 | 0.29 | 4.08* | 0.18* |
Total | 35 | 33 | 9.85 | 3.48 | 31.41 | 0.97 | 0.40 | 1.65 | 0.05 |
Analysis of molecular variance (AMOVA) testing for genetic differentiation between four populations of Pontoscolex corethrurus living in central Veracruz State, Mexico (A), and PhiPT pairwise comparisons (B).
Level of variation | d.f. | SS | MS | Est. Var. | Proportion (%) | |
---|---|---|---|---|---|---|
A) | Among Populations | 3 | 59.079 | 19.693 | 1.760 | 25% |
Within Populations | 29 | 154.497 | 5.327 | 5.327 | 75% | |
Total | 32 | 213.576 | 7.088 | 100% | ||
Population | Laguna Verde | Actopan | La Concepcion | Naolinco | ||
B) | Laguna Verde | 0.000 | ||||
Actopan | 0.229** | 0.000 | ||||
La Concepcion | 0.069 | 0.161** | 0.000 | |||
Naolinco | 0.335** | 0.347** | 0.199* | 0.000 |
Although some individuals cluster together according to site (e.g., animals in NA), most of the individuals scattered in a non-uniform clustering (Figure
A Principal Components Analysis, where colors indicate specimens of the population (A) and a Minimum Spanning Network where each node denotes a different MLG, with size matching the number of individuals. Edge thickness and color are proportional to absolute genetic distance. Edge lengths are arbitrary (B). Both analyses show the relationship between multilocus genotypes (MLGs) for four different earthworm populations of Pontoscolex corethrurus living in central Veracruz State, Mexico.
The Bayesian analysis of population structure estimated two distinct genetic clusters (K = 2, Ln P (D) = -557.89 ± 0.31) distributed across the four geographic locations (Figure
Estimated population genetic structure with a summary plot of Q estimates based on the ISSR data observed for four populations of Pontoscolex corethrurus in central Veracruz State, Mexico. Each individual is shown by a vertical line, which is partitioned into colored segments representing the fraction of the number of members in cluster K (%).
Genetic structure using ISSR data for 35 Pontoscolex corethrurus individuals based on discriminant analysis of principal components (DAPC). Proportion of eigenvalues in discriminant analysis (bottom left plot) and PCA eigenvalues (bottom right), with the first 12 significant principal components highlighted in black.
Classification of Pontoscolex corethrurus individuals according to a Bayesian assignment algorithm implemented in NEWHYBRIDS (
Soil texture and chemical composition are among the key environmental variables that play a role in the invasion process of earthworms (Hendrix and Bohlen 2006;
In this work, the study of genetic diversity and population structure of four populations living in Mexican tropical and temperate pasture was carried out through a molecular approach using ISSR markers. The use of ISSR markers is well supported by several studies since it produces a high percentage of polymorphic loci (
According to PhiPT values, there were significant differences between the LV, AC, and NA populations, meaning that most populations possess high genotypic diversity (Figure
As regards the movement of MLGs across the different sites, a conspicuous behavior was observed, particularly in LV, AC, and LC. Such observation may be explained only by an intense human-mediated transference of P. corethrurus through road networks or activities associated with agriculture (
Lineage A seems to be widespread, covering LC and LV sites (Figure
In contrast to lineage A, lineage B (mostly NA specimens) showed the best distinct cluster of individuals (Figure
On the other hand, sexual reproduction is a rare event in P. corethrurus (
In summary, the screening of genetic diversity is helpful to monitor the dynamics of population structure and its relationships to ecological and environmental features and to contribute valuable information about the isolation of invasive earthworm species. In this sense, our work provides evidence of the existence of two lineages of P. corethrurus in Veracruz State, Mexico, showing different distribution patterns according to the prevailing environmental conditions found in regions studied. Therefore, our data represent an relevant contribution to know the movement dynamics and diversification of P. corethrurus, which will be useful information for planning successful strategies aimed to control or prevent the biological invasion of this species in Mexico.
Despite being random, biological invasions are intriguing events, mainly because they involve populations of organisms with certain features and particular habits. Intriguingly, a parthenogenic species such as P. corethrurus has been successful in colonizing areas all over the world. The interaction of P. corethrurus genetics with the environment should drive its selection and distribution pattern.
In this work, we assessed populations of P. corethrurus inhabiting tropical and temperate pastures of Veracruz, Mexico, in terms of genetic variation through ISSR markers. Our results revealed the existence of at least two well-differentiated genetic clusters, corresponding to different lineages (lineages A and B). The lineages identified in this work likely correspond to lineages L1 and L3 identified previously by
We would like to thank to Norma Flores-Estévez and Wilberth A. Poot-Poot for the support and help at INBIOTECA, Ernesto Ormeño-Orrillo for his help at Ciencias Genómicas, and to Rogelio Lara González and Katia J. Martínez Velázquez for their help during fieldwork. We are thankful to Alejandra Velázquez Lobo for valuable comments and careful revision of the manuscript and Alberto Ortíz Brito for proving the sampling map. Diana Ortíz-Gamino was supported by a scholarship (No. 251818) by Consejo Nacional de Ciencia y Tecnología (CONACYT).
Figure S1
Data type: image
Explanation note: Linkage disequilibrium test using r̄ d as implemented in R package Poppr (