Research Article |
Corresponding author: Thembeka Clara Nxele ( tnxele@nmsa.org.za ) Academic editor: John Midgley
© 2020 Thembeka Clara Nxele, Jadwiga Danuta Plisko, Tarombera Mwabvu, Oliver Tendayi Zishiri.
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:
Nxele TC, Plisko JD, Mwabvu T, Zishiri OT (2020) Molecular phylogeny of Kazimierzus Plisko, 2006 (Clitellata, Kazimierzidae) from the Western and Northern Cape Province inferred from mitochondrial DNA sequences. African Invertebrates 61(2): 83-92. https://doi.org/10.3897/afrinvertebr.61.53380
|
Species identification of earthworms using morphology can be challenging and inconclusive as homoplasy in many characters is high. The use of molecular DNA technology, such as the use of conserved regions in mtDNA and nuclear DNA has unravelled the phylogenetic background of several earthworm species. The current study utilised the cytochrome c oxidase subunit I (COI) mitochondrial marker to reconstruct the phylogeny of Kazimierzus Plisko, 2006 species from the Western and Northern Cape provinces of South Africa. Phylogenetic reconstructions were implemented using Bayesian Inference, as well as Maximum Likelihood. Both tree building methods adhered to the monophyly of the majority of the taxa. Results showed that species fell into two clades and validated eleven currently known Kazimierzus species (K. circulatus (Plisko, 1998), K. franciscus (Pickford, 1975), K. guntheri (Pickford, 1975), K. hamerae (Plisko, 1998), K. kleinoodi Nxele & Plisko, 2017, K. nietvoorbiji Nxele & Plisko, 2017, K. nieuwoudtvillensis Nxele & Plisko, 2017, K. occidualis (Plisko, 1998), K. pearsonianus (Pickford, 1975), K. phumlani Nxele & Plisko, 2017, K. sophieae (Plisko, 2002)). Cryptic diversity is evident in K. occidualis with genetic divergence greater than 12% amongst populations. Kazimierzus franciscus and K. ljungstroemi (Pickford, 1975) have a low genetic variability suggesting close relatedness or probably conspecificity. A group of specimens from Clanwilliam are morphologically identical to K. sophieae, but are genetically distinct and may belong to undescribed species. This study demonstrates the importance of integrative taxonomy in earthworms in order to present reliable taxonomic and biogeographic data.
DNA, COI, mtDNA, taxonomy, earthworms, cryptic diversity
Earthworms constitute a large component of soil invertebrates and are regarded as soil engineers (
The use of DNA sequences has increased in the recent past, because it is less subjective than morphological characters, allows for the analysis of several characters (
According to
In order to obtain earthworms of Kazimierzus, qualitative sampling was carried out in 2011 and 2015 during the rainy season (July–September) in the Western and Northern Cape, South Africa. Besides the focus on type localities, potential sites other than the type localities were also sampled. Earthworms were collected by digging and hand sorting. Collected specimens were anaesthetised in 20% ethanol solution, fixed in 4% formalin solution and preserved in 75% ethanol for taxonomic purposes. A subsample was preserved in absolute ethanol for molecular analysis. All specimens were examined using a Wild Heerbrugg stereomicroscope and were identified according to the descriptions in
Tissue from the posterior section of the earthworm was used. All DNA extractions were performed using the ZR Genomic DNATM Tissue MicroPrep kit, following the manufacturer’s standard protocol. The concentration of DNA in each sample was estimated using the NanoDrop 2000 (Thermo Scientific). A fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using LCO1490 (5’ GGTCAACAAATCATAAAGATATTGG 3’) and HCO2198 (5’ TAAACTTCAGGGTGACCAAAAAATCA 3’) primers (
Sequencing of the 675 bp fragment of the COI mtDNA was conducted at Inqaba Biotechnical Industries (Pty) Ltd.
Identity of sequences was verified by the Basic Local Alignment Search Tool (BLAST) in the National Centre for Biotechnology (NCBI). Sequences of Amynthas minimus (Horst, 1893) and Amynthas corticis (Kinberg, 1867) were included as outgroup taxa. The sequences for outgroup taxa were obtained from GenBank (Accession nos: AB542509.1, AB542469.1) and current sequences are added as supplementary data. All specimens are at the KwaZulu-Natal Museum. The sequences were aligned using CLUSTAL X 2.1 (
Clade support was evaluated by 1000 bootstrap replicates for the ML analysis and posterior probability values for the Bayesian analysis. For Bayesian analyses, all MRBAYES analyses were run for 5000000 generations with a sampling frequency of 1000. The deviation of split frequencies was less than 0.01 at the conclusion of all analyses which confirmed that the MCMC chains had converged. The programme TRACER v1.5 (
Species | Specimen catalog # | Genbank # |
---|---|---|
K. circulatus | NMSA/OLIG.04942/3a,c | MN982467–68 |
K. franciscus | NMSA/OLIG.09659a,b,c | MN982469–71 |
K. guntheri | NMSA/OLIG.04986a,b,e | MN982474–75; MN982478 |
K. hamerae | NMSA/OLIG.04956a,b,c | MN982461–63 |
K. kleinoodi | NMSA/OLIG.04987 | MN982480 |
K. ljungstroemi | NMSA/OLIG.06960b,d,e | MN982472–73; MN982482 |
K. nietvoorbiji | NMSA/OLIG.04988a,c | MN982456–57 |
K. nieuwoudtvillensis | NMSA/OLIG.04990b,c,d | MN982458–60 |
K. occidualis | NMSA/OLIG.04958a,c,d; NMSA/OLIG.04962 | MN982464–66; MN982485 |
K. pearsonianus | NMSA/OLIG.04984 | MN982479 |
K. phumlani | NMSA/OLIG. 04951/2b,c,e | MN982453–55 |
K. sophieae | NMSA/OLIG.04950a,c; NMSA/OLIG.04963a,b | MN982483–84; MN982486–87 |
K. sp. | NMSA/OLIG.04989a,d,e | MN982476–77; MN982481 |
The sequences were 675 bp. Variable sites were 431 bp and conserved sites 233 bp showing great differentiation amongst taxa.
Most species pair comparisons showed a genetic distance above 13%, except for K. ljungstroemi and K. franciscus which have one percent genetic distance between them (Table
Pairwise p genetic distances (%) between the investigated Kazimierzus species/lineages.
Species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1. K. nietvoorbiji | ||||||||||||||||
2. K. phumlani | 21.1 | |||||||||||||||
3. K. hamerae | 22.3 | 20.1 | ||||||||||||||
4. K. sophieae | 25.4 | 25.5 | 16.9 | |||||||||||||
5. K. sp | 20.3 | 18.3 | 17.5 | 23.0 | ||||||||||||
6. K. sophieae | 23.1 | 20.1 | 21.5 | 25.5 | 19.0 | |||||||||||
7. K. nieuwoudtvillensis | 18.8 | 20.7 | 19.9 | 25.5 | 17.4 | 22.6 | ||||||||||
8. K. circulatus | 19.0 | 17.5 | 18.5 | 23.8 | 17.9 | 19.5 | 16.6 | |||||||||
9. K. occidualis | 18.7 | 15.5 | 20.9 | 25.2 | 18.2 | 19.6 | 17.7 | 15.8 | ||||||||
10. K. occidualis | 19.0 | 16.9 | 22.2 | 26.3 | 19.5 | 20.9 | 19.0 | 17.2 | 3.0 | |||||||
11. K. occidualis | 21.5 | 21.9 | 23.1 | 28.5 | 21.5 | 24.2 | 20.4 | 19.8 | 15.5 | 16.1 | ||||||
12. K. franciscus | 19.9 | 19.9 | 22.0 | 26.8 | 19.3 | 21.7 | 19.1 | 19.9 | 18.7 | 19.8 | 23.6 | |||||
13. K. ljungstroemi | 19.9 | 20.4 | 22.2 | 27.3 | 18.8 | 21.9 | 19.3 | 19.8 | 18.7 | 19.6 | 23.4 | 1.1 | ||||
14. K. guntheri | 13.6 | 21.1 | 21.1 | 26.6 | 20.7 | 23.1 | 19.3 | 20.4 | 19.5 | 18.7 | 19.3 | 21.4 | 21.5 | |||
15. K. kleinoodi | 13.2 | 21.4 | 23.9 | 28.5 | 21.9 | 23.8 | 20.9 | 22.5 | 19.5 | 20.7 | 22.6 | 22.6 | 22.8 | 15.0 | ||
16. K. pearsonianus | 14.0 | 22.6 | 23.8 | 27.3 | 23.4 | 25.4 | 22.2 | 22.0 | 21.7 | 22.3 | 23.6 | 22.2 | 22.0 | 15.0 | 15.8 |
The MI and BI trees were congruent, therefore, support values were annotated on to the branches of the most likely trees generated for each of the datasets analysed (ML run with no bootstrap, rooted using outgroup species (Amynthas minimus and Amynthas corticis).
Bootstrap values and posterior probabilities below 50% and 0.50, respectively, were not shown on the trees. Two clades, A and B, are distinct; clade A separates further to clades C and D whilst clade B separates to clades E and F (Fig.
Morphological examination revealed eleven currently known species (circulatus, franciscus, guntheri, hamerae, kleinoodi, nietvoorbiji, nieuwoudtvillensis, occidualis, pearsonianus, phumlani and sophieae). However, the current phylogenetic analysis resulted in additional lineages that suggest cryptic diversity (Fig.
Kazimierzus hamerae is similar in appearance to K. sophieae, but molecular data confirmed that they are separate species. The phylogenetic tree (Fig.
Cryptic diversity was observed in K. occidualis. Finding cryptic diversity is common in earthworms,
The specimens of Kazimierzus franciscus and Kazimierzus ljungstroemi were collected in two neighbouring forests, Duiwelbos and Koloniesbos in Marloth Nature Reserve, Swellendam. Duiwelbos is the type locality of K. franciscus, whilst the type locality for K. ljungstroemi is Great Winterhoek, Tulbagh District. Therefore, it is possible that all specimens belong to K. franciscus, but it would be of benefit to collect specimens from Winterhoek and compare them with the ones collected from Marloth Nature Reserve.
Although analysis of other conserved genomic regions in both mtDNA and nuclear DNA in the future would benefit the study of Kazimierzus species, the phylogenetic analysis of COI recovered several well-supported phylogenetic relationships, some of which are congruent with existing classification.
The KwaZulu-Natal Museum and the University of KwaZulu-Natal are acknowledged for their continued support given for the advancement of the study of earthworms in Southern Africa. The KZN Museum library staff is acknowledged for all their help. The research on the South African megadrile is financially supported in part by the National Research Foundation, South Africa, (Grant numbers:113989, 104846, 114024). Finally, we acknowledge comments of the referees, Samuel James, Csaba Csuzdi, Gabriela Cervantes and the fourth anonymous person.