FWS Conservation Genetics Labs

1. FWS Conservation Genetics Labs

2. FWS Genetics Capabilities Provide sampling guidance, genetic data generation, and interpretation of genetic data for management, conservation and policy issues. Address a variety of issues, such as management of hatchery broodstocks, characterization of population structure, and identification of management and conservation units. Establishing a Conservation Genetics Community of Practice An interactive forum to facilitate the exchange of information and technologies to strengthen the use and understanding of the conservation genetics within the FWS. http://www.fws.gov/ConservationGeneticsCOP

3. Wildlife Forensics:Wildlife Science Meets the Law The topic of “Crime Scene Investigation” has become fairly popular with the broadcast of shows like Law and Order, and CSI. So when you say the word “Forensics,” the first thing that people think of is investigators poring over a crime scene. But Forensic Science is much more than that – and I hope by the end of my talk, you come away with several messages: 1) Forensic Scientists do not always work in the dark; 2) Forensic Science does not always come up with the answers in less than a day; and 3) Wildlife Forensic Science is an area of applied science that is making a major impact on our ability to enforce management regulations to protect our living natural resources. I currently work with the U.S. Department of the Interior, Fish and Wildlife Service, at the Clark R. Bavin National Fish and Wildlife Forensic Laboratory in Ashland, Oregon. The center is named after the late Chief of the United States Fish and Wildlife Service’s Division of Law Enforcement who pioneered the Division’s highly effective covert investigations and “sting” operations. The topic of “Crime Scene Investigation” has become fairly popular with the broadcast of shows like Law and Order, and CSI. So when you say the word “Forensics,” the first thing that people think of is investigators poring over a crime scene. But Forensic Science is much more than that – and I hope by the end of my talk, you come away with several messages: 1) Forensic Scientists do not always work in the dark; 2) Forensic Science does not always come up with the answers in less than a day; and 3) Wildlife Forensic Science is an area of applied science that is making a major impact on our ability to enforce management regulations to protect our living natural resources. I currently work with the U.S. Department of the Interior, Fish and Wildlife Service, at the Clark R. Bavin National Fish and Wildlife Forensic Laboratory in Ashland, Oregon. The center is named after the late Chief of the United States Fish and Wildlife Service’s Division of Law Enforcement who pioneered the Division’s highly effective covert investigations and “sting” operations.

4. So in order to talk about Forensic Science, I feel we must begin with a quick discussion of definitions, and the first one we’ll tackle is “Science”. What is “science?” Science is that area of human endeavor which tries to organize and understand the connections among natural phenomena. It’s based in our need to know what and why about our world. So in order to talk about Forensic Science, I feel we must begin with a quick discussion of definitions, and the first one we’ll tackle is “Science”. What is “science?” Science is that area of human endeavor which tries to organize and understand the connections among natural phenomena. It’s based in our need to know what and why about our world.

5. So when we want to define “Forensic science,” we build on the definitions of science presented here – Forensic Science is really the application of natural science to matters of law. It includes a variety of different activities and specialties. In practice, forensic science draws upon the principles and methods of all the traditional sciences, such as physics, chemistry, and biology. When we talk about investigations in the natural sciences, we’re talking about natural phenomena – and we’re concerned with things and processes that are subject to observation, measurements, and experimentation. In addition, science can be distinguished from other disciplines on the basis of its combined theoretical and empirical approach, which is usually called the scientific method. So when we want to define “Forensic science,” we build on the definitions of science presented here – Forensic Science is really the application of natural science to matters of law. It includes a variety of different activities and specialties. In practice, forensic science draws upon the principles and methods of all the traditional sciences, such as physics, chemistry, and biology. When we talk about investigations in the natural sciences, we’re talking about natural phenomena – and we’re concerned with things and processes that are subject to observation, measurements, and experimentation. In addition, science can be distinguished from other disciplines on the basis of its combined theoretical and empirical approach, which is usually called the scientific method.

6. It is the scientific method then, which becomes the unifying concept when we connect science to law. It’s a way to merge a discipline which is really about uncertainty with a discipline that relies on the concrete, the certain, and getting beyond “reasonable doubt.” I occasionally like to launch into a quick discussion about the scientific method, because it reminds us about what we do and why we do it. It’s also a reminder to me that my preconceptions of what the answer may be, sometimes prevent me from pursuing very valuable lines of research. .It is the scientific method then, which becomes the unifying concept when we connect science to law. It’s a way to merge a discipline which is really about uncertainty with a discipline that relies on the concrete, the certain, and getting beyond “reasonable doubt.” I occasionally like to launch into a quick discussion about the scientific method, because it reminds us about what we do and why we do it. It’s also a reminder to me that my preconceptions of what the answer may be, sometimes prevent me from pursuing very valuable lines of research. .

7. The Scientific Method Observation The scientific method may be characterized as consisting of several more of less separate steps. Most scientists do follow this general procedure without really thinking about it, but it’s nice to be reminded now and then. Observations are made and data is collected. The significance of the observations is considered – conjecture – until some reasonable explanation consistent with all the data – the hypothesis – is arrived at. Experiments designed to test the hypothesis are then conceived and carried out. The new data thus obtained are used to refine the initial hypothesis as needed Thus, one would go through the cycle again, and again, and again if necessary! A modified hypothesis which has been verified by a good deal of rigorous testing may come to be called a theory. Sometimes, theories which have been extensively tested and verified by many scientists working independently may come to be regarded as natural laws.The scientific method may be characterized as consisting of several more of less separate steps. Most scientists do follow this general procedure without really thinking about it, but it’s nice to be reminded now and then. Observations are made and data is collected. The significance of the observations is considered – conjecture – until some reasonable explanation consistent with all the data – the hypothesis – is arrived at. Experiments designed to test the hypothesis are then conceived and carried out. The new data thus obtained are used to refine the initial hypothesis as needed Thus, one would go through the cycle again, and again, and again if necessary! A modified hypothesis which has been verified by a good deal of rigorous testing may come to be called a theory. Sometimes, theories which have been extensively tested and verified by many scientists working independently may come to be regarded as natural laws.

8. Dimensions of Forensic Science The dimensions of forensic science investigation may include any or all of three major activities in analyzing and interpreting physical evidence: Identification, Individualization, and Reconstruction. Identification is a process common to all the sciences and, in fact, to every day life. It may be regarded as a classification scheme, in which items are assigned to categories containing like items. Different items within a given category all have the same generic name. Class characteristics are the properties that all the members of a certain class of objects or substances have in common. In this way, botanists will identify plants by categorizing an naming them. Likewise, chemists identify chemical compounds. In forensic science, objects are identified by comparing their class characteristics with those of known standards or previously established criteria. Individualization is unique to forensic science; it refers to the demonstration that a particular sample is unique, even among members of the same class. It may also refer to the demonstration that a questioned piece of physical evidence and a similar known sample have a common origin. Thus, in addition to class characteristics, objects and materials possess individual characteristics that can be used to distinguish members of the same class. The nature of these individual characteristics varies from one type of evidence to another, but forensic scientists try to take advantage of them in efforts to individualize a piece of physical evidence by some type of comparison process. Reconstruction is fairly straightforward – being the process of assembling the evidence and reconstructing the events. The dimensions of forensic science investigation may include any or all of three major activities in analyzing and interpreting physical evidence: Identification, Individualization, and Reconstruction. Identification is a process common to all the sciences and, in fact, to every day life. It may be regarded as a classification scheme, in which items are assigned to categories containing like items. Different items within a given category all have the same generic name. Class characteristics are the properties that all the members of a certain class of objects or substances have in common. In this way, botanists will identify plants by categorizing an naming them. Likewise, chemists identify chemical compounds. In forensic science, objects are identified by comparing their class characteristics with those of known standards or previously established criteria. Individualization is unique to forensic science; it refers to the demonstration that a particular sample is unique, even among members of the same class. It may also refer to the demonstration that a questioned piece of physical evidence and a similar known sample have a common origin. Thus, in addition to class characteristics, objects and materials possess individual characteristics that can be used to distinguish members of the same class. The nature of these individual characteristics varies from one type of evidence to another, but forensic scientists try to take advantage of them in efforts to individualize a piece of physical evidence by some type of comparison process. Reconstruction is fairly straightforward – being the process of assembling the evidence and reconstructing the events.

9. To think about Forensics, you must think about the Forensic Triangle. Any one of these connections can be supported with scientific evidence to complete the forensic triangle. Because of policy and management decisions, when those decisions are made, this gives the opportunity to understand how to enforcement…along with monitoring and assessment, the enforcement sometimes gets left out. Example: Hunting season, heard shots on a refuge. 1. Agent sent leaf, with puddle of blood on the leaf. 2. unknown person suspected to be hunting geese on the refuge. 3. Agent went out to investigate and and sent blood to lab, is the victim a goose or not. 4. Test showed that blood originated from a woodpecker. 5. Reconstruction determined that most likely scenario was that bobcat killed the woodpecker and took it to a tree to eat it, blood drained out and fell on the leaf. 6. case closed. Both victim and suspect were wrong, there were both other animals.To think about Forensics, you must think about the Forensic Triangle. Any one of these connections can be supported with scientific evidence to complete the forensic triangle. Because of policy and management decisions, when those decisions are made, this gives the opportunity to understand how to enforcement…along with monitoring and assessment, the enforcement sometimes gets left out. Example: Hunting season, heard shots on a refuge. 1. Agent sent leaf, with puddle of blood on the leaf. 2. unknown person suspected to be hunting geese on the refuge. 3. Agent went out to investigate and and sent blood to lab, is the victim a goose or not. 4. Test showed that blood originated from a woodpecker. 5. Reconstruction determined that most likely scenario was that bobcat killed the woodpecker and took it to a tree to eat it, blood drained out and fell on the leaf. 6. case closed. Both victim and suspect were wrong, there were both other animals.

10. To exercise their function responsibly, courts have always sought ways to ascertain the facts surrounding particular cases. The relationship between law and science had developed out of a common interest in factual information. In order to prosecute an individual for a violation of law, our criminal justice system requires that the individual's guilt be established 'beyond a reasonable doubt.' Two ways that prosecutors can link the suspect to the victim and to the crime are to present to the court Eye Witness Testimony and Physical Evidence However, because the reliability of eye-witness testimony is often considered questionable at best, prosecutors often insist that investigators obtain linking physical evidence and relevant expert witness testimony before they are willing to take a case to court. Although the concepts of scientific fact and legal fact are often quite different, the application of scientific methods can often provide factual information that is relevant to a legal proceeding and would not be available without the intervention of science. To exercise their function responsibly, courts have always sought ways to ascertain the facts surrounding particular cases. The relationship between law and science had developed out of a common interest in factual information. In order to prosecute an individual for a violation of law, our criminal justice system requires that the individual's guilt be established 'beyond a reasonable doubt.' Two ways that prosecutors can link the suspect to the victim and to the crime are to present to the court Eye Witness Testimony and Physical Evidence However, because the reliability of eye-witness testimony is often considered questionable at best, prosecutors often insist that investigators obtain linking physical evidence and relevant expert witness testimony before they are willing to take a case to court. Although the concepts of scientific fact and legal fact are often quite different, the application of scientific methods can often provide factual information that is relevant to a legal proceeding and would not be available without the intervention of science.

11. What regulates the admissibility of scientific evidence in the courtroom? So how does scientific evidence get entered into the courtroom? Investigations conducted by law enforcement personnel may end up in court for litigation or sentencing, or lawyers may request the inclusion of scientific evidence with the case evidence when a case goes to trial. Ultimately, the judge is the “gatekeeper” and must determine the appropriateness of the evidence within the context of the trial. Scientific evidence is subjected to one of two standards of admissibility, either of which can be used to challenge a particular technique’s use in a trial. These are the Frye and Daubert criteria which relate to the scientific methods themselves – how appropriate are the methods for the evidence and the question in the trial? Are the methods used generally accepted among peer scientists? Does the method have a way of calculating an error rate – not whether it is right or wrong, but statistical error. How robust is the method?, etc. Forensic scientists are often called to be Expert Witnesses in court – in this way they can testify with respect to the conclusions drawn in the analysis, which are essentially interpretations based on the analyst’s knowledge, experience, and qualifications as a scientist. In contrast, a LAY witness can only testify to what was done and what was seen, but not what was interpreted. For example, if a gender test was conducted and an analyst was accepted as an expert witness in “voire dire”, they could testify that the banding pattern observed in the gender test was characteristic of a male mammal. A lay witness could only testify that two bands were observed on the gel for the gender test. Accreditation of the laboratory is an important addition to an analysis – accredited labs have gone through a rigorous series of qualifying steps in which protocols, technique validation, proficiency testing of analysts, and laboratory procedures are scrutinized. Currently the USFWS lab is accredited under the guidelines for ASCLD-LAB, but we will be working towards international accreditation qualifications under ISO 9000 in the next few years. Most importantly, chain of custody and evidence handling procedures are crucial to successful use of forensic science in the courtroom. Most challenges to criminal cases in court are made based on broken chain of custody or inappropriate evidence handling issues.So how does scientific evidence get entered into the courtroom? Investigations conducted by law enforcement personnel may end up in court for litigation or sentencing, or lawyers may request the inclusion of scientific evidence with the case evidence when a case goes to trial. Ultimately, the judge is the “gatekeeper” and must determine the appropriateness of the evidence within the context of the trial. Scientific evidence is subjected to one of two standards of admissibility, either of which can be used to challenge a particular technique’s use in a trial. These are the Frye and Daubert criteria which relate to the scientific methods themselves – how appropriate are the methods for the evidence and the question in the trial? Are the methods used generally accepted among peer scientists? Does the method have a way of calculating an error rate – not whether it is right or wrong, but statistical error. How robust is the method?, etc. Forensic scientists are often called to be Expert Witnesses in court – in this way they can testify with respect to the conclusions drawn in the analysis, which are essentially interpretations based on the analyst’s knowledge, experience, and qualifications as a scientist. In contrast, a LAY witness can only testify to what was done and what was seen, but not what was interpreted. For example, if a gender test was conducted and an analyst was accepted as an expert witness in “voire dire”, they could testify that the banding pattern observed in the gender test was characteristic of a male mammal. A lay witness could only testify that two bands were observed on the gel for the gender test. Accreditation of the laboratory is an important addition to an analysis – accredited labs have gone through a rigorous series of qualifying steps in which protocols, technique validation, proficiency testing of analysts, and laboratory procedures are scrutinized. Currently the USFWS lab is accredited under the guidelines for ASCLD-LAB, but we will be working towards international accreditation qualifications under ISO 9000 in the next few years. Most importantly, chain of custody and evidence handling procedures are crucial to successful use of forensic science in the courtroom. Most challenges to criminal cases in court are made based on broken chain of custody or inappropriate evidence handling issues.

12. So, where does all this forensic science occur? Why, at a Crime Laboratory, of course! The only real difference between our wildlife crime lab, and a 'typical' police crime laboratory is that our victim is an animal. And we must keep in mind that, every now and then, our suspect will turn out to be an animal also. For obvious reasons, it's important that we not confuse natural events with human violations of wildlife laws. Why do we need a Crime Laboratory for Animals? Prior to 1988, wildlife law enforcement officers at the federal and state levels were at a distinct disadvantage in taking their cases to a prosecutor because that had little or no access to wildlife-related forensics services. By providing wildlife-related crime laboratory services (analytical techniques and corresponding expert witness testimony) to these investigators, serious violations of wildlife laws can be successfully investigated and serious violators can be successfully prosecuted. The primary tasks of this laboratory are to examine, compare and/or identify the species-source of wildlife parts and products for law enforcement purposes; 2) to match suspect, victim (in our case, an animal) and crime scene together with physical evidence; and 3) to testify to our examination results in State and Federal Courts throughout the United States in support of Federal and State law enforcement investigations. So, where does all this forensic science occur? Why, at a Crime Laboratory, of course! The only real difference between our wildlife crime lab, and a 'typical' police crime laboratory is that our victim is an animal. And we must keep in mind that, every now and then, our suspect will turn out to be an animal also. For obvious reasons, it's important that we not confuse natural events with human violations of wildlife laws. Why do we need a Crime Laboratory for Animals? Prior to 1988, wildlife law enforcement officers at the federal and state levels were at a distinct disadvantage in taking their cases to a prosecutor because that had little or no access to wildlife-related forensics services. By providing wildlife-related crime laboratory services (analytical techniques and corresponding expert witness testimony) to these investigators, serious violations of wildlife laws can be successfully investigated and serious violators can be successfully prosecuted. The primary tasks of this laboratory are to examine, compare and/or identify the species-source of wildlife parts and products for law enforcement purposes; 2) to match suspect, victim (in our case, an animal) and crime scene together with physical evidence; and 3) to testify to our examination results in State and Federal Courts throughout the United States in support of Federal and State law enforcement investigations.

13. In performing this mission, we support the law enforcement efforts of our 200+ Special Agents and Wildlife Inspectors throughout the United States, all fifty State Fish & Game Commissions, and approximately 150 foreign countries who have signed the United Nation's CITES (Convention on International Trade in Endangered Species) Treaty. Any research that we conduct is specifically directed toward the species-source identification of wildlife parts and products. We do not receive any live animals at the laboratory, nor do we conduct any research regarding the habitat, behavior or management of wildlife. Primary scope is to aid in Dept. Interior cases-Park service, BIA, BLM, FWS, state cases sometimes, and fish or wildlife. Dogs, cats, horses, domestic animals-don’t do.In performing this mission, we support the law enforcement efforts of our 200+ Special Agents and Wildlife Inspectors throughout the United States, all fifty State Fish & Game Commissions, and approximately 150 foreign countries who have signed the United Nation's CITES (Convention on International Trade in Endangered Species) Treaty. Any research that we conduct is specifically directed toward the species-source identification of wildlife parts and products. We do not receive any live animals at the laboratory, nor do we conduct any research regarding the habitat, behavior or management of wildlife. Primary scope is to aid in Dept. Interior cases-Park service, BIA, BLM, FWS, state cases sometimes, and fish or wildlife. Dogs, cats, horses, domestic animals-don’t do.

15. Genetics Team

16. Lab Capabilities Forensic scientists vary from being generalists to specialists in specific fields. In our lab, tasks are divvied up by several areas of expertise. Genetics: primarily species identification of animal parts, caviar, crafted items; gender determination, also individualization and population assignment for a variety of species including wolves, black bear, elk, white-tailed deer, mule deer, moose, white sturgeon, eagle and other raptors. Morphology : species identification of crafted items, animal carcasses, bones, capes, skulls including mammals, reptiles, birds. I do fish. Chemistry: Species identification with Mass Spectrometry of hemoglobin, identification and quantification of pesticides in animal death cases, identification of species of plant (ginseng) and animals in asian medicinals, identification of species origin of gall bladders from bile analysis Pathology : Cause of death analyses Digital Data : Computer siezures, computer data analysis for search warrants, audio and video analysis, preparation of courtroom visuals Ballistics: identification of caliber, rifle source, bullet vs arrow point, also work with NIBIN = National Integrated Ballistic Information Network sponsored by Department of Justice Fingerprints: Latent print development, part of CJIS = Criminal Justice Information Systems sponsored by FBI Because, it turns out that an individual involved in a wildlife crime is likely to have a criminal record!Forensic scientists vary from being generalists to specialists in specific fields. In our lab, tasks are divvied up by several areas of expertise. Genetics: primarily species identification of animal parts, caviar, crafted items; gender determination, also individualization and population assignment for a variety of species including wolves, black bear, elk, white-tailed deer, mule deer, moose, white sturgeon, eagle and other raptors. Morphology : species identification of crafted items, animal carcasses, bones, capes, skulls including mammals, reptiles, birds. I do fish. Chemistry: Species identification with Mass Spectrometry of hemoglobin, identification and quantification of pesticides in animal death cases, identification of species of plant (ginseng) and animals in asian medicinals, identification of species origin of gall bladders from bile analysis Pathology : Cause of death analyses Digital Data : Computer siezures, computer data analysis for search warrants, audio and video analysis, preparation of courtroom visuals Ballistics: identification of caliber, rifle source, bullet vs arrow point, also work with NIBIN = National Integrated Ballistic Information Network sponsored by Department of Justice Fingerprints: Latent print development, part of CJIS = Criminal Justice Information Systems sponsored by FBI Because, it turns out that an individual involved in a wildlife crime is likely to have a criminal record!

17. The Morphology Section is involved in a number of different areas of investigation, primarily centered around species groups. The common thread is that the forensic scientists use observable physical characteristics to make their identifications. The mammologists look at carcasses, hides, capes, bones, hairs and recognizable body parts to make species identifications in comparison to known species standards. The species standards consist of bone collections, hide collections, hair collections, and mounted specimens. One of the most important identifications made by the mammologists was a method to discriminate the natural fibers in shatoosh shawls from other fibers in cashmere and synthetic shawls. Shatoosh shawls are made from the hairs of the endangered Tibetan antelope – which must be killed to obtain its hair. 3 antelopes are needed for each shawl – the last case that went to trial involved a group that was importing over 500 shawls in violation of CITES treaties, in addition to Tibetan wildlife laws. Reptiles – Our herpetologists deal mainly with illegal animals involved in live trade activities, products made from reptile skins and shells, clothing items, and the odd food items, such as this bottle of snake wine. Birds – Our ornithologist works with identification of bird species that appear in crafted items both as feathers and carvings, identification of sport-caught species for which feathers and carcasses are available, and identification of birds involved in oil pond violations. Oil ponds are common near active drilling operations and are supposed to be protected to prevent accidental capture of birds looking for water bodies. Violators are prosecuted particularly for deaths of migratory birds, raptors and songbirds. Ivory – Ivory is still a big item in the commercial trade, and the concern is that crafted items are originating from endangered species – elephants and marine mammals. In addition to determining if crafted items are coming from elephant tusk or rhino horn, our morphology group has developed a way to determine if ivory is from elephant or mammoth. Mammoth ivory is legal in trade and can be discriminated from elephant ivory by the angle of the “Schreager lines” that appear in SEM images.The Morphology Section is involved in a number of different areas of investigation, primarily centered around species groups. The common thread is that the forensic scientists use observable physical characteristics to make their identifications. The mammologists look at carcasses, hides, capes, bones, hairs and recognizable body parts to make species identifications in comparison to known species standards. The species standards consist of bone collections, hide collections, hair collections, and mounted specimens. One of the most important identifications made by the mammologists was a method to discriminate the natural fibers in shatoosh shawls from other fibers in cashmere and synthetic shawls. Shatoosh shawls are made from the hairs of the endangered Tibetan antelope – which must be killed to obtain its hair. 3 antelopes are needed for each shawl – the last case that went to trial involved a group that was importing over 500 shawls in violation of CITES treaties, in addition to Tibetan wildlife laws. Reptiles – Our herpetologists deal mainly with illegal animals involved in live trade activities, products made from reptile skins and shells, clothing items, and the odd food items, such as this bottle of snake wine. Birds – Our ornithologist works with identification of bird species that appear in crafted items both as feathers and carvings, identification of sport-caught species for which feathers and carcasses are available, and identification of birds involved in oil pond violations. Oil ponds are common near active drilling operations and are supposed to be protected to prevent accidental capture of birds looking for water bodies. Violators are prosecuted particularly for deaths of migratory birds, raptors and songbirds. Ivory – Ivory is still a big item in the commercial trade, and the concern is that crafted items are originating from endangered species – elephants and marine mammals. In addition to determining if crafted items are coming from elephant tusk or rhino horn, our morphology group has developed a way to determine if ivory is from elephant or mammoth. Mammoth ivory is legal in trade and can be discriminated from elephant ivory by the angle of the “Schreager lines” that appear in SEM images.

18. The Forensic Chemists divide their tasks by area of analysis rather than by species. They use chromatography to identify species of bear gall bladders that show up in trade. Species identity of components of Asian medicinals is a common investigation – medicinals that claim to have rhino and tiger bone are popular in Asian markets. Sometimes we see grooming products such as shampoos and lotions that claim to contain whale and caviar extracts. Identification of pesticides involved in animal poisonings is an important task of the chemistry group. Ranchers desperate to protect herds of sheep and cows will sometimes lace piles of bait with agricultural pesticides to get rid of “nuisance” species. Such “nuisance species” include coyotes, wolves, eagles and other animals that eat carrion. MALDI-TOF is a new technology that is being implemented in criminalistics to identify species according to hemoglobin profiles. It’s also being used for identification of the presence of heavy metals such as mercury and lead. And the use of mass spectrometry for analysis of fibers is a technology being developed – some questions we get involving illegal game hunting include whether or not rope fibers can be matched between a suspect and physical evidence.The Forensic Chemists divide their tasks by area of analysis rather than by species. They use chromatography to identify species of bear gall bladders that show up in trade. Species identity of components of Asian medicinals is a common investigation – medicinals that claim to have rhino and tiger bone are popular in Asian markets. Sometimes we see grooming products such as shampoos and lotions that claim to contain whale and caviar extracts. Identification of pesticides involved in animal poisonings is an important task of the chemistry group. Ranchers desperate to protect herds of sheep and cows will sometimes lace piles of bait with agricultural pesticides to get rid of “nuisance” species. Such “nuisance species” include coyotes, wolves, eagles and other animals that eat carrion. MALDI-TOF is a new technology that is being implemented in criminalistics to identify species according to hemoglobin profiles. It’s also being used for identification of the presence of heavy metals such as mercury and lead. And the use of mass spectrometry for analysis of fibers is a technology being developed – some questions we get involving illegal game hunting include whether or not rope fibers can be matched between a suspect and physical evidence.

19. We have two Veterinary Pathologists on staff who conduct necropsy analyses on animal carcasses that arrive at the lab. Their questions are primarily concerned with cause of death of just about any animal of legal concern, except for skunks. So they will look for evidence of vehicular death, intentional death by poisoning, arrow vs. bullet wounds which are important if a hunting season is regulated by type of firearm. They also look for evidence of electrocution, trap wounds, or effects of environmental pollution (oil ponds)We have two Veterinary Pathologists on staff who conduct necropsy analyses on animal carcasses that arrive at the lab. Their questions are primarily concerned with cause of death of just about any animal of legal concern, except for skunks. So they will look for evidence of vehicular death, intentional death by poisoning, arrow vs. bullet wounds which are important if a hunting season is regulated by type of firearm. They also look for evidence of electrocution, trap wounds, or effects of environmental pollution (oil ponds)

20. Digital forensics is becoming an increasingly important part of our suite of investigative capabilities with the increased use of computer technology to transmit messges and archive data. Our computer forensics team is involved with computer seizures and software analyses primarily to address the needs of investigative warrants. We also have audio and video analytical capabilities to use in analysis of video surveillance and audio tapes involved in covert operations. We also have graphics capabilities for preparation of courtroom displays.Digital forensics is becoming an increasingly important part of our suite of investigative capabilities with the increased use of computer technology to transmit messges and archive data. Our computer forensics team is involved with computer seizures and software analyses primarily to address the needs of investigative warrants. We also have audio and video analytical capabilities to use in analysis of video surveillance and audio tapes involved in covert operations. We also have graphics capabilities for preparation of courtroom displays.

21. Our fingerprinting and ballistics capabilities are probably the most traditional of all the analytical capabilites we have at the Lab. The fingerprinting analyses are used for covert operations and to link suspects to the physical evidence. Our lab is a cooperator in the CJIS = Criminal Justice Information Systems sponsored by FBI. Ballistics includes analysis of bullets, bullet fragments, and firearm comparisons. They are cooperators in the NIBIN = National Integrated Ballistic Information Network sponsored by Department of Justice Because, it turns out that an individual involved in a wildlife crime is likely to have a criminal record! Our fingerprinting and ballistics capabilities are probably the most traditional of all the analytical capabilites we have at the Lab. The fingerprinting analyses are used for covert operations and to link suspects to the physical evidence. Our lab is a cooperator in the CJIS = Criminal Justice Information Systems sponsored by FBI. Ballistics includes analysis of bullets, bullet fragments, and firearm comparisons. They are cooperators in the NIBIN = National Integrated Ballistic Information Network sponsored by Department of Justice Because, it turns out that an individual involved in a wildlife crime is likely to have a criminal record!

22. In the Genetics Section, in contrast to Morhpology, but similar to Chemistry, our activities are defined by the technology, rather than the species. DNA is present in all animals, so the method of extracting and analyzing DNA is similar as well. We use different techniques to answer different questions. For Species identification, we often use DNA sequencing of mitochondrial DNA loci. This is because mitochondria are abundant in cells and as such, there are more targets available from trace amounts of evidence, such as blood splatters and caviar eggs. The drawback is that mtDNA is maternally inherited, so we cannot detect hybrids readily. We also conduct tests for gender determination – this primarily targets questions about illegal hunting when seasons are segragated by gender.In the Genetics Section, in contrast to Morhpology, but similar to Chemistry, our activities are defined by the technology, rather than the species. DNA is present in all animals, so the method of extracting and analyzing DNA is similar as well. We use different techniques to answer different questions. For Species identification, we often use DNA sequencing of mitochondrial DNA loci. This is because mitochondria are abundant in cells and as such, there are more targets available from trace amounts of evidence, such as blood splatters and caviar eggs. The drawback is that mtDNA is maternally inherited, so we cannot detect hybrids readily. We also conduct tests for gender determination – this primarily targets questions about illegal hunting when seasons are segragated by gender.

23. Genetics is the only section that conducts individual matching on animal species. CLICK for Individual matching: For this we use nuclear DNA markers to detect the biparentally-inherited genetic material. Individual matching is a valuable technique in this respect to complete the forensic triangle that links the suspect to the victim to the crime scene. In this case, the meat in the suspect’s freezer came from the same animal as the body parts left at a campsite. Because we know the mode of inheritance and mutation rate of these multilocus markers, we can model how they are distributed among populations in our reference database. This allows us to use robust statistical analyses to determine whether or not a suspect or animal can be excluded from consideration in a comparison. It’s important to note, in these analyses, we are not looking to match individual items of physical evidence, but rather, in the spirit of the scientific method, we are trying to determine the probability of exclusion. The statistics in that way are interpreted as “given that 8 independent genetic markers are the same, the chance of these two items originated from two different individuals is 1 in more than 10,000.” CLICK for Population assignment: Using the same multilocus markers, we can conduct analyses to determine population of origin of most species for which we can determine individual identity. This includes Elk, Wolves, Eagles, White-tailed and Mule Deer, Bears, and White sturgeon. White sturgeon is our most challenging anlaysis that is currently under development because the question is whether we can match caviar, or single eggs, back to a known female sturgeon. The two main challenges are that eggs only have a single complete nuclear genome, which makes amplification of multilocus markers a challenge, and that the White sturgeon nuclear genome is tetraploid – some of the loci of interest are diploid, some are tetraploid, and some are octaploid, which makes interpretation a challenge! CLICK for Doina – But we do have to acknowledge one person in particular on our staff. Proper reference standards for species comparisons are absolutely critical to what we do. Doina, our standards technician, is in charge of obtaining appropriate reference material as well as maintenance of our tissue standards collection. Without Doina, our jobs would be infinitely harder to carry out.Genetics is the only section that conducts individual matching on animal species. CLICK for Individual matching: For this we use nuclear DNA markers to detect the biparentally-inherited genetic material. Individual matching is a valuable technique in this respect to complete the forensic triangle that links the suspect to the victim to the crime scene. In this case, the meat in the suspect’s freezer came from the same animal as the body parts left at a campsite. Because we know the mode of inheritance and mutation rate of these multilocus markers, we can model how they are distributed among populations in our reference database. This allows us to use robust statistical analyses to determine whether or not a suspect or animal can be excluded from consideration in a comparison. It’s important to note, in these analyses, we are not looking to match individual items of physical evidence, but rather, in the spirit of the scientific method, we are trying to determine the probability of exclusion. The statistics in that way are interpreted as “given that 8 independent genetic markers are the same, the chance of these two items originated from two different individuals is 1 in more than 10,000.” CLICK for Population assignment: Using the same multilocus markers, we can conduct analyses to determine population of origin of most species for which we can determine individual identity. This includes Elk, Wolves, Eagles, White-tailed and Mule Deer, Bears, and White sturgeon. White sturgeon is our most challenging anlaysis that is currently under development because the question is whether we can match caviar, or single eggs, back to a known female sturgeon. The two main challenges are that eggs only have a single complete nuclear genome, which makes amplification of multilocus markers a challenge, and that the White sturgeon nuclear genome is tetraploid – some of the loci of interest are diploid, some are tetraploid, and some are octaploid, which makes interpretation a challenge! CLICK for Doina – But we do have to acknowledge one person in particular on our staff. Proper reference standards for species comparisons are absolutely critical to what we do. Doina, our standards technician, is in charge of obtaining appropriate reference material as well as maintenance of our tissue standards collection. Without Doina, our jobs would be infinitely harder to carry out.

24. DNA in Forensic Science DNA analysis plays a unique role in forensic science DNA analysis and identification in itself is an exclusionary test. PID is the most common statistic used for individualization cases – this probability of identity is the estimate of the probability that two different individuals share the same observed genotype, and expressed as a number “1 in over #######” DNA analysis plays a unique role in forensic science DNA analysis and identification in itself is an exclusionary test. PID is the most common statistic used for individualization cases – this probability of identity is the estimate of the probability that two different individuals share the same observed genotype, and expressed as a number “1 in over #######”

25. DNA in Forensic Science What do we get the dna FROM?What do we get the dna FROM?

26. Wildlife trade on the Internet The Internet is growing at an amazing rate E-commerce is currently the fastest growing segment of the economy at between 300% and 700% per year. It’s the largest growth industry in the history of the world and criminals are also grabbing some of the market share. Criminals are using the Internet to find new and more efficient ways to commit crimes. Every form of commerce, including illegal, is using the internet, and that includes the wildlife trade.The Internet is growing at an amazing rate E-commerce is currently the fastest growing segment of the economy at between 300% and 700% per year. It’s the largest growth industry in the history of the world and criminals are also grabbing some of the market share. Criminals are using the Internet to find new and more efficient ways to commit crimes. Every form of commerce, including illegal, is using the internet, and that includes the wildlife trade.

27. Scope of the Trade In the past year, USFWS has worked on Internet-related cases involving: Tiger and leopard skins and skin products from the UK; Rhino horn from the UK; Elephant ivory products from Europe and Asia; Bird feather trade from Europe and South America; Live animals Horn, elephant ivory, bushmeat, handcrafts/crafted itemsHorn, elephant ivory, bushmeat, handcrafts/crafted items

28. Scale of the Trade Arowanas for Sale on a popular Fish trade Website The Red Arowana is arguably the most prized aquarium fish in the world. It is probably the most expensive fish in the world on the basis of the highest dollar amount ever paid for a single fish. Its price variables include size, condition of body parts and overall body shape, color, and wealth with motivation on the part of the buyer. A large Red Arowana will usually command tens of thousands of dollars and in some cases hundreds of thousands. Use genetics to identify species-some are okay to import, some aren’tThe Red Arowana is arguably the most prized aquarium fish in the world. It is probably the most expensive fish in the world on the basis of the highest dollar amount ever paid for a single fish. Its price variables include size, condition of body parts and overall body shape, color, and wealth with motivation on the part of the buyer. A large Red Arowana will usually command tens of thousands of dollars and in some cases hundreds of thousands. Use genetics to identify species-some are okay to import, some aren’t

30. The impact of bushmeat hunting in Africa has accelerated as a result of the direct and indirect impact of human population growth. Bushmeat is a generic term for any wildlife harvested, cooked, and sold commercially. There has been an increase in the appearance of bushmeat in North America, driven by the demand for ethnic foods by foreign nationals and immigrants. Can’t tell what it is, so genetics commonly used The forensic concerns are that endangered species are being exploited in the bushmeat trade. There are also disease issues surrounding rodent and primate meats. And, maybe more importantly, there is a concern in conservation circles about overexploitation of indigenous wildlife and endemic populations that are being indiscriminantly targeted in third world nations.The impact of bushmeat hunting in Africa has accelerated as a result of the direct and indirect impact of human population growth. Bushmeat is a generic term for any wildlife harvested, cooked, and sold commercially. There has been an increase in the appearance of bushmeat in North America, driven by the demand for ethnic foods by foreign nationals and immigrants. Can’t tell what it is, so genetics commonly used The forensic concerns are that endangered species are being exploited in the bushmeat trade. There are also disease issues surrounding rodent and primate meats. And, maybe more importantly, there is a concern in conservation circles about overexploitation of indigenous wildlife and endemic populations that are being indiscriminantly targeted in third world nations.

31. Some of you have seen bushmeat and others have not. Well it can be in pretty bad shape Sometimes it’s just hung out to dry….like gallbladders. A lot of it however is cooked and hence destroys or degrades the dnaSome of you have seen bushmeat and others have not. Well it can be in pretty bad shape Sometimes it’s just hung out to dry….like gallbladders. A lot of it however is cooked and hence destroys or degrades the dna

32. Bushmeat Species Identified Anas Asia Water Buffalo Cane Rat Badger Bay Duiker Burmese Python Cow Deer Goat Hartebeest Hystrix Mangabey Maxwell’s Duiker Nutria Pig/Wild Boar Rat Reticulated Python Serow Sheep Sequencing to identify species. Varies by sample, sometimes not a lot of samples available. D-loop, cyt-b, snakes and crocs-cyt b, 16SSequencing to identify species. Varies by sample, sometimes not a lot of samples available. D-loop, cyt-b, snakes and crocs-cyt b, 16S

33. With few exceptions, population abundance of most sturgeon species worldwide is at historically low levels. The top 3 caviar producers shown here are flirting with extinction in their native habitats. Sturgeon are large, long-lived fish which tend to mature late and spawn infrequently. Substantial declines in abundance over the last century cannot be counterbalanced by these species reproductive characteristics. Species status: Beluga (Huso Huso)- prohibited from commercial shipments Stellatus (sevruga –middle grade)-can be imported with proper paperwork Russian sturgeon (Gue…-lower grade)-can be imported with proper paperwork Siberian-prohibited from commercial shipments Russian sturgeon caviar-allowed to come into country, but some hybrid sturgeon with Siberian due to import to some areas for caviar production, so With few exceptions, population abundance of most sturgeon species worldwide is at historically low levels. The top 3 caviar producers shown here are flirting with extinction in their native habitats. Sturgeon are large, long-lived fish which tend to mature late and spawn infrequently. Substantial declines in abundance over the last century cannot be counterbalanced by these species reproductive characteristics. Species status: Beluga (Huso Huso)- prohibited from commercial shipments Stellatus (sevruga –middle grade)-can be imported with proper paperwork Russian sturgeon (Gue…-lower grade)-can be imported with proper paperwork Siberian-prohibited from commercial shipments Russian sturgeon caviar-allowed to come into country, but some hybrid sturgeon with Siberian due to import to some areas for caviar production, so

34. In order to address the myriad of issues surrounding the caviar industry, US and International cooperation is needed to stem illiegal trade, enforce management regulations set up by the ESA and CITES, and assist fishermen and the industry in efforts to develop sustainable fisheries and low-impact commercial aquacultural operations. From a law enforcement standpoint, we have the capability of determining the species identity of caviar from a single egg. We are currently developing the capability of determining individual identity and the ability to match individual eggs to a single female sturgeon. Our most difficult conservation problem is with hybrids at this time. We know that past escapement from aquacultural facilities in the Caspian Sea has resulted in a background rate of hybrid detection of Siberian sturgeon in Russian sturgeon of around 20%. It is the loss of species that certainly will do the most damage – the health of aquatic communities depends on the health of its animal components. The loss of one or more constituents leads to instability and a lack of resilience in the face of changing environments. We see the results in invasive species, dead zones, and incidence of disease. Escapement-deals with Russian sturgeon and Siberian sturgeonIn order to address the myriad of issues surrounding the caviar industry, US and International cooperation is needed to stem illiegal trade, enforce management regulations set up by the ESA and CITES, and assist fishermen and the industry in efforts to develop sustainable fisheries and low-impact commercial aquacultural operations. From a law enforcement standpoint, we have the capability of determining the species identity of caviar from a single egg. We are currently developing the capability of determining individual identity and the ability to match individual eggs to a single female sturgeon. Our most difficult conservation problem is with hybrids at this time. We know that past escapement from aquacultural facilities in the Caspian Sea has resulted in a background rate of hybrid detection of Siberian sturgeon in Russian sturgeon of around 20%. It is the loss of species that certainly will do the most damage – the health of aquatic communities depends on the health of its animal components. The loss of one or more constituents leads to instability and a lack of resilience in the face of changing environments. We see the results in invasive species, dead zones, and incidence of disease. Escapement-deals with Russian sturgeon and Siberian sturgeon

35. This map shows the location of the remainiing wild populations of commercially important sturgeons in Eurasia. Currently, ship sturgeon and beluga sturgeon are prohibited in international commerce. Populations of the others are dangerously low, due in combination to continued commercial exploitation, an intense black market, and degraded habitats. Russian and Siberian sturgeon hybridizing in the Caspian Sea. Export implications due to regulations prohibiting some sturgeon imports are the increased pressure on NA sturgeon : white, paddlefish, and Pallid and shovelnoseThis map shows the location of the remainiing wild populations of commercially important sturgeons in Eurasia. Currently, ship sturgeon and beluga sturgeon are prohibited in international commerce. Populations of the others are dangerously low, due in combination to continued commercial exploitation, an intense black market, and degraded habitats. Russian and Siberian sturgeon hybridizing in the Caspian Sea. Export implications due to regulations prohibiting some sturgeon imports are the increased pressure on NA sturgeon : white, paddlefish, and Pallid and shovelnose

36. In the wake of the decline of Eurasian sturgeon species, the attention of commercial and black market interests has shifted to North American sturgeon and paddlefish as replacements in this multi-million dollar industry. The market for caviar from Columbia River white sturgeon is said to be second only to that of Russian sturgeon. White sturgeon have become a popular replacement species in the caviar industry. On the black market, white sturgeon eggs can fetch over $100 per pound, and a mature female can carry up to 200 pounds in eggs alone. Between 1985 and 1990, poachers killed over 2000 adults white sturgeon to obtain 1.5 kilos of caviar, which was turned around and sold to “undiscriminating palates” as Beluga caviar – not even as white sturgeon caviar! The distributor was a well-known and respected member of the caviar industry. Last year, a Russian mother and son team was sentenced with jail time and monetary fines for purchasing sturgeon from fishermen on the Sacramento River and processing it into caviar which was then sold on the black market in Vancouver, WA and Portland, OR. This, all in violation of the Lacey Act which prohibits interstate transport of illegally obtained wildlife. Genetics question: are sturgeon eggs coming from Sacramento River or Columbia River…which state could recoup fines based on origin of sturgeon, feds involved due to Lacey Act. Caviar being sold in California. D-loop and Cyt b for population assessment, using STRs now but not as good for population assignment yet. In the wake of the decline of Eurasian sturgeon species, the attention of commercial and black market interests has shifted to North American sturgeon and paddlefish as replacements in this multi-million dollar industry. The market for caviar from Columbia River white sturgeon is said to be second only to that of Russian sturgeon. White sturgeon have become a popular replacement species in the caviar industry. On the black market, white sturgeon eggs can fetch over $100 per pound, and a mature female can carry up to 200 pounds in eggs alone. Between 1985 and 1990, poachers killed over 2000 adults white sturgeon to obtain 1.5 kilos of caviar, which was turned around and sold to “undiscriminating palates” as Beluga caviar – not even as white sturgeon caviar! The distributor was a well-known and respected member of the caviar industry. Last year, a Russian mother and son team was sentenced with jail time and monetary fines for purchasing sturgeon from fishermen on the Sacramento River and processing it into caviar which was then sold on the black market in Vancouver, WA and Portland, OR. This, all in violation of the Lacey Act which prohibits interstate transport of illegally obtained wildlife. Genetics question: are sturgeon eggs coming from Sacramento River or Columbia River…which state could recoup fines based on origin of sturgeon, feds involved due to Lacey Act. Caviar being sold in California. D-loop and Cyt b for population assessment, using STRs now but not as good for population assignment yet.

37. SO lastly, I’d like to talk about our developing work with individual matching of eagles and other raptors. This stems from a case we received in 2002 in response to a major animal theft at the Santa Barbara, CA Zoo. Two eagles, one golden and one bald eagle, were stolen from their zoo cage and presumed killed. Investigative work uncovered a black market ring that was dealing in eagle feathers. None of the principle suspects was a card-carrying Native American. There are specific rules regarding possession of eagle feathers, and they are only to be used for ceremonial purposes. SO lastly, I’d like to talk about our developing work with individual matching of eagles and other raptors. This stems from a case we received in 2002 in response to a major animal theft at the Santa Barbara, CA Zoo. Two eagles, one golden and one bald eagle, were stolen from their zoo cage and presumed killed. Investigative work uncovered a black market ring that was dealing in eagle feathers. None of the principle suspects was a card-carrying Native American. There are specific rules regarding possession of eagle feathers, and they are only to be used for ceremonial purposes.

38. Once the suspects home was searched and evidence was seized, investigators found hundreds of crafted items, mostly Native American prayer sticks and ceremonial pieces, as well as bags and bags of loose feathers. The main question was whether any of the eagle feathers in the collection came from either of the Santa Barbara Zoo animals. The complicating factors were that the only reference material the Zoo had in its possession was an immunological slide from the Golden eagle, and a single shed feather from the eagle cage!Once the suspects home was searched and evidence was seized, investigators found hundreds of crafted items, mostly Native American prayer sticks and ceremonial pieces, as well as bags and bags of loose feathers. The main question was whether any of the eagle feathers in the collection came from either of the Santa Barbara Zoo animals. The complicating factors were that the only reference material the Zoo had in its possession was an immunological slide from the Golden eagle, and a single shed feather from the eagle cage!

39. So we sampled a collection of the feathers that our Ornithologist identified as either Bald or Golden eagle feathers. It’s actually not hard to extract DNA from flight and blood feathers because of the amount of tissue that sticks to the feather shaft. Shed feathers and downy feathers were another story. The immunological slide wasn’t very hopeful either. So we chose to look at multi-locus nuclear DNA markers (microsatellites), some of which could discriminate bald and golden eagle based on single allele differences. That gave us the ability to segregate individuals feathers by species. Of the 20 markers tested, 4 turned out to be valuable for discrimination at the individual level. One of those is shown here: CLICK –highlights Feather 8, 10, 5 Click – Feather 18, 12 CLICK – Feather 28, 4 In the end, we were not able to conclusively determine that any of the feathers matched the cage feather or the immunological slide. We were, however able to conclusively say that the feathers tested for DNA matching came from not 2, but at least 16 different individual bald or golden eagles. The suspects were convicted of illegal possession of the feathers of protected birds, and they were able to increase the counts and subsequent fines and jail time by the number of different birds detected in DNA analysis. Used STRs. Couldn’t prove if birds were from the zoo, because didn’t have reference samples from zoo for those individuals.So we sampled a collection of the feathers that our Ornithologist identified as either Bald or Golden eagle feathers. It’s actually not hard to extract DNA from flight and blood feathers because of the amount of tissue that sticks to the feather shaft. Shed feathers and downy feathers were another story. The immunological slide wasn’t very hopeful either. So we chose to look at multi-locus nuclear DNA markers (microsatellites), some of which could discriminate bald and golden eagle based on single allele differences. That gave us the ability to segregate individuals feathers by species. Of the 20 markers tested, 4 turned out to be valuable for discrimination at the individual level. One of those is shown here: CLICK –highlights Feather 8, 10, 5 Click – Feather 18, 12 CLICK – Feather 28, 4 In the end, we were not able to conclusively determine that any of the feathers matched the cage feather or the immunological slide. We were, however able to conclusively say that the feathers tested for DNA matching came from not 2, but at least 16 different individual bald or golden eagles. The suspects were convicted of illegal possession of the feathers of protected birds, and they were able to increase the counts and subsequent fines and jail time by the number of different birds detected in DNA analysis. Used STRs. Couldn’t prove if birds were from the zoo, because didn’t have reference samples from zoo for those individuals.

40. So, what do we see in the future? Certainly an increase in work as more and more of our analyses are admitted into court and more violators are successfully prosecuted. Currently we’re working on ways to increase the successful typing of individual caviar eggs to address the issue of illegal take of White sturgeon and other North American sturgeons. We will also use this technique to work with typing caviar claimed to be of aquacultural or hybrid origin. We have an upcoming courtroom challenge in the next month in which our analysts will participate in a “Daubert-Frye” hearing to determine the scientific worthiness of DNA typing of Elk populations in courtroom testimony. We are also getting more involved in a technique that is becoming more popular with folks working on genome typing and whole genome sequencing – that is the use of “single nucleotide polymorphisms” or “SNPs” And finally, other emerging issues such as coral reef investigations, and addressing typing of shark fins and other endangered species issues. Forensics component is more complex than just CSI. Keep the lab in mind.So, what do we see in the future? Certainly an increase in work as more and more of our analyses are admitted into court and more violators are successfully prosecuted. Currently we’re working on ways to increase the successful typing of individual caviar eggs to address the issue of illegal take of White sturgeon and other North American sturgeons. We will also use this technique to work with typing caviar claimed to be of aquacultural or hybrid origin. We have an upcoming courtroom challenge in the next month in which our analysts will participate in a “Daubert-Frye” hearing to determine the scientific worthiness of DNA typing of Elk populations in courtroom testimony. We are also getting more involved in a technique that is becoming more popular with folks working on genome typing and whole genome sequencing – that is the use of “single nucleotide polymorphisms” or “SNPs” And finally, other emerging issues such as coral reef investigations, and addressing typing of shark fins and other endangered species issues. Forensics component is more complex than just CSI. Keep the lab in mind.