Research Article |
Corresponding author: Laurent Granjon ( laurent.granjon@ird.fr ) Academic editor: Raquel López-Antoñanzas
© 2016 Arame Ndiaye, Caroline Tatard, William Stanley, Laurent Granjon.
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:
Ndiaye A, Tatard C, Stanley W, Granjon L (2016) Taxonomic hypotheses regarding the genus Gerbillus (Rodentia, Muridae, Gerbillinae) based on molecular analyses of museum specimens. ZooKeys 566: 145-155. https://doi.org/10.3897/zookeys.566.7317
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Methodological improvements now allow routine analyses of highly degraded DNA samples as found in museum specimens. Using these methods could be useful in studying such groups as rodents of the genus Gerbillus for which i) the taxonomy is still highly debated, ii) collection of fresh specimens may prove difficult. Here we address precise taxonomic questions using a small portion of the cytochrome b gene obtained from 45 dry skin/skull museum samples (from 1913 to 1974) originating from two African and three Asian countries. The specimens were labelled G. gerbillus, G. andersoni, G. nanus, G. amoenus, G. perpallidus and G. pyramidum, and molecular results mostly confirmed these assignations. The close relationship between G. nanus (Asian origin) and G. amoenus (African origin) confirmed that they represent vicariant sibling species which differentiated in allopatry on either side of the Red Sea. In the closely related G. perpallidus and G. pyramidum, specimens considered as belonging to one G. pyramidum subspecies (G. p. floweri) appeared closer to G. perpallidus suggesting that they (G. p. floweri and G. perpallidus) may represent a unique species, distributed on both sides of the Nile River, for which the correct name should be G. floweri. Furthermore, the three other G. pyramidum subspecies grouped together with no apparent genetic structure suggesting that they may not yet represent genetically differentiated lineages. This study confirms the importance of using these methods on museum samples, which can open new perspectives in this particular group as well as in other groups of interest.
Cytochrome b, degraded DNA, synonymy, systematics, vicariance
DNA sequences have proven useful in taxonomic studies, and they now represent a primary source of information when it comes to the delimitation of species (
Gerbils of the genus Gerbillus represent a good example where such an approach can be expected to bring significant information. The systematics of this genus, as well as the one of the Gerbillinae subfamily to which it belongs, is still intensely debated, at various taxonomic levels (
Is the differentiation between Gerbillus amoenus (from Africa) and Gerbillus nanus (from Asia), recently evidenced by
What are the evolutionary relationships between various purported G. pyramidum subspecies and other Gerbillus species, such as G. perpallidus, G. andersoni and G. gerbillus?
Do the subspecies of Gerbillus pyramidum listed by
Forty-five tissue samples were obtained from dry fragments that were still present on the skulls and skins of Gerbillus specimens from Egypt and Asia, that are housed at the Field Museum of Natural History, Chicago, USA (Suppl. material
DNA was extracted in the Labex CeMEB degraded DNA platform (Montpellier, France) using the QiaAmp DNA micro kit (Qiagen). Due to the degradation of DNA in museum samples, we amplified a short fragment of cytb by designing two new primers named GERBCYTB-F2 (5’- GCA AAC GGA GCC TCA ATA TT - 3’) and GERBCYTB-R3 (5’-CAT TCT ACR ATT GTT GGG CCA - 3’). These primers are respectively located at positions 250 and 488 of the cytb gene, delimiting a 239 base pair (bp) fragment. The 25μl reaction solution was prepared by mixing 14.5μl of DNase-RNase free water (Qiagen), 2.5μl of buffer (1X concentration), 2μl MgCl2 (2mM), 2.5μl dNTP (250μM; Sigma), 0.5μl of each primer (0.5μM), 0.5μl of AmpliTaq Gold (2.5 units; Applied biosystems). 1μl and 2μl of DNA aliquots of the extracted samples were amplified separately, and used for further comparisons. The cytb amplification was done at the CBGP molecular biology platform (Montferrier-sur-Lez, France) using PCR programs on a Master Cycle rep Gradient (eppendorf), including an activation step of 95 °C for 10 min followed by 55 cycles comprising a first denaturation at 94 °C for 30 s, hybridization at 50 °C for 30 s and elongation at 72 °C for 45s. The last step was a final extension at 72 °C for 7min. Three negative controls were used to check for contamination during DNA extraction, preparation of the mix and DNA distribution. In the first control (extraction control), no tissue was added to the tube; the second control (PCR mix control) was a closed tube, with only the PCR mix; the last control (DNA distribution control) was a tube with only the PCR mix, which was open during the entire process of DNA distribution, in order to check for the presence of DNA in the air. We verified the size and quality of each amplified DNA sequence fragment by performing an electrophoretic migration on a 2% agarose gel. The PCR products obtained at both DNA concentrations were sent to Eurofins MWG (Germany) for sequencing, and the results were compared among individuals to ensure that we obtained the same amplified sequence fragment.
The sequences were then checked, aligned and edited with BioEdit v.7.1.3.0 (
No contamination was recorded during this series of experiments, as testified by examination of the content of the control tubes. DNA could not be amplified from only one of the samples. Additionally, a comparison of the results obtained using amplified PCR products from two DNA concentrations showed that the obtained sequences were identical in all but seven individuals, making us suspect the presence of nuclear copies of mitochondrial DNA (Numt). Unambiguous sequences (239 bp) of 37 individuals were obtained, to which we added the sequences of 40 “reference” individuals of different, well-characterized, Gerbillus species taken from GenBank and a representative of the outgroup Sekeetamys calurus, leading to a final cytb dataset comprising 77 individuals.
The phylogenetic tree (Fig.
Phylogenetic reconstruction based on 239bp sequences of the cytochrome b gene using Neighbor-Joining. Values on nodes correspond to bootstraps / posterior probabilities respectively while “-” refer to places where both methods of reconstruction did not agree. Colored clades include museum specimens for which original sequences were obtained for the purpose of this study.
K2P genetic distances between and within (in italic) taxa based on cyt b sequences.
G. pyramidum floweri + G. perpallidus | G. pyramidum | G. gerbillus | G. amoenus | G. nanus | G. andersoni | |
G. pyramidum floweri + G. perpallidus | 0.004 | |||||
G. pyramidum | 0.017 | 0.004 | ||||
G. gerbillus | 0.105 | 0.109 | 0.013 | |||
G. amoenus | 0.135 | 0.139 | 0.146 | 0.011 | ||
G. nanus | 0.112 | 0.116 | 0.138 | 0.037 | 0.025 | |
G. andersoni | 0.113 | 0.114 | 0.126 | 0.103 | 0.106 | 0.006 |
We removed 7 individuals from the analyses, for which minor differences between sequences obtained from the amplified DNA coming from the two different concentrations of extracted DNA were observed. This observation strongly suggests the presence of nuclear copies of the target sequence, as is sometimes recorded in gerbilline rodents (
Overall, the labelling of the museum samples was remarkably in line with current taxonomy, and all corresponding specimens appear coherently placed in the phylogenetic tree produced (but see below for further details). This validates a posteriori the “mini-barcode” method used. Indeed, the sequences obtained, although short (239bp) made it possible to obtain generally robust reconstructions of the phylogenetic relationships between the study specimens, thus confirming the findings of
Museum specimens of Gerbillus gerbillus and G. andersoni from Egypt clustered unambiguously with “fresh” specimens of the same species from other origins. As for the distinction between Asian G. nanus and African G. amoenus, even if supported here by non-optimal BP values, it confirms the findings of
The series of museum specimens of large-sized and hairy-footed gerbils referred to as G. perpallidus and G. pyramidum ssp. were distributed into two moderately well supported genetic clades: the first one includes all G. pyramidum floweri and G. perpallidus samples, together with a reference specimen of G. perpallidus (namely Gperp1, underlined in Fig.
In addition to G. p. floweri,
Reevaluated Gerbillus floweri distribution area based on the results presented here (see text; horizontal lines: G. perpallidus; vertical lines: G. floweri distributions according to the IUCN Red List of Threatened Species. Version 2015.2. www.iucnredlist.org). Black circles and black star: specimens of G. perpallidus and G. p. floweri, respectively, used in the present study.
In conclusion, we show here that molecular analysis of historic museum samples of the genus Gerbillus, up to more than 100 years after their collection, may give useful information, and address testable hypotheses, about the systematics of the genus. This could aid in the completion of the taxonomic understanding of this complex and speciose genus, which is well represented in museum collections worldwide. The new primers specifically designed here, may prove useful for this purpose.
This study was funded by the “Service de Coopération et d’Action Culturelle” of the French Embassy in Senegal. We thank Christelle Tougard and the LabEX CeMEB for allowing us to use their degraded DNA platform, as well as Marie Pagès and Jean-François Cosson for useful advice in the beginning of this study. We also thank Joy Peplinski for help with sampling dried tissue from skulls at the Field Museum and Sylvain Piry for help in obtaining the map used in this study. Corrections and comments by two referees, especially by Bader Alhajeri, enabled us to greatly improve the final version of this paper.
List of study specimens with identification numbers, geographic origin, collector name and collection date
Data type: Information of study specimens and cytb sequences