Freshwater invasive alien crayfish species in South Africa: testing eDNA assessment and DNA barcoding
- Mdidimba, Nonkazimulo Dorothea
- Authors: Mdidimba, Nonkazimulo Dorothea
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424413 , vital:72151
- Description: The expansion of the wild populations of the two invasive alien crayfish species, i.e., Cherax quadricarinatus and Procambarus clarkii in South Africa is of high concern. Given this, innovative and improved monitoring methods are required to be explored in order to effectively detect and manage their spread. Environmental DNA (eDNA) has shown promising and reliable results to detect and monitor alien crayfish in freshwater systems even at low densities. This research study aimed to field-test the application of eDNA for detecting and monitoring invasive alien freshwater crayfish species (IAFCs) in South African freshwater systems (Chapter 2). Further, this study also aimed to confirm the true taxonomic identity of the IAFCs in South African freshwater systems by using DNA barcoding to avoid potential misidentifications with morphological identification alone (Chapter 3). Also, ectocommensal temnocephalans and leeches attached to the crayfishes were also identified with DNA barcoding. The mitochondrial COI gene fragment of the crayfishes and the ectocommensal temnocephalans and leeches was amplified, sequenced, and used for species identification. For eDNA monitoring of C. cainii, C. quadricarinatus and P. clarkii, water samples were collected from Eastern Cape, Free State and KwaZulu Natal sites that are known to house these species. The current study did not detect the eDNA of the wild populations of both C. quadricarinatus and P. clarkii. This failure was encountered despite employing several methods, such as, end-point polymerase chain reaction (PCR), quantitative PCR (qPCR) and next-generation sequencing (NGS). The eDNA assessment to detect IAFCs in the wild populations is discussed in detail, and corrective measures suggested (Chapter 2). However, only C. cainii was able to be detected by eDNA. Cherax cainii is housed/farmed in an aquaculture facility (Vaughn Bursey’s Stock Farm in Eastern Cape) with large number of individuals per pond. For this, NGS was able to detect C. cainii but the other two methods, end-point PCR and qPCR were not able to detect the species. Based on the results, refinement of eDNA monitoring methods for the South African freshwater systems is recommended. Evaluation of different DNA collection methods, preservation, DNA extraction kits and primer design to obtain effective eDNA detection is discussed as possible sources of error. Interestingly, the newly established population of P. clarkii in Mimosa Dam in Free State was infested with alien leeches, that were confirmed by DNA barcoding to be Helobdella europaea and Helobdella octatestica. Consequently, this study reports for the first time in South Africa the presence of this alien freshwater leeches. The presence of alien leeches and P. clarkii suggests a double invasion in Mimosa Dam, P. clarkii seems to continue expanding its range in the country. However, the long-established population of P. clarkii in Driehoek Farm in Mpumalanga, which has been in the country for over 30 years did not have any leech or temnocephalan infestation. This could suggest several interesting ecological phenomena, including environmental filtering over time of the co-invaders, differential source populations, and post-introduction adaptation. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Mdidimba, Nonkazimulo Dorothea
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424413 , vital:72151
- Description: The expansion of the wild populations of the two invasive alien crayfish species, i.e., Cherax quadricarinatus and Procambarus clarkii in South Africa is of high concern. Given this, innovative and improved monitoring methods are required to be explored in order to effectively detect and manage their spread. Environmental DNA (eDNA) has shown promising and reliable results to detect and monitor alien crayfish in freshwater systems even at low densities. This research study aimed to field-test the application of eDNA for detecting and monitoring invasive alien freshwater crayfish species (IAFCs) in South African freshwater systems (Chapter 2). Further, this study also aimed to confirm the true taxonomic identity of the IAFCs in South African freshwater systems by using DNA barcoding to avoid potential misidentifications with morphological identification alone (Chapter 3). Also, ectocommensal temnocephalans and leeches attached to the crayfishes were also identified with DNA barcoding. The mitochondrial COI gene fragment of the crayfishes and the ectocommensal temnocephalans and leeches was amplified, sequenced, and used for species identification. For eDNA monitoring of C. cainii, C. quadricarinatus and P. clarkii, water samples were collected from Eastern Cape, Free State and KwaZulu Natal sites that are known to house these species. The current study did not detect the eDNA of the wild populations of both C. quadricarinatus and P. clarkii. This failure was encountered despite employing several methods, such as, end-point polymerase chain reaction (PCR), quantitative PCR (qPCR) and next-generation sequencing (NGS). The eDNA assessment to detect IAFCs in the wild populations is discussed in detail, and corrective measures suggested (Chapter 2). However, only C. cainii was able to be detected by eDNA. Cherax cainii is housed/farmed in an aquaculture facility (Vaughn Bursey’s Stock Farm in Eastern Cape) with large number of individuals per pond. For this, NGS was able to detect C. cainii but the other two methods, end-point PCR and qPCR were not able to detect the species. Based on the results, refinement of eDNA monitoring methods for the South African freshwater systems is recommended. Evaluation of different DNA collection methods, preservation, DNA extraction kits and primer design to obtain effective eDNA detection is discussed as possible sources of error. Interestingly, the newly established population of P. clarkii in Mimosa Dam in Free State was infested with alien leeches, that were confirmed by DNA barcoding to be Helobdella europaea and Helobdella octatestica. Consequently, this study reports for the first time in South Africa the presence of this alien freshwater leeches. The presence of alien leeches and P. clarkii suggests a double invasion in Mimosa Dam, P. clarkii seems to continue expanding its range in the country. However, the long-established population of P. clarkii in Driehoek Farm in Mpumalanga, which has been in the country for over 30 years did not have any leech or temnocephalan infestation. This could suggest several interesting ecological phenomena, including environmental filtering over time of the co-invaders, differential source populations, and post-introduction adaptation. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
The enigmatic subterranean amphipod genus (sternophysinx: sternophysingidae): conservation and systematics
- Authors: Mlungu, Zizile
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424424 , vital:72152
- Description: Sternophysingidae Holsinger, 1992 is a monotypic subterranean amphipod family endemic to Namibia and South Africa. It currently has eight described species under the genus Sternophysinx. The species in this family are considered narrow endemics, having only been identified using morphological characters. However, the apparent wide distribution of certain species, reported in several subterranean waters in different provinces, suggests that our current taxonomic understanding of these species needs to be completed, thus needing revision. Further, detailed knowledge of the distribution and phylogenetic relationship between species of this taxon still needs to be improved. This study aimed to use integrative taxonomy to analyse both morphology and molecular biology to study hidden diversity and to further our understanding of the distribution in this taxon. The available morphological keys were used to identify species, and 12S rRNA was also used to investigate the relationships of Sternophysinx species and delimit species boundaries. The study primarily aimed to use mitochondrial cytochrome c oxidase subunit I (COI). However, due to technical issues such as getting the correct primers, this study's aims were altered, and 12S was used instead of CO1. However, the process of getting CO1 sequences is still ongoing, and the results will be made available elsewhere. The current study aimed to visit all the type localities of the seven species of Sternophysinx reported in South Africa, including all known localities in which these amphipods have been recorded in the past. Nevertheless, this study did not get samples from Nelspruit, De Hoop Nature Reserve, and Nash Nature Reserve, which cover type localities of S. travaleensis, Matlapitse and Chaos cave that harbours S. calceola. Instead, the current study added five new localities Graaff-Reinet, Beaufort West, Hogsback, Makhambathi Nature Reserve and Ntsikeni Nature Reserve, for the distribution, including two new provinces (i.e., Eastern Cape and KwaZulu-Natal) that had records of this genus in the country. Three delimitation methods, Assemble Species by Automatic Partitioning (ASAP), Bayesian implementation of the Poison Tree Processor (bPTP) and Automatic Barcode Gap Discovery (ABGD), were used to identify cryptic species within Sternophysinx. Phylogenetic analyses and three delimitation methods (ASAP, bPTP and ABGD) revealed considerable hidden cryptic diversity within this Sternophysinx. Preliminary morphological examination revealed at least two species as undescribed from Bakwena and Sterkfontein caves, and several that were morphologically identified using the current taxonomic keys did not match with the molecular analysis. For example, S. basilobata from three different systems, Boesmansgat cave (Northern Cape), Beaufort West (Western Cape) and Potchefstroom (North West), was in genetically different putative species. The same is true for S. robertsi and S. alca in Ficus and Peppercorn cave in Limpopo. Overall, the results of the current study demonstrate the need for a comprehensive integrative taxonomic revision of Sternophysinx. These findings suggest that S. basilobata is a complex of distinct lineages and requires further study. On the other hand, species delimitation methods suggested that there are 12- 14 candidate species (Figure 2.5). Lastly, this study investigated the threat posed by the invasive koi fish introduced to an important cave, Boesmansgat cave in the Northern Cape, which is a type locality of two endemic species (S. megacheles and S. basilobata). Stable isotope (δ13C and δ15N) and gut content analyses of the fish were investigated to establish whether this invasive fish feed on these critical creatures, thus endangering their long-term survival. Stable isotope signatures and the stomach gut content proved to be helpful in determining the diet of the koi fish, revealing it to be an omnivorous feeder with high invertebrate preference, including amphipods. These results indicate that the introduction of koi fish in the Boesmansgat cave threatens the long-term sustainability of these amphipods and other invertebrate assemblages in this cave. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Mlungu, Zizile
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424424 , vital:72152
- Description: Sternophysingidae Holsinger, 1992 is a monotypic subterranean amphipod family endemic to Namibia and South Africa. It currently has eight described species under the genus Sternophysinx. The species in this family are considered narrow endemics, having only been identified using morphological characters. However, the apparent wide distribution of certain species, reported in several subterranean waters in different provinces, suggests that our current taxonomic understanding of these species needs to be completed, thus needing revision. Further, detailed knowledge of the distribution and phylogenetic relationship between species of this taxon still needs to be improved. This study aimed to use integrative taxonomy to analyse both morphology and molecular biology to study hidden diversity and to further our understanding of the distribution in this taxon. The available morphological keys were used to identify species, and 12S rRNA was also used to investigate the relationships of Sternophysinx species and delimit species boundaries. The study primarily aimed to use mitochondrial cytochrome c oxidase subunit I (COI). However, due to technical issues such as getting the correct primers, this study's aims were altered, and 12S was used instead of CO1. However, the process of getting CO1 sequences is still ongoing, and the results will be made available elsewhere. The current study aimed to visit all the type localities of the seven species of Sternophysinx reported in South Africa, including all known localities in which these amphipods have been recorded in the past. Nevertheless, this study did not get samples from Nelspruit, De Hoop Nature Reserve, and Nash Nature Reserve, which cover type localities of S. travaleensis, Matlapitse and Chaos cave that harbours S. calceola. Instead, the current study added five new localities Graaff-Reinet, Beaufort West, Hogsback, Makhambathi Nature Reserve and Ntsikeni Nature Reserve, for the distribution, including two new provinces (i.e., Eastern Cape and KwaZulu-Natal) that had records of this genus in the country. Three delimitation methods, Assemble Species by Automatic Partitioning (ASAP), Bayesian implementation of the Poison Tree Processor (bPTP) and Automatic Barcode Gap Discovery (ABGD), were used to identify cryptic species within Sternophysinx. Phylogenetic analyses and three delimitation methods (ASAP, bPTP and ABGD) revealed considerable hidden cryptic diversity within this Sternophysinx. Preliminary morphological examination revealed at least two species as undescribed from Bakwena and Sterkfontein caves, and several that were morphologically identified using the current taxonomic keys did not match with the molecular analysis. For example, S. basilobata from three different systems, Boesmansgat cave (Northern Cape), Beaufort West (Western Cape) and Potchefstroom (North West), was in genetically different putative species. The same is true for S. robertsi and S. alca in Ficus and Peppercorn cave in Limpopo. Overall, the results of the current study demonstrate the need for a comprehensive integrative taxonomic revision of Sternophysinx. These findings suggest that S. basilobata is a complex of distinct lineages and requires further study. On the other hand, species delimitation methods suggested that there are 12- 14 candidate species (Figure 2.5). Lastly, this study investigated the threat posed by the invasive koi fish introduced to an important cave, Boesmansgat cave in the Northern Cape, which is a type locality of two endemic species (S. megacheles and S. basilobata). Stable isotope (δ13C and δ15N) and gut content analyses of the fish were investigated to establish whether this invasive fish feed on these critical creatures, thus endangering their long-term survival. Stable isotope signatures and the stomach gut content proved to be helpful in determining the diet of the koi fish, revealing it to be an omnivorous feeder with high invertebrate preference, including amphipods. These results indicate that the introduction of koi fish in the Boesmansgat cave threatens the long-term sustainability of these amphipods and other invertebrate assemblages in this cave. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
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