Corresponding author: Emmanuel Matamba ( ematamba@sun.ac.za ) Academic editor: Clara Stefen
© 2021 Emmanuel Matamba, Leigh R. Richards, Michael I. Cherry, Ramugondo V. Rambau.
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:
Matamba E, Richards LR, Cherry MI, Rambau RV (2021) DNA barcoding of the mesic adapted striped mouse, Rhabdomys dilectus in the Eastern Cape and KwaZulu-Natal provinces of South Africa. Vertebrate Zoology 71: 503-515. https://doi.org/10.3897/vz.71.e68897
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Abstract
South African small mammals are under-represented in DNA barcoding efforts, particularly from the eastern forested regions of the country. This study reports DNA barcoding of Rhabdomys taxa from previously unsampled parts of the Eastern Cape and KwaZulu-Natal provinces of South Africa. The complete mitochondrial DNA cytochrome oxidase I (COI) gene was sequenced for 101 Rhabdomys sp. individuals from 16 localities from all three main forest groups (coastal, mistbelt, and scarp forests). Molecular data were supplemented with external morphological measurements, including those deemed potential taxonomically diagnostic characters. Findings indicate the area to be inhabited solely by Rhabdomys dilectus chakae. Haplotypes distributed across the three forest groups were separated by shallow sequence divergences ranging from 0.001–0.015 (Kimura 2-parameter model) and displayed very little population genetic structure (FST= 0.071787). Morphological data revealed some regional metric differences in external morphology, but all the head-and-body to tail (HB: tail) ratios match that of R. d. chakae, and consequently, molecular and morphological data are congruent. These data confirm a range extension of R. d. chakae, supporting the utility of COI barcodes in the identification of small mammalian species.
COI barcodes, forest fragmentation, morphology, Rhabdomys dilectus
The four-striped grass mouse genus Rhabdomys (Thomas, 1916), is distributed throughout southern Africa, occurring in a variety of habitats with ample grass cover (
In the Eastern Cape, there are broad forest group types that extend to the neighboring KwaZulu-Natal and Western Cape provinces: Southern mistbelt, Scarp, and Southern coastal forest groups (
Barcoding provides a rapid way to identify organisms without the need for morphological expertise (
Here we present cytochrome oxidase subunit I (COI) barcodes from the Eastern Cape and parts of KwaZulu-Natal forests, to address the distribution gaps highlighted in recent publications (du Toit et al. 2012;
Animals were collected from three forest types in the Eastern Cape and southern parts of the KwaZulu-Natal Province of South Africa (Figure
Sample localities of Rhabdomys sp. in the Eastern Cape and southern KwaZulu-Natal provinces. These localities encompass the mistbelt forest (A-H), coastal group (letters P and O), and Scarp group (I-N). Letters represent the study locations as shown in Table
Summary of sample details including the study localities with corresponding letters (#) matching the map in Figure
# | Locality, geographic co-ordinates | Forest type | Sample sizes (Haplotype number) | Voucher number only for collected specimens (all released animals not shown) |
A | Fort Fordyce, -31.68237, 26.482653 | Amatole mistbelt | 8 (Hap 8, 9, 17, 18 ,19, 20, 21,22) | DM15041, DM15240, EM10070118, EM29070118 |
B | Hogsback, -32.613906, 26.972192 | Amatole mistbelt | 3 (Hap 28, 29) | DM15266, DM15267 |
C | Isidenge, -33.69975, 26.359898 | Amatole mistbelt | 11 (Hap 4, 8, 21, 26 28,30, 31,33,34) | DM4253, DM4249, DM15016, DM15024, DM15024, DM15252, DM15253, DM15254, DM15255 |
D | Kologha, -32.535330, 27.36258 | Amatole mistbelt | 6 (Hap 11, 25, 26, 27,32) | DM15273, DM15274, DM15275 |
E | Baziya, -31.547255, 28.437503 | Transkei mistbelt | 12 (Hap 7, 8, 9, 10, 11, 45, 46) | LRR180212BSF_RP1, LRR180212BSF_RP2, LRR180212BSF_RP3, LRR180212BSF_RP4, LRR180212BSF_RP5, LRR180213BSF_RP7 |
F | Gomo, -31.013194, 29.344585 | Transkei mistbelt | 5 (Hap 9, 11, 23, 24) | EM73031217, EM84041217, EM9021217 |
G | Ngeli, -30.542827, 29.680979 | Eastern mistbelt | 6 (Hap 11, 28, 42, 43, 44) | LRR180314NSF_RD1, LRR180315NSF_RD1, LRR180315NSF_RD2, LRR180317NSF_RD1, LRR181001NSF_RD1, LRR181001NSF_RD2 |
H | Nxumeni, -29.927042, 29.844179 | Eastern mistbelt | 14 (Hap 10, 11, 12, 13, 44, 47, 48) | LRR180321BF_RD1, LRR180321BF_RD2, LRR180321BF_RD3, LRR180321BF_RD6, LRR180321BF_RD8, LRR180324BF_RD2, LRR180324BF_RD5, LRR180325BF_RD1 |
I | Umtamvuna, -31.007467, 30.094205 | Pondoland scarp | 4 (Hap 49, 54, 55, 56) | DM1174, DM1177, DM1178, DM1182 |
J | Mkambati, -31.247618, 29.990551 | Pondoland scarp | 4 (Hap 10, 28, 39) | DM151163, DM15164, DM15165, DM15166 |
K | Mbotyi, -31.418887, 29.723978 | Pondoland scarp | 5 (Hap 11, 28, 37, 38) | DM15176, DM15177 |
L | Silaka, -31.657010, 29.502839 | Pondoland carp | 5 (Hap 28, 50, 51, 52, 53) | EM83151217, EM89111217, EM85151217, EM87111217, EM241151217, |
M | Dwesa, -32.445594, 28.60766 | Transkei scarp | 3 (Hap 1, 14, 15) | DM15190, DM15191, DM15192 |
N | Manubi, -33.990792, 25.360209 | Transkei scarp | 6 (Hap 9, 10, 16, 35, 36) | DM15214, DM15215, DM15216 |
O | Morgans Bay, -32.701243, 28.353646 | Eastern Cape dune | 3 (Hap 40, 41) | EM201, EM207 |
P | Alexandria, -33.699756, 26.359898 | Albany | 6 (Hap 1, 2, 3, 4, 5, 6) | No vouchers. |
16 study localities | 7 Forest types | 101(56 haplotypes) | Haplotype diversity, Hd: 0.972 Nucleotide diversity (per site), Pi: 0.01126 | |
LRR=Not catalogued, EM=Stellenbosch university vouchers, DM=Durban Natural Science Museum catalogued specimens (includes historic specimen material) |
DNA was extracted using the NucleoSpin (R) Tissue Kit technique (Macherey-Nagel) following the manufacturer’s manual. The COI gene fragment was isolated using L1490 (5`-GGT CAA CAA ATC ATA AAG ATA TTG G-3`) and H2198 (5`-TAA CTT CAG GGT GAC CAA AAA ATC A-3`) primers (
Morphological analyses of 78 Rhabdomys dilectus specimens across different forest types. Standard measurements including total length (TL), tail length (T), hindfoot length (with the claw, HF) and ear length (E), to the nearest 1 mm were taken in the field; the head and body length (HB) was calculated as the difference of tail and the total length of the animal. Sample sizes are presented in parentheses. Descriptive statistics (mean, standard deviation) and Kruskal-Wallis tests were conducted in SPSS 27.0 (IBM Corp., 2020).
Mistbelt | Scarp | Coastal | The combined average for all groups | P-value | |
Total length (mm) | 196.0±5.09 (42) | 188.6±7.47 (25) | 186.9±7.75 (11) | 192.4±5.09 (78) | < 0.001 |
Head-and-body (mm) | 103.1±3.40 (42) | 99.0±4.83 (25) | 97.6±4.52 (11) | 101.1±3.40 (78) | < 0.001 |
Tail length (mm) | 92.9±2.33 (42) | 89.6±3.07 (25) | 89.3±3.47 (11) | 91.3±2.33 (78) | < 0.001 |
Ear (mm) | 12.0±0.14 (42) | 12.1±0.44 (25) | 12.5±0.52 (11) | 12.1±0.38 (78) | 0.010 |
Hindfoot (mm) | 20.4±0.92 (42) | 18.9±1.20 (25) | 18.5±1.63 (11) | 19.5±1.41 (78) | < 0.001 |
HB:Tail ratio | 1.11±0.03 (42) | 1.10±0.03 (25) | 1.09±0.02 (11) | 1.11±0.03 (78) | 0.123 |
Sequences generated were edited and assembled manually in BioEdit v7.0.9 (
A haplotype dataset was created using DnaSP v6 (Rozas et al. 2017) from aligned sequences and it was subsequently used to generate a network tree using TCS networks (
All sequences named R. dilectus and R. pumilio retrieved from GenBank were aligned with sequences generated from this study. Phylogenetic trees were reconstructed using Neighbour-joining (NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML) based on the best substitution model which was identified using MODELTEST using MEGA version X (
The Barcoding Gap is defined as the difference between the greatest intraspecific variation and the smallest interspecific variation in a group of organisms and is used as the basis for species delimitation (
The COI gene fragment (658 bp) was successfully amplified for all specimens (N=101). The nucleotide content of the gene fragment comprised 30.3% of thymine, 25.6% adenine, 27.2% cytosine and 16.9% guanine. Of the 658 bp, only 13.98% was variable (92/658 bp) while 8.5% of sites were parsimony informative and retrieved 56 haplotypes (Table
Modeltest selected the HKY+G+I (Hasegawa-Kashino-Yano with Gamma and invariant) for the COI dataset. However, because of barcoding analysis preference, phylogenetic reconstructions were undertaken using the Kimura 2-parameter model (
A Neighbour-joining (NJ) phylogram illustrating phylogenetic relationships among haplotypes generated from a total of 280 Rhabdomys COI sequences. The dataset includes 101 sequences from this study with other Rhabdomys sequences downloaded from GenBank. The included outgroups were two sequences of Praomys species and Myomyscus species, and a single sequence of Rattus rattus, Apodemus flavicollis and Otomys irroratus. The tree is condensed, while the insert shows R. d. chakae which includes all samples from this study. The Neighbour-joining Maximum Likelihood (ML), and Maximum Parsimony (MP) analyses were drawn using the Kimura 2-Parameter model (
The mean p-distance among and within forest types for R. dilectus varied from 0.001±0.001 to 0.017±0.003 (Table
The mean p-distances among and within (in bold) forest types in the Eastern Cape and KwaZulu-Natal provinces of South Africa for COI (658bp) for Rhabdomys dilectus. The number of base substitutions per site, derived from the mean of all sequence pairs between groups, is shown with the standard error estimate(s) next to each. Analyses were conducted using the Kimura 2-parameter model (
Albany | Transkei mistbelt | Eastern mistbelt | Transkei scarp | Amatole mistbelt | Pondoland scarp | Eastern Cape dune | |
Albany | 0.011±0.003 | ||||||
Transkei Mistbelt | 0.008±0.002 | 0.004±0.001 | |||||
Eastern Mistbelt | 0.012±0.002 | 0.008±0.001 | 0.011±0.002 | ||||
Transkei Scarp | 0.011±0.002 | 0.008±0.002 | 0.012±0.002 | 0.012±0.002 | |||
Amatole Mistbelt | 0.011±0.002 | 0.008±0.001 | 0.012±0.002 | 0.012±0.002 | 0.011±0.002 | ||
Pondoland Scarp | 0.014±0.002 | 0.012±0.002 | 0.015±0.002 | 0.015±0.002 | 0.015±0.002 | 0.017±0.003 | |
Eastern Cape Dune | 0.009±0.003 | 0.008±0.003 | 0.012±0.003 | 0.010±0.003 | 0.011±0.003 | 0.013±0.003 | 0.001±0.001 |
Tajima’s D: –2.60563 | P<0.001 | Va = 92.81 | Vb = 7.19 | Fixation Index | FST: 0.071787 | P<0.001 |
The median-joining networks for COI (Figure
Median-joining haplotype network generated from COI of 101 specimens of R. dilectus from forests in the Eastern Cape and southern KwaZulu-Natal provinces of South Africa. The haplotype colors represent the forest groups from which the individuals were collected. Forest groups are indicated in the insert (also in Figure
The groupings as retrieved above by the phylogenetic tree were used for calculating divergences between and within Rhabdomys taxa (Table
Barcode gap analysis of Rhabdomys taxa generated by Automatic Barcode Discovery Gap Discovery (
The number of base substitutions per site in Rhabdomys taxa. These were derived from the mean of all sequence pairs between and within species, with standard error estimate(s) shown next to each. Analyses were conducted using the Kimura 2-parameter model (
R. bechuane | R. d. dilectus | R. d. chakae | R. intermedius | R. pumilio coastal A | R. pumilio Coastal B | |
R. bechuane | 0.009±0.002 | |||||
R. d. dilectus | 0.115±0.014 | 0.023±0.004 | ||||
R. d. chakae | 0.123±0.015 | 0.053±0.001 | 0.008±0.002 | |||
R. intermedius | 0.079±0.011 | 0.105±0.002 | 0.113±0.002 | 0.004±0.001 | ||
R. pumilio coastal A | 0.131±0.016 | 0.086±0.001 | 0.099±0.002 | 0.115±0.002 | 0.007±0.002 | |
R. pumilio Coastal B | 0.118±0.015 | 0.094±0.002 | 0.098±0.002 | 0.114±0.002 | 0.050±0.002 | 0.005±0.002 |
The average total length of specimens was 186.9±7.75 mm for coastal forest, 188.6±7.47 mm in the scarp forest and 196.0±5.09 mm for mistbelt forests; mean head-and-body length was 97.6±4.52 mm for coastal forest, 99.0±4.83 in the scarp forest and 103.1±3.40 mm for mistbelt forests; and mean tail length was 89.3±3.47 mm for coastal forest, 89.6±3.07 in the scarp forest and 92.9±2.33 mm in the mistbelt forests (Table
Based on DNA barcoding, all samples in this study were attributed to R. d. chakae. The two sub-species of R. dilectus (R. d. chakae and R. d. dilectus) have long since been recognized: R. d. chakae is endemic to the country, while the geographic distribution of R. d. dilectus extends beyond South Africa (
Our combined (sequence divergence, ABGD and phylogenetic analyses) findings are largely congruent, and suggest six groups may be assigned to two sub-species of R. dilectus; two lineages of R. pumilio; and two species R. bechuanae and R. intermedius. These findings are consistent with the previous findings relating to this genus (du Toit et al. 2012;
The reticulate tree (Figure
Although a wide range of rodents are good candidate taxa for phylogeographical analysis (
The utility of DNA barcoding in identifying Rhabdomys taxa has been supported. This study focused on supplying information on the distribution of the taxa in the Eastern Cape and southern parts of KwaZulu-Natal provinces and confirmed that all samples included in this study were of R. d. chakae. These findings are consistent with previous revisions of this genus (du Toit et al. 2012,
The project was funded by the Foundational Biodiversity Information Programme (FBIP 98871) of the Department of Science and Technology of South Africa through the National Research Foundation. The student extension funding was provided by the National Research Foundation and the Stellenbosch University. We express gratitude to the Durban Natural Science Museum and eThekwini Municipality for co-funding field trips and provisioning equipment. We express gratitude to private land-owners, the former Department of Environment, the former Department of Agriculture, Forestry and Fisheries, Eastern Cape Province: Department of Economic Development Environmental Affairs and Tourism, The Eastern Cape Parks and Tourism Agency, Ezemvelo KZN Wildlife, and South African National Parks and private landowners for permits and facilitating sample collection. We also acknowledge the staff of the Durban Natural Science Museum and fellow research students for assistance during sample collection; as well as the collection of samples by Vusi Martins and Dale Wright. The capturing and handling of animals was approved by the ethics committee of the University of Stellenbosch (protocol number 1285) and followed the guidelines from the Animal Care and Use Committee of the American Society of Mammalogists (Sikes and the Animal Care and Use Committee of the American Society of Mammalogists 2016).