Research Article |
Corresponding author: Michael J. Jowers ( michaeljowers@hotmail.com ) Academic editor: Antonio M. de Frias Martins
© 2024 Michael J. Jowers, José Liétor, Antonio R. Tudela, Pedro A. Jódar, Inés Galán-Luque, Gregorio Moreno-Rueda.
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:
Jowers MJ, Liétor J, Tudela AR, Jódar PA, Galán-Luque I, Moreno-Rueda G (2024) Phylogenetic evidence suggests the non-validity of the Iberian land snail genus Tartessiberus and confirms its synonymy with Iberus (Helicidae). ZooKeys 1201: 219-231. https://doi.org/10.3897/zookeys.1201.117318
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The monospecific genus Tartessiberus was described in the year 2021 including a single species (T. cilbanus). However, its description relied solely on morphological and anatomical data. In the present work, we use a fraction of the mitochondrial DNA cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA) and the nuclear large ribosomal subunit (LSU) to clarify its validity through phylogenetic positioning. Knowledge of the distribution of this species is also improved by citing new locations and expanding the geographical range to approximately 200 km2. Additionally, a morphometric analysis of 259 shells is presented for comparisons with shells of the Iberus marmoratus complex and testing the power of conchological features as a tool for specimen identification. The relatively high conchological variability found for T. cilbanus, together with the discovery of populations with intermediate conchological features between T. cilbanus and other closely related taxa, suggest that the determination of this species should be based on genetic criteria. Our molecular analyses demonstrate that T. cilbanus belongs to the Iberus genus, and thus, we proceed to update its taxonomic status to Iberus cilbanus comb. nov., and, thus, to consider Tartessiberus from now on as a junior synonym of Iberus.
Andalusia, Gastropoda, Helicidae, Iberian Peninsula, land snails, morphometrics, new combination, Spain, Tartessiberus, taxonomy
The Iberian Peninsula is unquestionably a flora and fauna biodiversity hotspot (
Despite the importance of carrying out genetic analysis for species delimitation, still several land snail species, or even genera, are described solely based on anatomical and/or morphological approaches. A recent example is the description of Tartessiberus cilbanus Altaba & Ríos Jiménez (2021), a new monospecific genus endemic from southern Spain. The description and delimitation of this genus were entirely based on morphological and anatomical traits (genitalia, shell, and radula morphology), in comparison to closely related species of the tribe Allognathini. The fact that the morphology of this new species was intermediate between those of the genera Iberus Montfort, 1810 and Allognathus Pilsbry, 1888 directed the authors to create a new genus for the species. Despite morphological characters being useful for discerning within-population variance, they should be complemented with molecular analyses to complete taxonomic evidence when possible.
Tartessiberus cilbanus is linked to a number of snail populations located in the Sierra de Grazalema Natural Park (Cadiz Province, southwestern Spain), which were traditionally assigned to Iberus loxanus (A. Schmidt, 1855) because their shells fit within the pattern of variation of this species. However, I. loxanus exhibits a great conchological variation (
The objective of this work is to analyse the phylogenetic position of T. cilbanus, providing molecular analyses of specimens sampled in various locations of its potential distribution area. The determination of its validity has important implications for cataloguing the Iberian land snail diversity and understanding the speciation processes in gastropods in the Iberian Peninsula.
We carried out a field sampling systematically covering all the calcareous mountain ranges of the potential distribution area of T. cilbanus, according to
Top left: Map of the western provinces of Andalusia (Southern Spain) showing the geographic location (in red-filled circles) of the two known populations for T. cilbanus. Acronyms on map: SG (Sierra de Grazalema Natural Park, Cadiz), LA (Los Alcornocales Natural Park, Cadiz), SU (Sierra de la Utrera, Malaga). Right: maximum likelihood tree of Iberus. Values by nodes represent bootstrap values for the ML analyses (> 75%) and BI posterior probabilities (BPP = 1) (represented by yellow-filled circles) are shown for all major clades and for T. cilbanus and closely related taxa. T. cilbanus clade is shown in red.
We measured 259 T. cilbanus shells. Measurements of shell morphometrics were conducted following
We carried out statistical comparisons between morphometric measurements with those of the two taxa closely related both phylogenetically and geographically (I. marmoratus loxanus and I. marmoratus marmoratus) with ANOVA tests when the variables were homoscedastic and normally distributed, otherwise using the Kruskal-Wallis test. In addition, a Principal Components Analysis (PCA) was carried out to determine the overlap in the morphospace between the populations of the described species and those of both I. marmoratus ssp. The variables used to place each population into the morphospace were the averages of the largest Ø and the height of the shells along with the average percentage of the total surface of the shells occupied by the peristome. These variables were shown to be adequate because more than 92% of the variance of the grouped data was explained by accumulating the first two principal components (PC).
Three specimens (codes A2, A3, and AH1) were sacrificed by drowning and a tissue sample was extracted for molecular analyses, stored in absolute ethanol and maintained at -20 °C. Specimen A3 was collected 660 m north from the type locality shown by
Genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The total alignment comprises all known Iberus sequences from Genbank (N = 141) including Iberellus sp. and two outgroup taxa, (Rossmaessleria sicanoides (Kobelt, 1881) and Eremina dillwyniana (L. Pfeiffer, 1853) (Suppl. material
We firstly used the primers LCO and HCO (
Phylogenetic tree reconstructions for the three concatenated gene fragments (total length 1984 bp) were performed using maximum likelihood (ML) and Bayesian inference (BI), through RAxML v.7.0.4 (
From the BI analysis, two independent runs (each with four Markov chains for 10 × 107 generations) were performed. Trees and parameters were sampled every 1000 generations. A majority-rule consensus tree was estimated by combining results from duplicated analyses, after discarding 25% of the total samples as burn-in. ML searches were conducted under GTRGAMMA and support was assessed by using 1000 bootstrapped replicates. All phylogenetic analyses were performed in the CIPRES platform (
The phylogenetic analyses recovered three well-supported clades for the genus Iberus with Tartessiberus included within the tree topology, a clear indication that this later genus cannot be valid. Sequences of T. cilbanus specimens were grouped in the centre clade, with I. rositai, I. loxanus, I. marmoratus and Iberus sp. (Fig.
Genetic divergence between T. cilbanus and the rest of the closely associated taxa remained high, with a minimum divergence of 7.5% and a maximum of 10.9% for the COI and 3% and 5.8% for the 16S rRNA gene fraction (Table
P-uncorrected distances for the taxa of the clade closely associated with T. cilbanus, COI (lower matrix) and 16S rRNA (upper matrix).
T. cilbanus | I. cobosi | I. loxanus | I. marmoratus | I. rositai/loxanus | |
---|---|---|---|---|---|
T. cilbanus | – | 5.80% | 3.07% | 4.99% | 3.25% |
I. cobosi | 10.90% | – | 4.74% | 5.76% | 5.04% |
I. loxanus | 10.45% | 12.73% | – | 4.28% | 2.58% |
I. marmoratus | 8.23% | 11.13% | 10.65% | – | 4.19% |
I. rositai/loxanus | 7.48% | 10.09% | 10.18% | 7.73% | – |
As expected, most locations for T. cilbanus were from the Cadiz Province. Nevertheless, a new locality was found in the Sierra de la Utrera massif (province of Malaga, southern Spain), a karstic habitat ecologically analogous to that of its main distribution region in the Grazalema Natural Park (Fig.
Suppl. material
The first factor of the PCA (PC1, Fig.
Distribution of T. cilbanus (8 localities), I. marmoratus loxanus (35 localities) and I. marmoratus marmoratus (36 localities) in the bidimensional space generated by the two first PC of a PCA analysis. Each point in the graph represents a single sampling locality. Coordinates of centroids for each species have been calculated as the average X and Y coordinates of the points included in the corresponding clouds. T. cilbanus cloud has been highlighted in light red.
Suppl. material
During the sampling, we found populations composed of dwarf-sized specimens with intermediate conchological characteristics between T. cilbanus and other taxa of the I. marmoratus complex that surround the Grazalema Natural Park. These populations were found in the distribution margins of T. cilbanus, pointing to possible genetic introgression in the north (Algodonales, Cadiz Province), as well as in the south (Casares, Malaga Province). Fig.
Photographic series of intermediate specimens between T. cilbanus and I. marmoratus ssp. A Hermitage of Virgencita, Algodonales, Sierra de Lijar (Cadiz Province) B Sierra Crestellina, Casares (Malaga Province). Below each photographic series, a tentative composition with the parents and an intermediate specimen in a central position is displayed. Selected shells of I. marmoratus ssp. come from the closest locations where sampling material was available: Cueva del Gato, Benaojan (Malaga Province) for series A and Gaucin Castle (Malaga Province) for series B.
The position of I. cilbanus as an independent lineage rules out that this clade could be mistaken for any of its closely related species. Our findings, consequently, provide a study case highlighting the importance of genetic analysis to correctly assign taxonomic value when describing species or even genera, although
In addition to the phylogenetic position, we rely on genetic divergence to ascertain the high genetic differentiation between I. cilbanus and its sister clade (Fig.
The existence of the genus Tartessiberus would not only imply an unusually young genus (~ 5 Ma versus Iberus at 18. 5 Ma;
Our field observations and captive breeding trials (unpublished data) have found that individuals and populations of different species of the genus Iberus tend to show dwarfism tendencies as a possible consequence of hybridization. Further studies will be necessary to determine if the smaller population of Sierra de la Utrera is undergoing a process of introgression by I. marmoratus marmoratus or, alternatively, if the small size is a local adaptive response or a symptom of phenotypic plasticity. Further genetic sequencing will corroborate possible hybridization between these species.
We are most grateful to Mohammed Bakkali for his valuable support concerning the genetic work. Special thanks to Martin Haase and Thomas von Rintelen for their reviews. Thanks to Amelia Abromaitis for graphic design assistance of figure 1. This study has been carried out in accordance with both Spanish and Andalusian legislation (Law 8/2003) for the protection of wild fauna and flora in the case of invertebrate species not included in the National (Royal Decree 139/2011) and Andalusian (Decree 23/2012) catalogue of protected species. The collection of the live specimens and shells mentioned in this article was authorized by the Dirección General de Política Forestal y Biodiversidad de la Consejería de Sostenibilidad, Medio Ambiente y Economía Azul de la Junta de Andalucía.
The authors have declared that no competing interests exist.
No ethical statement was reported.
Work supported by the European Union’s Horizon 2020 Research and Innovation Programme under the Grant Agreement Number 857251.
Conceptualization: JL, MJJ, GMR. Data curation: MJJ, PAJ, ART, JL, IGL. Formal analysis: IGL, MJJ, JL. Funding acquisition: GMR, MJJ. Methodology: IGL, MJJ, JL, ART, PAJ. Supervision: JL. Validation: MJJ. Writing - original draft: MJJ, GMR. Writing - review and editing: ART, IGL, JL, MJJ, GMR.
Michael J. Jowers https://orcid.org/0000-0001-8935-5913
José Liétor https://orcid.org/0009-0009-5877-6550
Antonio R. Tudela https://orcid.org/0000-0002-9402-6345
Pedro A. Jódar https://orcid.org/0009-0001-1691-745X
Inés Galán-Luque https://orcid.org/0000-0003-2356-9374
Gregorio Moreno-Rueda https://orcid.org/0000-0002-6726-7215
All data generated or analysed during this study are included in this published article (Supporting information).
Supporting information
Data type: docx
Explanation note: figure S1. Photographic series showing the range of variability for the shells of T. cilbanus (Cadiz): 1–11: Grazalema town ring road, Grazalema Natural Park; 12–20: Next to the Caldereto neighborhood, Ubrique, Grazalema Natural Park; 21–24: Llanos del Apeo, Los Alamos, Grazalema Natural Park; 25–39: `El Cintillo´ viewpoint, Benaocaz, Grazalema Natural Park; (Malaga); 40: Sierra de la Utrera, Manilva, Casares. table S1. Sampling locations for T. cilbanus. table S2. Samples used in the phylogenetic analyses. GenBank voucher abbreviations, species names, localities, coordinates and GenBank accessions. table S3. Morphometric parameters and ratios measured for T. cilbanus (N = 259). table S4. Morphometric comparisons between T. cilbanus and the two taxa of the marmoratus complex which inhabit the surrounding areas. K: Kruskal Wallis plus 2-tailed multiple comparison H test; A: one-way ANOVA plus post hoc Tukey test (HSD) for the comparisons between T. cilbanus and I. marmoratus marmoratus and I. marmoratus loxanus, respectively; ns: non-significant.