Population genetic structure and phenotypic diversity of Aspidodera raillieti (Nematoda: Heterakoidea), a parasite of Didelphini marsupials in Brazil’s South and Southeast Atlantic Forest

Background: Population genetics of parasites may be influenced by host specificity, life-cycle, geographical distance, evolutionary history, and host-populations structure. The nematode Aspidodera raillieti infects different marsupial and rodent hosts in the Nearctic and Neotropical regions, implying a presumably significant gene flow among populations. However, niche diversification of A. raillieti main hosts in superimposed areas may provide conditions for population genetic structuring within this parasite species. We examined the genetic structuring of A. raillieti infecting three marsupial species co-occurring along South and Southeast Brazilian Atlantic Forest, a hotspot of biodiversity.Methods: We employed morphometric analyses and partial mitochondrial cytochrome c oxidase I gene sequences (MT-CO1) to characterize populations via phylogenetic and phylogeographic analyses.Results: Among 175 A. raillieti specimens recovered from marsupial hosts Didelphis aurita, D. albiventris, and Philander quica, we identified 99 MT-CO1 haplotypes forming four groups in phylogenetic trees and networks. Clades I and II encompassed parasites of D. albiventris from the South region, Clade III comprised parasites of D. aurita from the South and Southeast regions, and Clade IV encompassed parasites of D. aurita and D. albiventris from the South and Southeast regions and parasites of Philander quica from the South region. High genetic differentiation between clades, with a high fixation index and greater genetic variation in the analysis of molecular variance (AMOVA), indicated low gene flow between clades. Haplotypes shared among host species revealed a lack of host specificity. Significant correlation in the Mantel test, suggested parasite isolation by distance, although there was no evidence of geographic structure between populations. Negative values in neutrality tests for Clades III and IV suggested recent population expansion. Morphometric differentiation between A. raillieti specimens recovered from different host species, as well as from different localities, was more evident in males.Conclusion: The genetic structure of A. raillieti populations in the South and Southeast Atlantic Forest resulted from historical events rather than from current geographical distribution or host specificity. We also demonstrate morphometric variation associated with host species and localities, suggesting phenotypic plasticity to host attributes and to spatial variables.

Phylogeography of the oyster borer (Haustrum scobina)

<p>An effective investigation of the underlying ecological processes that shape genetic diversity and connectivity typically requires comparisons among phylogeographic studies of multiple species. Phylogeographic studies of New Zealand’s coastal marine benthos have historically relied on post hoc speculation rather than directed research questions to investigate ecological processes. There has also been a lack of studies on direct developing marine molluscs. Direct developers are expected to have a low potential for dispersal and thus show a pattern of genetic isolation by distance across their distributions. Recent research indicates that this assumption may frequently be violated by instances of long distance dispersal/translocation. The oyster borer (Haustrum scobina) is an endemic direct-developing marine mollusc found in high abundances at rocky intertidal environments across the entirety of New Zealand. This distribution and life history makes H. scobina an ideal target to study genetic connectivity in a species expected to show low realised dispersal and high population genetic structuring. This thesis research used 379 new DNA sequences from the mitochondrial gene cytochrome c oxidase subunit 1 (COI) to investigate the phylogeography of H. scobina across the southern North Island. In addition 16 new COI sequences were inadvertently sequenced from the morphologically similar congener Haustrum albomarginatum. Results from both species support the recently proposed division of H. scobina and H. albomarginatum as separate species. H. scobina populations show significant geographic structure and a lack of haplotype diversity across the south-eastern North Island concordant with results of another previous study of a direct developer. This finding suggests that ecological processes may be producing similar population genetic structures for direct developers generally. Contrast between high and low haplotype diversities in northern and southern H. scobina populations respectively, indicates that southern H. scobina populations may have originated via recolonisation from northern populations following a range contraction during the Last Glacial Maximum. Evidence of multiple long distance dispersal/translocation events was found indicating that long distance dispersal via rafting and/or inadvertent human-mediated translocations may have occurred frequently. Results are then discussed with a view to inform further research in to New Zealand direct developers.</p>

Phylogeography of the oyster borer (Haustrum scobina)

<p>An effective investigation of the underlying ecological processes that shape genetic diversity and connectivity typically requires comparisons among phylogeographic studies of multiple species. Phylogeographic studies of New Zealand’s coastal marine benthos have historically relied on post hoc speculation rather than directed research questions to investigate ecological processes. There has also been a lack of studies on direct developing marine molluscs. Direct developers are expected to have a low potential for dispersal and thus show a pattern of genetic isolation by distance across their distributions. Recent research indicates that this assumption may frequently be violated by instances of long distance dispersal/translocation. The oyster borer (Haustrum scobina) is an endemic direct-developing marine mollusc found in high abundances at rocky intertidal environments across the entirety of New Zealand. This distribution and life history makes H. scobina an ideal target to study genetic connectivity in a species expected to show low realised dispersal and high population genetic structuring. This thesis research used 379 new DNA sequences from the mitochondrial gene cytochrome c oxidase subunit 1 (COI) to investigate the phylogeography of H. scobina across the southern North Island. In addition 16 new COI sequences were inadvertently sequenced from the morphologically similar congener Haustrum albomarginatum. Results from both species support the recently proposed division of H. scobina and H. albomarginatum as separate species. H. scobina populations show significant geographic structure and a lack of haplotype diversity across the south-eastern North Island concordant with results of another previous study of a direct developer. This finding suggests that ecological processes may be producing similar population genetic structures for direct developers generally. Contrast between high and low haplotype diversities in northern and southern H. scobina populations respectively, indicates that southern H. scobina populations may have originated via recolonisation from northern populations following a range contraction during the Last Glacial Maximum. Evidence of multiple long distance dispersal/translocation events was found indicating that long distance dispersal via rafting and/or inadvertent human-mediated translocations may have occurred frequently. Results are then discussed with a view to inform further research in to New Zealand direct developers.</p>

Population genetic structure of the Natterjack toad (Epidalea calamita) in Ireland: implications for conservation management

Molecular methods can play a crucial role in species management and conservation. Despite the usefulness of genetic approaches, they are often not explicitly included as part of species recovery plans and conservation practises. The Natterjack toad (Epidalea calamita) is regionally Red-Listed as Endangered in Ireland. The species is declining and is now present at just seven sites within a highly restricted range. This study used 13 highly polymorphic microsatellite markers to analyse the population genetic diversity and structure. Genetic diversity was high with expected heterozygosity between 0.55 and 0.61 and allelic richness between 4.77 and 5.92. Effective population sizes were small (Ne < 100 individuals), but not abnormal for pond breeding amphibians. However, there was no evidence of historical or contemporary genetic bottlenecks or high levels of inbreeding. We identified a positive relationship between Ne and breeding pond surface area, suggesting that environmental factors are a key determinant of population size. Significant genetic structuring was detected throughout the species’ range, and we identified four genetic entities that should be considered in the species’ conservation strategies. Management should focus on preventing further population declines and future loss of genetic diversity overall and within genetic entities while maintaining adequate local effective population size through site-specific protection, human-mediated translocations and head-start programs. The apparent high levels of genetic variation give hope for the conservation of Ireland’s rarest amphibian if appropriately protected and managed.

Population Genetics of New Zealand Scampi (Metanephrops challengeri)

<p>A fundamental goal of fisheries management is sustainable harvesting and the preservation of properly functioning populations. Therefore, an important aspect of management is the identification of demographically independent populations (stocks), which is achieved by estimating the movement of individuals between areas. A range of methods have been developed to determine the level of connectivity among populations; some measure this directly (e.g. mark-recapture) while others use indirect measures (e.g. population genetics). Each species presents a different set of challenges for methods that estimate levels of connectivity. Metanephrops challengeri is a species of nephropid lobster that supports a commercial fishery and inhabits the continental shelf and slope of New Zealand. Very little research on population structure has been reported for this species and it presents a unique set of challenges compared to finfish species. M. challengeri have a short pelagic larval duration lasting up to five days which limits the dispersal potential of larvae, potentially leading to low levels of connectivity among populations. The aim of this study was to examine the genetic population structure of the New Zealand M. challengeri fishery. DNA was extracted from M. challengeri samples collected from the eastern coast of the North Island (from the Bay of Plenty to the Wairarapa), the Chatham Rise, and near the Auckland Islands. DNA from the mitochondrial CO1 gene and nuclear ITS-1 region was amplified and sequenced. The aligned dataset of DNA sequences was then used to estimate levels of both genetic diversity and differentiation, and examine demographic history. Analyses of population structure indicate that M. challengeri from the Auckland Islands region are genetically distinct from M. challengeri inhabiting the Chatham Rise, and those collected from waters off the eastern coast of the North Island. There appears to be gene flow among the sampling sites off the eastern coast of the North Island and on the Chatham Rise, but some isolation by distance was detected. These results indicate that some of these populations may be demographically uncoupled. Genetic diversity estimates combined with Bayesian skyline plots and demographic history parameters suggest that M. challengeri populations have recently undergone a size expansion. The genetic structuring between the Auckland Islands site and all others may be due to a putative habitat disjunction off the Otago shelf. In contrast, a largely continuously distributed population along the eastern coast of the North Island and the Chatham Rise most likely promotes gene flow as larvae can be transported limited distances by oceanic currents. Historical changes in climate may have influenced the patterns of present-day structure and genetic diversity of M. challengeri, by altering habitat availability and other characteristics of their environment. This study provides evidence that species which appear to have limited dispersal potential can still maintain connected populations, but there are situations where large breaks in suitable habitat appear to limit gene flow. The results of this study will help inform stock structure of the M. challengeri fishery, which will enable stock assessments to be more precisely aligned to natural population boundaries.</p>

Population Genetics of New Zealand Scampi (Metanephrops challengeri)

<p>A fundamental goal of fisheries management is sustainable harvesting and the preservation of properly functioning populations. Therefore, an important aspect of management is the identification of demographically independent populations (stocks), which is achieved by estimating the movement of individuals between areas. A range of methods have been developed to determine the level of connectivity among populations; some measure this directly (e.g. mark-recapture) while others use indirect measures (e.g. population genetics). Each species presents a different set of challenges for methods that estimate levels of connectivity. Metanephrops challengeri is a species of nephropid lobster that supports a commercial fishery and inhabits the continental shelf and slope of New Zealand. Very little research on population structure has been reported for this species and it presents a unique set of challenges compared to finfish species. M. challengeri have a short pelagic larval duration lasting up to five days which limits the dispersal potential of larvae, potentially leading to low levels of connectivity among populations. The aim of this study was to examine the genetic population structure of the New Zealand M. challengeri fishery. DNA was extracted from M. challengeri samples collected from the eastern coast of the North Island (from the Bay of Plenty to the Wairarapa), the Chatham Rise, and near the Auckland Islands. DNA from the mitochondrial CO1 gene and nuclear ITS-1 region was amplified and sequenced. The aligned dataset of DNA sequences was then used to estimate levels of both genetic diversity and differentiation, and examine demographic history. Analyses of population structure indicate that M. challengeri from the Auckland Islands region are genetically distinct from M. challengeri inhabiting the Chatham Rise, and those collected from waters off the eastern coast of the North Island. There appears to be gene flow among the sampling sites off the eastern coast of the North Island and on the Chatham Rise, but some isolation by distance was detected. These results indicate that some of these populations may be demographically uncoupled. Genetic diversity estimates combined with Bayesian skyline plots and demographic history parameters suggest that M. challengeri populations have recently undergone a size expansion. The genetic structuring between the Auckland Islands site and all others may be due to a putative habitat disjunction off the Otago shelf. In contrast, a largely continuously distributed population along the eastern coast of the North Island and the Chatham Rise most likely promotes gene flow as larvae can be transported limited distances by oceanic currents. Historical changes in climate may have influenced the patterns of present-day structure and genetic diversity of M. challengeri, by altering habitat availability and other characteristics of their environment. This study provides evidence that species which appear to have limited dispersal potential can still maintain connected populations, but there are situations where large breaks in suitable habitat appear to limit gene flow. The results of this study will help inform stock structure of the M. challengeri fishery, which will enable stock assessments to be more precisely aligned to natural population boundaries.</p>

Inferring the late Quaternary phylogeography of Pseudopanax crassifolius using microsatellite analysis

<p>Geologic processes have shaped the New Zealand archipelago throughout its existence. The last major geologic event was the Pleistocene glaciations beginning around 2.5 million years ago. This cold period left its mark in the phylogeography (the geographic distribution of genetic variation) of New Zealand’s globally significant biota. Studies into the phylogeography of New Zealand have largely focused on species with limited distributions through rarity or ecological preferences. This study focuses on the ubiquitous species Pseudopanax crassifolius (Sol. Ex A. Cunn) K. Koch, also known commonly as Horoeka or Lancewood. This species is widespread and almost continuously distributed throughout New Zealand giving a broad scale look at the patterns and processes that have influenced the formation of New Zealand’s natural history. Seven microsatellite loci and two rps4 chloroplast haplotypes were utilised to study 247 Pseudopanax crassifolius and nine P. chathamicus individuals sampled from populations from around New Zealand. Pseudopanax crassifolius was found to have levels of genetic diversity and overall differentiation consistent with common widespread trees. The genetic structuring suggests P. crassifolius is not a single homogenous population across a southern cluster. The geographic structuring of genetic variation within these clusters is poor. The genetic patterns and the spatial distribution of these patterns may reflect the response of Pseudopanax crassifolius to changing environmental conditions during the late Quaternary following the maximum extent of the last glacial maximum (LGM) period. During the maximally cold periods of the LGM, P. crassifolius is likely to have been eliminated or at least greatly reduced in the south and west coast of the South Island. In the remainder of the South Island and throughout the North Island it remained widespread. The heterogeneous pattern of genetic variation with little geographic correlation in the northern cluster may reflect either the extent of the historic distribution of the species or the effect of gene flow between populations acting to inhibit population structuring from establishing. The reduction in genetic diversity and the homogeneity of structure in the south indicate a pattern of leading edge re-colonisation into southern areas as conditions became more favourable following the LGM. The leading edge mode is supported by asymmetric introgression of rps4 haplotype seen between P. crassifolius and P. ferox along the east coast of the South Island. This study also investigated levels of differentiation between Pseudopanax crassifolius and P. chathamicus. There is limited evidence of differentiation based on microsatellite markers. There is therefore no strong genetic evidence for either the support or rejection of the current species delimitation of the crassifolius group of Pseudopanax species. The two species are morphologically different and geographically isolated. This, alongside evidence from previous studies suggest that P. chathamicus is possibly an example of a group undergoing incipient allopatric speciation. A recent founder event is proposed with enough potential diversity carried in two individual fruit to account for the diversity seen in P. chathamicus.</p>

Inferring the late Quaternary phylogeography of Pseudopanax crassifolius using microsatellite analysis

<p>Geologic processes have shaped the New Zealand archipelago throughout its existence. The last major geologic event was the Pleistocene glaciations beginning around 2.5 million years ago. This cold period left its mark in the phylogeography (the geographic distribution of genetic variation) of New Zealand’s globally significant biota. Studies into the phylogeography of New Zealand have largely focused on species with limited distributions through rarity or ecological preferences. This study focuses on the ubiquitous species Pseudopanax crassifolius (Sol. Ex A. Cunn) K. Koch, also known commonly as Horoeka or Lancewood. This species is widespread and almost continuously distributed throughout New Zealand giving a broad scale look at the patterns and processes that have influenced the formation of New Zealand’s natural history. Seven microsatellite loci and two rps4 chloroplast haplotypes were utilised to study 247 Pseudopanax crassifolius and nine P. chathamicus individuals sampled from populations from around New Zealand. Pseudopanax crassifolius was found to have levels of genetic diversity and overall differentiation consistent with common widespread trees. The genetic structuring suggests P. crassifolius is not a single homogenous population across a southern cluster. The geographic structuring of genetic variation within these clusters is poor. The genetic patterns and the spatial distribution of these patterns may reflect the response of Pseudopanax crassifolius to changing environmental conditions during the late Quaternary following the maximum extent of the last glacial maximum (LGM) period. During the maximally cold periods of the LGM, P. crassifolius is likely to have been eliminated or at least greatly reduced in the south and west coast of the South Island. In the remainder of the South Island and throughout the North Island it remained widespread. The heterogeneous pattern of genetic variation with little geographic correlation in the northern cluster may reflect either the extent of the historic distribution of the species or the effect of gene flow between populations acting to inhibit population structuring from establishing. The reduction in genetic diversity and the homogeneity of structure in the south indicate a pattern of leading edge re-colonisation into southern areas as conditions became more favourable following the LGM. The leading edge mode is supported by asymmetric introgression of rps4 haplotype seen between P. crassifolius and P. ferox along the east coast of the South Island. This study also investigated levels of differentiation between Pseudopanax crassifolius and P. chathamicus. There is limited evidence of differentiation based on microsatellite markers. There is therefore no strong genetic evidence for either the support or rejection of the current species delimitation of the crassifolius group of Pseudopanax species. The two species are morphologically different and geographically isolated. This, alongside evidence from previous studies suggest that P. chathamicus is possibly an example of a group undergoing incipient allopatric speciation. A recent founder event is proposed with enough potential diversity carried in two individual fruit to account for the diversity seen in P. chathamicus.</p>

Speciation, Connectivity and Self-Recruitment Among Mollusc Populations from Isolated Oceanic Islands

<p>The conventional view that marine populations are demographically ‘open’ and exchange migrants (juveniles or adults, but mostly larvae) has been challenged by recent genetic studies and the discovery of significant genetic subdivision among populations on small geographic scales. Despite the numerous publications on the matter, the extent to which some/all marine populations rely on self-recruitment and whether this reliance is stable in time and space currently remains unanswered. This is particularly true for populations from isolated oceanic archipelagos, such as the New Zealand (NZ) subantarctic islands and the Kermadec Islands. The specific objectives of this thesis were to: 1) assess the genetic diversity, phylogeography and contemporary levels of dispersal and self-recruitment in populations of the Cellana strigilis limpet complex, endemic to the NZ subantarctic islands; 2) conduct a morphometric analysis of the C. strigilis complex to complement its molecular investigation; 3) develop and optimize specific microsatellite markers for Nerita melanotragus, a marine gastropod of the Kermadec Islands and New Zealand North Island rocky shores; 4) assess the genetic structuring and levels of connectivity of N. melanotragus populations within the Kermadec Islands, within NZ North Island, and between the Kermadec Islands and NZ; and 5) compare the genetic structuring of N. melanotragus at the Kermadec Islands to that of NZ North Island populations, to test for any “island effect” on connectivity levels, and test for possible gene flow between the two groups. Genetic investigation of the C. strigilis complex confirmed the presence of two distinct lineages, separated by their sister species Cellana denticulata. Morphometric analyses were congruent with molecular analyses, and were used to provide a new taxonomic description of the C. strigilis limpet complex: two species were recognized, Cellana strigilis and Cellana oliveri. The role of the subantarctic islands during the last glacial maximum was highlighted, and the colonisation history of the islands by the two Cellana species was explained. Contemporary levels of connectivity (gene flow) among the different populations of the two lineages were low, or non-existant, revealing their high reliability on self-recruitment. However, the analysis detected a recent migration event in one of the two lineages. Considering the geographical distance of the islands and the life history of the Cellana species, the use of mediated dispersal means (e.g., rafting on a natural substrate such as kelp) seems very likely. Ten novel polymorphic microsatellite loci were developed for N. melanotragus, and seven of those were used to investigate the levels of connectivity and self-recruitment in six populations from the Kermadec Islands, and nine populations from the east coast of NZ North Island. According to what can be expected for a species with a long pelagic larval duration (PLD), genetic homogeneity was recorded for the Kermadec Islands populations. A lack of genetic structuring was also found for the nine populations on the NZ North Island, which is congruent with the literature in this geographic area. However, what was surprising was the high level of genetic homogeneity found between the Kermadec Islands and the NZ North Island, meaning that the two groups are effectively exchanging individuals. Hence, the Kermadec archipelago can be considered “open” at the scale of the South Pacific, for N. melanotragus populations. This Ph.D. highlights the importance of having the correct taxonomy for conservation and connectivity studies, and gives a better understanding of the historical and contemporary patterns of genetic connectivity in the NZ offshore islands. It illustrated how historical events, such as the last glacial maximum, can shape local genetic diversity, and how this historical pattern can be maintained because of limited contemporary gene exchange. Also, this thesis demonstrated that remote populations could be strongly connected to mainland populations, contributing to the resilience of both systems and confirming the necessity of integrating remote oceanic habitats in the creation of effective Marine Protected Areas (MPA) networks to protect the marine environment.</p>

Speciation, Connectivity and Self-Recruitment Among Mollusc Populations from Isolated Oceanic Islands

<p>The conventional view that marine populations are demographically ‘open’ and exchange migrants (juveniles or adults, but mostly larvae) has been challenged by recent genetic studies and the discovery of significant genetic subdivision among populations on small geographic scales. Despite the numerous publications on the matter, the extent to which some/all marine populations rely on self-recruitment and whether this reliance is stable in time and space currently remains unanswered. This is particularly true for populations from isolated oceanic archipelagos, such as the New Zealand (NZ) subantarctic islands and the Kermadec Islands. The specific objectives of this thesis were to: 1) assess the genetic diversity, phylogeography and contemporary levels of dispersal and self-recruitment in populations of the Cellana strigilis limpet complex, endemic to the NZ subantarctic islands; 2) conduct a morphometric analysis of the C. strigilis complex to complement its molecular investigation; 3) develop and optimize specific microsatellite markers for Nerita melanotragus, a marine gastropod of the Kermadec Islands and New Zealand North Island rocky shores; 4) assess the genetic structuring and levels of connectivity of N. melanotragus populations within the Kermadec Islands, within NZ North Island, and between the Kermadec Islands and NZ; and 5) compare the genetic structuring of N. melanotragus at the Kermadec Islands to that of NZ North Island populations, to test for any “island effect” on connectivity levels, and test for possible gene flow between the two groups. Genetic investigation of the C. strigilis complex confirmed the presence of two distinct lineages, separated by their sister species Cellana denticulata. Morphometric analyses were congruent with molecular analyses, and were used to provide a new taxonomic description of the C. strigilis limpet complex: two species were recognized, Cellana strigilis and Cellana oliveri. The role of the subantarctic islands during the last glacial maximum was highlighted, and the colonisation history of the islands by the two Cellana species was explained. Contemporary levels of connectivity (gene flow) among the different populations of the two lineages were low, or non-existant, revealing their high reliability on self-recruitment. However, the analysis detected a recent migration event in one of the two lineages. Considering the geographical distance of the islands and the life history of the Cellana species, the use of mediated dispersal means (e.g., rafting on a natural substrate such as kelp) seems very likely. Ten novel polymorphic microsatellite loci were developed for N. melanotragus, and seven of those were used to investigate the levels of connectivity and self-recruitment in six populations from the Kermadec Islands, and nine populations from the east coast of NZ North Island. According to what can be expected for a species with a long pelagic larval duration (PLD), genetic homogeneity was recorded for the Kermadec Islands populations. A lack of genetic structuring was also found for the nine populations on the NZ North Island, which is congruent with the literature in this geographic area. However, what was surprising was the high level of genetic homogeneity found between the Kermadec Islands and the NZ North Island, meaning that the two groups are effectively exchanging individuals. Hence, the Kermadec archipelago can be considered “open” at the scale of the South Pacific, for N. melanotragus populations. This Ph.D. highlights the importance of having the correct taxonomy for conservation and connectivity studies, and gives a better understanding of the historical and contemporary patterns of genetic connectivity in the NZ offshore islands. It illustrated how historical events, such as the last glacial maximum, can shape local genetic diversity, and how this historical pattern can be maintained because of limited contemporary gene exchange. Also, this thesis demonstrated that remote populations could be strongly connected to mainland populations, contributing to the resilience of both systems and confirming the necessity of integrating remote oceanic habitats in the creation of effective Marine Protected Areas (MPA) networks to protect the marine environment.</p>