Identification of Potential Fish Hosts from Wild Populations of State-Threatened East Texas Freshwater Mussels Using a Molecular Identification Dataset

Six species of freshwater mussel are of conservation concern throughout their range in East Texas, (Texas pigtoe (Fusconaia askewi), triangle pigtoe (Fusconaia lananensis), southern hickorynut (Obovaria jacksoniana), sandbank pocketbook (Lampsilis satura), Louisiana pigtoe (Pleurobema riddellii), and Texas heelsplitter (Potamilus amphichaenus)). These species warranted listing in Texas due, in part, to their restricted distributions and low abundances. These mussels, like most unionids, exhibit an unusual life cycle, unique to Unionidae, in which their larvae, called glochidia, are obligate ectoparasites on fish. Knowledge of host fish species is severally lacking for many mussels, as many hosts are unknown or have not been verified. Such natural history data is critical to the conservation of unionids. The purpose of this study was to evaluate if host fish identified in the laboratory act as hosts in natural populations. In addition, new species will be assessed as possible hosts by sampling naturally parasitized fish in the wild. Morphological identification of glochidia to the species level is very difficult due to the small size of glochidia (50-500 µm), therefore a species molecular identification dataset utilizing the sequence of the ND1 gene was developed prior to sampling naturally parasitized fish. A molecular identification dataset designed from sequences of 37 mussel species found in East Texas was successfully designed and utilized to identify encysted glochidia on wild-caught fish. A total of 151 glochidia were successfully identified from eight mussel species. New potential fish hosts were identified for two state-threatened species, Fusconaia askewi (Texas pigtoe) and Pleurobema riddellii (Louisiana pigtoe). Glochidia abundance and diversity was found to differ over the sampling season within the Sabine River. Ecological niche modeling in Maxent supported the results found in fish host use of naturally encysted glochidia. These findings are critical for understanding the complex relationships between mussels and their fish hosts, which is necessary for conservation planning

Design and Implementation of Environmental DNA Metabarcoding Methods for Monitoring the Southern California Marine Protected Area Network

Marine protected areas (MPAs) are important tools for maintaining biodiversity and abundance of marine species. However, key to the effectiveness of MPAs is monitoring of marine communities. Current monitoring methods rely heavily on SCUBA-based visual observations that are costly and time consuming, limiting the scope of MPA monitoring. Environmental DNA (eDNA) metabarcoding is a promising cost effective, rapid, and automatable alternative for marine ecosystem monitoring. However, as a developing tool, the utility of eDNA metabarcoding requires improved bioinformatic techniques and reference barcode databases. Furthermore, it is important to understand how eDNA metabarcoding performs relative to visual surveys to better understand the strengths and limitations of each approach. This thesis improves eDNA metabarcoding approaches to survey the nearshore rocky reef and kelp forest ecosystems within the Southern California MPA network. It then tests the effectiveness of eDNA metabarcoding against visual surveys conducted by the Channel Islands National Park Service Kelp Forest Monitoring Program and Reef Check California. In Chapter 1, I develop FishCARD, a 12S reference barcode database specific to fishes of the California Current ecosystem. FishCARD improves eDNA metabarcoding taxonomic assignments, resulting in the identification of a broader array of marine vertebrate diversity, including invasive, endangered, and mobile species frequently missed by visual surveys. In Chapter 2, I compare eDNA metabarcoding and visual underwater survey methods inside, on the edge of, and outside the Scorpion State Marine Reserve off Santa Cruz Island. We demonstrate that eDNA captures a broader range of fish taxa than visual surveys and detects fine-scale spatial differences in fish communities. In Chapter 3, I demonstrate that eDNA metabarcoding and visual underwater surveys capture similar biogeographic patterns of fish communities across 44 sites within the Southern California Bight. Importantly, eDNA methods distinguished fish communities inside and outside of Southern California MPAs, finding a greater abundance of target species inside MPAs matching patterns observed through visual surveys. These results built off the collaborative development of the Anacapa Toolkit metabarcoding pipeline. Together I demonstrate the utility of eDNA metabarcoding for monitoring MPAs, providing an important complementary tool to visual methods, helping expand MPA monitoring across space, time, and depth

Forensic Examination of Electronic Documents

[Purpose] The purpose of the study is to reveal the concept and essence of forensic prevention of crimes of forgery of electronic documents, to identify problems in the use of information to establish a system of countering crime and document management. [Methodology] The following approaches were used in the work: system-structural, dialectical, empirical. Forgery of electronic documents and their use is investigated not only within the framework of a single criminal case but also by a set of crimes committed depending on the mechanism that is the main one in the structure of criminal technologies. [Findings] Lack of skills and knowledge about the latest forms of documents, methods of their forgery and use in the field of forensic investigations determine the reasons for the development of this condition. The analysis of investigative and judicial practice shows that cases of forgery of electronic documents are moved to separate proceedings due to the inability to fix the person who committed the crime. In some cases, court procedures are returned for additional investigation, since investigators cannot establish mechanisms for falsification tools and bring appropriate charges. [Practical Implications] The practical significance lies in the formation of proposals for improving or making changes to the legislation, effectively improving the activities of law enforcement agencies involved in countering or combating the forgery of documents.[Purpose] The purpose of the study is to reveal the concept and essence of forensic prevention of crimes of forgery of electronic documents, to identify problems in the use of information to establish a system of countering crime and document management. [Methodology] The following approaches were used in the work: system-structural, dialectical, empirical. Forgery of electronic documents and their use is investigated not only within the framework of a single criminal case but also by a set of crimes committed depending on the mechanism that is the main one in the structure of criminal technologies. [Findings] Lack of skills and knowledge about the latest forms of documents, methods of their forgery and use in the field of forensic investigations determine the reasons for the development of this condition. The analysis of investigative and judicial practice shows that cases of forgery of electronic documents are moved to separate proceedings due to the inability to fix the person who committed the crime. In some cases, court procedures are returned for additional investigation, since investigators cannot establish mechanisms for falsification tools and bring appropriate charges. [Practical Implications] The practical significance lies in the formation of proposals for improving or making changes to the legislation, effectively improving the activities of law enforcement agencies involved in countering or combating the forgery of documents.[Purpose] The purpose of the study is to reveal the concept and essence of forensic prevention of crimes of forgery of electronic documents, to identify problems in the use of information to establish a system of countering crime and document management. [Methodology] The following approaches were used in the work: system-structural, dialectical, empirical. Forgery of electronic documents and their use is investigated not only within the framework of a single criminal case but also by a set of crimes committed depending on the mechanism that is the main one in the structure of criminal technologies. [Findings] Lack of skills and knowledge about the latest forms of documents, methods of their forgery and use in the field of forensic investigations determine the reasons for the development of this condition. The analysis of investigative and judicial practice shows that cases of forgery of electronic documents are moved to separate proceedings due to the inability to fix the person who committed the crime. In some cases, court procedures are returned for additional investigation, since investigators cannot establish mechanisms for falsification tools and bring appropriate charges. [Practical Implications] The practical significance lies in the formation of proposals for improving or making changes to the legislation, effectively improving the activities of law enforcement agencies involved in countering or combating the forgery of documents.[Purpose] The purpose of the study is to reveal the concept and essence of forensic prevention of crimes of forgery of electronic documents, to identify problems in the use of information to establish a system of countering crime and document management. [Methodology] The following approaches were used in the work: system-structural, dialectical, empirical. Forgery of electronic documents and their use is investigated not only within the framework of a single criminal case but also by a set of crimes committed depending on the mechanism that is the main one in the structure of criminal technologies. [Findings] Lack of skills and knowledge about the latest forms of documents, methods of their forgery and use in the field of forensic investigations determine the reasons for the development of this condition. The analysis of investigative and judicial practice shows that cases of forgery of electronic documents are moved to separate proceedings due to the inability to fix the person who committed the crime. In some cases, court procedures are returned for additional investigation, since investigators cannot establish mechanisms for falsification tools and bring appropriate charges. [Practical Implications] The practical significance lies in the formation of proposals for improving or making changes to the legislation, effectively improving the activities of law enforcement agencies involved in countering or combating the forgery of documents

IonSeq Genome Sequencing

The emergence of advanced DNA sequencing methods has presented disruptive opportunities in biotechnology, establishing the foundation for the personalized medicine industry. Since the completion of the Human Genome Project, the number of genomes sequenced has grown exponentially and the sequencing price has dropped precipitously. To make personalized medicine a reality, there is a need for a large collection of sequenced genomes in order to link specific genes to diseases. IonSeq seeks to be the leading DNA sequencing service, employing new semiconductor- based sequencing technology offered by Ion Torrent, to help pharmaceutical companies generate these libraries of genomes for their drug-development processes. To support sequencing reliability and throughput, IonSeq will explore such technical details such as chip configuration, insertion kinetics, signal generation, base-calling methods, and accuracy metrics. IonSeq will prove a 40 genome/day output, made possible by the massively parallel procedure employed by the sequencers. IonSeq will sequence each genome at a price of $2,000 while the cost of ‘manufacture’ will only be$645. Series A will consist of a $3,682,886 investment and will yield the investors a MIRR of 102.98$4,510,491 and result in a 93.43% MIRR over a three period. The NPV by the time of liquidation or acquisition event will be $39,322,347, at a conservative projected growth rate of 5%

Forensic Examination of Electronic Documents

[Purpose] The purpose of the study is to reveal the concept and essence of forensic prevention of crimes of forgery of electronic documents, to identify problems in the use of information to establish a system of countering crime and document management. [Methodology] The following approaches were used in the work: system-structural, dialectical, empirical. Forgery of electronic documents and their use is investigated not only within the framework of a single criminal case but also by a set of crimes committed depending on the mechanism that is the main one in the structure of criminal technologies. [Findings] Lack of skills and knowledge about the latest forms of documents, methods of their forgery and use in the field of forensic investigations determine the reasons for the development of this condition. The analysis of investigative and judicial practice shows that cases of forgery of electronic documents are moved to separate proceedings due to the inability to fix the person who committed the crime. In some cases, court procedures are returned for additional investigation, since investigators cannot establish mechanisms for falsification tools and bring appropriate charges. [Practical Implications] The practical significance lies in the formation of proposals for improving or making changes to the legislation, effectively improving the activities of law enforcement agencies involved in countering or combating the forgery of documents

DNA-based methods: technology solutions to evaluate ecosystem function (Part of 'Understanding ecosystems and resilience using DNA: Chief Scientist’s Group report')

Isolation of genetic material from environmental samples (soil, water sediment), so called eDNA is now routine. These samples provide insight into the macro and micro ecology of the ecosystem from which they are isolated. Targeting specific sequences from specific species allows non-invasive sampling of organisms of interest. Information derived from DNA can be used to support conservation efforts and it also allows the tracking of pathogens and invasive species. More generic approaches allow us to profile bacterial, fungal, algal, plant or animal species. From these data we can perform multi-dimensional analysis, addressing key environmental questions. For example, a bacterial profile can report on the presence of pathogens or the impact of pollutants. Quantitative genetic approaches and Next Generation Sequencing technology that supports the use of eDNA, represents a mature technology with validated applications throughout the healthcare industry. New innovations will provide additional, enhanced utility and cost efficiencies, supporting increased spatial and temporal resolution monitoring with the potential to provide real time surveillance. Currently, these eDNA approaches have been deployed as adjuncts to established approaches limiting the potential benefits gained. Our recommendation is for a new generation of biomonitoring approaches to be adopted based on the full potential proved by eDNA. The potential of these novel eDNA approaches combined with good ecological knowledge and interpretation will provide the tools needed to realise ‘A Green Future’, and deliver the “Plan to Improve the Environment”. Since the technology is well established, the major hurdles to exploiting eDNA tools are in transferring from research tools to regulatory and industrial implementation. Our recommendation therefore are focused around a four step process to fast-track this objective: 1. Define and specify the explicit need or question. 2. Establish a transparent ‘AGILE’ assessments and validation processes. 3. Liaise between UKRI and BEIS to establish funding pipeline from research concept to product. 4. Engage with the industrial sectors to deliver products and services for the environmental sector

Integrative approach unravels the evolutionary history of Western Mediterranean small cicadas (Hemiptera: Cicadettini)

Tese de mestrado em Biologia Evolutiva e Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2017As cigarras (Hemiptera: Cicadidae) são um grupo bem conhecido pelo seu canto estival. Apenas o macho produz som e fá-lo para atrair a fêmea para o acasalamento. A produção de som é feita com o auxílio dos tímbalos. O canto produzido pelos machos tem valor taxonómico na distinção das espécies e que tem atraído o interesse de inúmeros cientistas. Outra característica igualmente interessante associada ás cigarras é a duração do seu ciclo de vida que pode chegar a 17 anos na fase ninfal. Tanto a produção de som pelos machos como os longos ciclos de vida, são características que tornam as cigarras um modelo de estudo tão interessante como desafiante. Assim sendo, dado que o canto emitido pelo macho é único de cada espécie, é possível um diagnóstico rápido apenas com recurso a esta característica. Esta informação é crítica na identificação, tanto mais que na sua ausência apenas um taxonomista treinado consegue distinguir espécies de cigarras. No entanto, as últimas investigações têm apontado a necessidade de camadas adicionais de informação. Assim, em meados do último século, a análise de dados de acústica começou a ser grandemente utilizada e inúmeras espécies novas são descritas. Até então, a descrição unicamente morfológica ignorava esta camada informativa, levando à aglutinação de várias espécies morfologicamente semelhantes numa só. A biodiversidade de cigarras em Portugal e Espanha foi amplamente subestimada, até finais do século XX. Nas últimas décadas várias espécies foram descritas/redescritas, com o auxílio da análise de dados de acústica. É o caso de uma série de espécies do género Tettigetta que em 2010 foram atribuídas a um novo género, Tettigettalna. Estas cigarras, ainda que de pequeno tamanho e de cores apagadas, possuem uma grande diversidade de cantos, reconhecendo-se actualmente oito espécies de Tettigettalna e duas subespécies na Península Ibérica e que estão essencialmente limitadas à porção sul da região. No entanto, Tettigettalna estrellae ocorre apenas no centro e norte de Portugal e Tettigettalna argentata possui uma distribuição generalizada na Península Ibérica (não tendo sido ainda encontrada na cordilheira Bética) estendendo-se até França, Itália, Suiça e este da Eslóvenia. Esta riqueza de espécies de Tettigettalna no sul da Península Iberica, despertou grande interesse, nomeadamente por levantar questões relativas à sua origem e em particular a possível ocorrência na área a sul do Mar Mediterrâneo, nomeadamente em Marrocos. Ainda que existam várias descrições de espécies de cigarras dadas para este país, estas descrições não possuem qualquer informação relativamente ao som produzido pelos machos, sendo baseadas em exemplares de museu muito antigos. Como tal, em 2014 efectuou-se uma expedição, financiada pela Linnean Society e pela FCT, que permitiu explorar o Rife e o Médio Atlas. Nesta expedição foram recolhidos vários espécimes de cigarras, assim como várias gravações de som e vídeo de espécies não conhecidos/identificados. Estes novos dados permitiram efectuar o trabalho desenvolvido nesta tese. A primeira parte do trabalho foca-se na descrição com uma abordagem integrativa (dados de morfologia, genética e acústica) de morfótipos seleccionados de cigarras de Marrocos. Assim, a identificação inicial dos exemplares foi efectuada com recurso à morfologia externa e da genitália. Nesta abordagem inicial confrontou-se a descrição morfológica de 68 espécies e subespécies dadas para o Oeste Mediterrânico. Seguidamente, a análise acústica efectuada às gravações de som de ambas espécies revelou que os exemplares de Tettigettalna sp. (atribuídos a este género pela análise morfológica) possuíam uma estrutura de sinal acústico diferente das espécies congenéricas e como tal, poder-se-ia tratar duma espécie nova. Os morfótipos do outro grupo (sem correspondência morfológica a um género já estabelecido) revelaram uma estrutura da som muito distinto e peculiar com modulação em frequência. Ou seja, o sinal acústico possui dois máximos de amplitude distintos nas frequências de ≈14 kHz e ≈8 kHz, curiosamente fazendo lembrar o som dum flato. Finalmente, a análise genética com o fragmento do gene mitocondrial Citocromo C Oxidase I, (COI-Lep). apoiam a monofilia de cada espécie, bem como a inclusão da nova espécie de Tettigettalna na base do género. A abordagem integrativa destes dados permitiu assim a descrição da agora designada Tettigettalna afroamissa Costa et al, 2017, cujo epíteto significa “a deixada em África”. Adicionalmente, o outro grupo de espécimes foram atribuídos a um novo género Berberigetta, significando “a cigarra dos Berbéres”, pertencentes à espécie Berberigetta dimelodica Costa et al, 2017, cujo epíteto, “duas melodias”, se refere ao curioso som emitido. Os resultados obtidos foram publicados na revista internacional indexada Zootaxa, líder em publicações de taxonomia e sistemática. Com a descrição formal da primeira espécie de Tettigettalna a ocorrer naturalmente fora da Europa foram levantadas novas questões sobre a história, origem e diversificação deste género e que foram analisadas e discutidas na segunda parte da presente tese. Em particular, a questão de como T. afroamissa ocorre em África e se este padrão actual de distribuição das Tettigettalna se deve a dispersão a partir da Península Ibérica ou de Marrocos ou se algum evento vicariante está na base desta separação. Por forma a responder a esta pergunta, optou-se por uma abordagem multilocus, sequenciando-se em adição ao COI outros quatro fragmentos, dois mitocondriais (COI-CTL e ATPase) e dois nucleares (Elongation factor 1α e Calmodulin), com uma maior amostragem. As árvores filogenéticas obtidas por máxima verossimilhança e inferência Bayesiana são largamente congruentes apoiando e reforçando resultados previamente obtidos com apenas o fragmento COI-Lep. Estas análises multilocus apoiam: (1) a monofilia do género Tettigettalna com a inclusão de T. afroamissa; (2) a posição basal de T. josei; (3) uma politomia formada pelas três espécies mais recentes: T. argentata, T.mariae e T. aneabi e; (4) T. armandi e T. defauti como táxones irmãos e subsequente estruturação entre as populações amostradas de ambas espécies. Para estimar os tempos de divergência entre espécies, recorreu-se à estimação de relógios moleculares através do programa *BEAST. Assim, recorrendo-se à calibração de relógios moleculares para este grupo as árvores obtidas apoiam a posição basal de T. josei e a inclusão de T. afroamissa num “clade” em conjunto com a linhagem de espécies de Tettigettalna europeias. A posição basal de T. josei também é suportada por estudos prévios que, visando a morfologia e acústica desta espécie, colocaram T. josei como o táxon mais divergente do grupo. As estimativas obtidas relativamente à separação de T. josei e T. afroamissa da linhagem de Tettigettalna europeias são coincidentes com o início do Messiniano e com a crise salínica do Messiniano (5.97-5.33 Ma). Durante este período uma extensa ponte terrestre entre a Europa e o Norte de África, que cortou a ligação do Atlântico ao mar Mediterrâneo resultando na dessecação quase total deste. A reconstrução dos eventos que levaram à diversificação de Tettigettalna inicia-se no Tortoniano (11 -7 Ma). Nesta altura, uma população ancestral estaria dispersa pelo sul do maciço Ibérico. No início do período do Messiniano (7 Ma), esta população ter-se-á expandindo para Sul, pela zona que agora corresponde à cordilheira Bética. O isolamento e especiação de T. josei no sul de Portugal, estará associado a este fenómeno. A restante população terá colonizado o Norte de África durante a crise salínica (5.9 Ma – 5.3 Ma). Com a abertura do Estreito de Gibraltar , as populações em ambas as margens ficaram isoladas, originando T. afroamissa no Norte de África e as restantes espécies de Tettigettalna na Península Ibérica. Assim, o cenário de vicariância é o mais provável para explicar a distribuição de T. afroamissa. As distribuições actuais das Tettigettalna que compõem a linhagem europeia (excl. T. josei) são concomitantes com os múltiplos refúgios glaciais encontrados na Península Ibérica para flora e fauna. Durante as glaciações, as populações poderão ter ficado isoladas em vales e evoluído separadamente das restantes, dando origem ao actual padrão de distribuição reticulado destas cigarras. Resumindo, com o exercício da tese descreveu-se duas novas espécies de cigarras marroquinas, Berberigetta dimelodica e Tettigettalna afroamissa com o uso integrativo de três camadas informativas: morfologia, acústica e genética. Também conseguiu-se explicar a distribuição trans-Mediterrânica da espécie africana, T. afroamissa, relacionando o seu isolamento e especiacão com a crise salínica do Messiniano. Concluindo, esta tese permitiu melhorar o conhecimento sobre a biodiversidade críptica que é característica do género Tettigettalna, e em particular revelar e descobrir a origem deste grupo.Cicadas are no strangers to people in summer. Despite being difficult to spot among the vegetation they are well-known for the loud songs males produce to attract a potential mate and which are useful to tell species apart. They are also well-known for their long nymph stages that may last up to 17 years. In Portugal and Spain the diversity of cicadas has been long underestimated until last decades. It is the case of the formal description of several species of Tettigettalna in 2010. These cicadas are morphologically very similar, therefore only with the recent inclusion of acoustics and genetics data several taxa were unraveled from a single taxon. Tettigettalna is a genus of small and dull-colored cicadas, composed of nine taxa (eight species and two subspecies) and are present mainly in the Iberian Peninsula. Several of these species are endemic to the southern portion of the Iberian Peninsula, which led us to ask if this genus could also occur in Morocco. Morocco has a rather large number of recorded cicada species, but these species are the result of only a handful of expeditions, that only delivered morphological data. Therefore, in the summer of 2014, the CoBIG2 group, organized an expedition to the Rif and Middle Atlas of Morocco, in order to collect and record these poorly-known group of insects. This trip yelded several unidentified morphotypes, two of which were of particular interest. These two morphotypes were studied with an integrative taxonomic approach, with the inclusion of morphology, genetics and acoustic data. One of these morphotypes was identified as belonging to the genus Tettigettalna. Acoustic and genetic analyses confirmed this taxa as an acoustically-distinct and monophyletic biological entity. This Tettigettalna, T. afroamissa, was named as “afroamissa” which can be translated as “the one left in Africa”, because this is the first cicada of this genus to be found outside Europe. The second taxa could not be directly ascribed to a known genus on the grounds of morphologic analyses alone, so a new genus had to be erected. Berberigetta, the Berberian cicada, its type species has a curious and rather unique calling song. The calling song has two distinct call pitches, one of which can be roughly compared to the sound of “blowing a raspberry”, thus the name B. dimelodica, meaning “two melodies”. This frequency modulation of the calling song is an unlikely pattern to be found amongst Mediterranean cicadas, which may be of interest to further investigate. The description of T. afroamissa as the first Tettigettalna outside Europe raised important questions on the origin and diversification of this group of cicadas. To ascertain the evolutionary history of these species, sampling was geographically expanded and five genetic loci were sequenced. We followed a multilocus approach alongside a Bayesian framework to generate a robust species tree alongside estimates for species divergences. The resulting species tree supports the inclusion of T. afroamissa while placing T. josei as the basal taxon of the group. Divergence estimates of the separations of T. afroamissa and T. josei from the remainder of the European Tettigettalna lineage are mostly concurrent to the early Messinian and the Salinity Crisis (5.97-5.33 Ma) when extensive land bridges formed between North Africa and Europe closing the connection between the Mediterranean sea and the Atlantic ocean. We suggest that during this period, T. josei was separated from other lineages by the Guadalquivir basin and T. afroamissa is the remnant of a large population that occurred between both continents that divided with the opening of the Gibraltar Strait, separating the T.afroamissa lineage from the European Tettigettalna lineages. By the end of the Messinian there were (at least) three separately evolving lineages. In conclusion, this theses allowed a better knowledge on the cryptic diversity of Iberian cicadas and contributed to unravel its origins and rediscover the Moroccan cicadas, that only now we have begun to listen