Species delineation in the southern African endemic catshark genus Haploblepharus

Van Staden, Michaela (2018-11)

Thesis (MScAgric)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Accurate species identification is paramount for the effective implementation of conservation and management plans. Species identification in the genus Haploblepharus has historically been problematic due to the high degree of morphological conservatism between congeners, further complicated by the possibility of interspecific hybridisation. The research presented in this thesis addresses crucial knowledge gaps on species delineation in southern African endemic scyliorhinids by developing and applying molecular markers to assess species divergence in a morphologically conserved and threatened genus. Firstly, this study investigated the apparent lack of mitochondrial DNA sequence divergence previously reported among Haploblepharus species, using newly assembled mitochondrial genomes for Haploblepharus edwardsii, Haploblepharus pictus, Halaelurus natalensis and Poroderma pantherinum. The mitogenome assemblies for H. edwardsii and H. pictus contained single nucleotide polymorphism sequence variants in various mitochondrial genes. Following haplotype separation, interspecific sequence divergence was assessed for each protein-coding gene. Interestingly, divergence estimates between the mitogenome haplotypes recovered from a single Haploblepharus specimen met previously proposed species delineation thresholds. Accordingly, this study describes the presence of heteroplasmy in elasmobranchs, with evidence hinting at hybridisation and paternal leakage as possible factors responsible for the phenomenon. The phylogenetic reconstruction performed in this study illustrated that Scyliorhinidae, as presently recognised, is paraphyletic. Furthermore, the clustering of co-distributed southern African endemic scyliorhinids with alternate scyliorhinid species displaying distributions endemic to the northern Pacific provides valuable insight into the origin of these species. The observed genetic divergence between P. pantherinum and the rest of the study species suggests the occurrence of two separate colonisation events of the southern African coastline. In an attempt to provide evidence for the suspected hybridisation among Haploblepharus species, the second aspect of this project assessed species differentiation using novel microsatellite markers. Species-specific microsatellite markers were developed for a South African endemic catshark, H. edwardsii. Subsequently, the cross-species utility of these markers was assessed in H. fuscus, H. pictus and H. natalensis. A high cross-species amplification rate of success was observed, suggesting that these markers may also be useful in future population genetic studies for catsharks. In this study the null hypothesis of panmixia was rejected in only one of the study species, H. pictus, where genetic discontinuity was evident due to geographic distance. Accurate species assignment for H. natalensis illustrated the utility of these markers for species discrimination. In contrast, some of the Haploblepharus specimens presented a more complex assignment pattern. Interspecific genetic differentiation was statistically significant between all species; however, the level of differentiation between H. fuscus and H. pictus was low in comparison and seemed to be at a population level rather than at a species level. The index of admixture in Bayesian analysis has been used to identify introgression, and by implementing qi thresholds this study was able to confidently identify pure and admixed individuals. While the presence of admixture among Haploblepharus taxa was evident, distinct genetic clusters were also present. Approximately 59% of 88 specimens genotyped were unambiguously assigned to a distinct genetic cluster that confirmed accurate taxonomic assignment. Accordingly, the research presented in this thesis provided valuable insights into the evolutionary relationships, genetic diversity and population connectivity of southern African endemic scyliorhinids.

AFRIKAANSE OPSOMMING: Akkurate spesie identifikasie is noodsaaklik vir die effektiewe implementasie van bewarings- en bestuursplanne. Spesies identifikasie in die genus Haploblepharus was voorheen problematies te danke aan die hoë mate van morfologiese ooreenstemming tussen soortgelyke spesies. Hierdie tendens word verder gekompliseerd deur die moontlikheid van inter-spesie hibridisasie. Die navorsing in hierdie tesis het merkwaardige tekortkominge in kennis aangespreek in die spesie verwantskappe van endemiese kathaaie (“scyliorhinids”) in suidelike Afrika. Dit was bereik deur die ontwikkeling en toepassing van molekulêre merkers om spesie diversifikasie in morfologies eenderse en bedreigde spesies te bestudeer. Die studie het eerstens ondersoek ingestel op die sogenaamde tekort van mitokondriese DNA volgorde divergensie, gevind in die Haploblepharus spesies, d.m.v. nuut bepaalde mitokondriese genome vir Haploblepharus edwardsii, Haploblepharus pictus, Halaelurus natalensis en Poroderma pantherinum te gebruik. Die mitogenomiese samestelling vir H. edwardsii en H. pictus het enkele nukleotied polimorfiese volgorde variasies in die mitokondriale gene bevat. Na haplotipe skeiding is interspesifieke volgorde divergensie vir elke proteïenkoderende geen geassesseer. Interessant genoeg stem die mate van divergensie tussen die mitogenoom haplotipes vanaf 'n enkele Haploblepharus individu ooreen met voorheen voorgestelde spesie-delinerings drempels. Verder beskryf hierdie studie die teenwoordigheid van heteroplasmie in kraakbeenvis met bewyse wat hibridisasie en “paternal leakage” as moontlike faktore identifiseer vir die verskynsel. Die filogenetiese rekonstruksie wat in hierdie studie uitgevoer is, het geïllustreer dat Scyliorhinidae, soos tans erken word, parafileties is. Daarbenewens bied die groepering van die endemiese kathaaie van suidelike Afrika met die vanaf die noordelike Stille Oseaan, waardevolle insig in die oorsprong van hierdie spesies. Die waargenome genetiese divergensie tussen P. pantherinum en die res van die studie spesies dui op die voorkoms van twee afsonderlike kolonisasies van die suidelike Afrikaanse kus. In ‘n poging om bewyse voor te bring wat dui op die verdagte hibridisasie tussen Haploblepharus spesies het die tweede deel van die navorsing, spesie verskille geassesseer deur gebruik te maak van nuwe mikrosatelliet merkers. Spesiespesifieke mikrosatelliet merkers was ontwikkel vir ‘n skaam-haai spesie, H. edwardsii, endemies tot Suid Afrika. Gevolglik was die merkers getoets in H. fuscus, H. pictus en H. natalensis. 'n Hoë sukses van merker amplifisering in ander spesies is waargeneem, wat daarop dui dat hierdie merkers ook nuttig kan wees in toekomstige populasie genetiese studies vir ander kathaaie. In hierdie studie is die nulhipotese van panmiksia in slegs een van die studie spesies, H. pictus, verwerp, waar genetiese diskontinuïteit vanweë geografiese afstand duidelik was. Akkurate spesie toekenning vir H. natalensis het die nut van hierdie merkers vir spesie diskriminasie verder geïllustreer. In kontras, was daar sommige van die Haploblepharus individue wat ‘n meer komplekse patroon van toedelings vertoon het. Inter-spesifieke genetiese differensiasie was statisties beduidend tussen alle spesies; nietemin, die vlak van genetiese differensiasie tussen H. fuscus en H. pictus in vergelyking was laag en was meer in lyn met ‘n populasie eerder as op spesie-vlak. Die indeks van vermenging in Bayesiaanse analise is gebruik om introgressie te identifiseer, en deur die implementering van qi-drempels was hierdie studie in staat om individue te identifiseer wat suiwer en vermeng is. Terwyl die teenwoordigheid van vermenging tussen Haploblepharus taxa duidelik was, was daar ook verskillende genetiese groepe teenwoordig. Ongeveer 59% van die 88 individue is onvoorwaardelik toegeken aan 'n spesifieke genetiese groep wat ook akkurate taksonomiese toekenning bevestig het. Gevolglik het die navorsing wat in hierdie proefskrif aangebied word, waardevolle insig gegee in die evolusionêre verwantskappe, genetiese diversiteit en populasie konnektiwiteit van Suider-Afrikaanse endemiese kathaaie.

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