The Tn7 transposon is a  mobile genetic element  found in many prokaryotes such as  Escherichia coli  ( E. coli ), and was first discovered as a DNA sequence in bacterial chromosomes and naturally occurring  plasmids  that encoded resistance to the antibiotics  trimethoprim  and  streptomycin . [ 19 ] [ 20 ]  Specifically classified as a  transposable element  (transposon), the sequence can duplicate and move itself within a genome by utilizing a self-encoded recombinase enzyme called a transposase, resulting in effects such as creating or reversing mutations and changing genome size. The Tn7 transposon has developed two mechanisms to promote its propagation among prokaryotes. [ 21 ]  Like many other bacterial transposons, Tn7 transposes at low-frequency and inserts into many different sites with little to no site-selectivity. Through this first pathway, Tn7 is preferentially directed into conjugable  plasmids , which can be replicated and distributed between bacteria. However, Tn7 is unique in that it also transposes at high-frequency into a single specific site in bacterial chromosomes called attTn7. [ 22 ]  This specific sequence is an essential and highly conserved gene found in many strains of bacteria. However, the recombination is not deleterious to the host bacterium as Tn7 actually transposes downstream of the gene after recognizing it, resulting in a safe way to propagate the transposon without killing the host. This highly evolved and sophisticated target-site selection pathway suggests this pathway evolved to promote coexistence between the transposon and it host, as well as Tn7's successful transmission into future generations of bacterium. [ 21 ]


The Tn7 transposon is 14 kb long and codes for five enzymes. [ 21 ]  The ends of the DNA sequence consists of two segments that the Tn7 transposase interacts with during recombination. The left segment (Tn7-L) is 150 bp long and the right sequence (Tn7-R) is 90 bp long. Both ends of the transposon contain a series of 22 bp binding sites that the Tn7 transposase recognizes and binds to. Within the transposon are five discrete genes encoding for proteins that make up the transposition machinery. In addition, the transposon contains an  integron , a DNA segment containing several  cassettes  of genes encoding for antibiotic-resistance. [ 21 ]


The Tn7 transposon codes for five proteins: TnsA, TnsB, TnsC, TnsD, and TnsE. [ 21 ]  TnsA and TnsB interact together to form the Tn7 transposase enzyme TnsAB. The enzyme specifically recognizes and binds to the ends of the DNA sequence of the transposon, and excises it by introducing double-stranded DNA breaks to each end. The excised sequence is then inserted to another target DNA site. Much like other characterized transposons, the mechanism for Tn7 transposition involves cleavage of the 3' ends from the donating DNA by the TnsA protein of the TnsAB transposase. However, Tn7 is also uniquely cleaved near the 5' ends, about 5 bp from the 5' end towards the Tn7 transposon, by the TnsB protein of TnsAB. After the insertion of the transposon into the target DNA site, the 3' ends are covalently linked to the target DNA, but the 5 bp gaps are still present at the 5' ends. As a result, repair of these gaps leads to a further 5 bp duplication at the target site. The TnsC protein interacts with the transposase enzyme and the target DNA to promote the excision and insertion processes. The ability of TnsC to activate the transposase depends on its interaction with a target DNA along with its appropriate targeting protein, TnsD or TnsE. The TnsD and TnsE proteins are alternative target selectors that are also DNA binding  activators  that promote excision and insertion of Tn7.  Their ability to interact with a particular target DNA is key to the target-site selection of Tn7. The proteins TnsA, TnsB, and TnsC thus form the core machinery of Tn7: TnsA and TnsB interact together to form the transposase, while TnsC functions as a  regulator  of the transposase's activity, communicating between the transposase and TnsD and TnsE.  When the TnsE protein interacts with the TnsABC core machinery, Tn7 preferentially directs insertions into conjugable plasmids. When the TnsD protein interacts with TnsABC, Tn7 preferentially directs insertions downstream into a single essential and highly conserved site in the bacterial chromosome. This site, attTn7, is specifically recognized by TnsD. [ 21 ]


Transposon mutagenesis , or  transposition mutagenesis , is a  biological  process that allows genes to be transferred to a host organism's  chromosome , interrupting or modifying the function of an  extant   gene  on the chromosome and causing  mutation . [ 1 ]  Transposon mutagenesis is much more effective than  chemical mutagenesis , with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate  selectable markers  in strain construction, and being able to recover genes after mutagenesis. [ 2 ]  Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.


Transposon mutagenesis was first studied by  Barbara McClintock  in the mid-20th century during her Nobel Prize-winning work with corn. McClintock received her BSc in 1923 from Cornell’s College of Agriculture. By 1927 she had her PhD in botany, and she immediately began working on the topic of  maize  chromosomes. [ 3 ]  In the early 1940s, McClintock was studying the  progeny  of self-pollinated maize plants which resulted from  crosses  having a broken chromosome 9. These plants were missing their  telomeres . This research prompted the first discovery of a  transposable element , [ 4 ]  and from there transposon mutagenesis has been exploited as a biological tool.


In the case of  bacteria , transposition mutagenesis is usually accomplished by way of a  plasmid  from which a  transposon  is extracted and inserted into the host chromosome. This usually requires a set of  enzymes  including  transposase  to be  translated . The transposase can be expressed either on a separate plasmid, or on the plasmid containing the gene to be integrated. Alternatively, an injection of transposase  mRNA  into the host cell can induce translation and  expression . [ 5 ]  Early transposon mutagenesis experiments relied on  bacteriophages  and conjugative bacterial plasmids for the insertion of sequences. These were very non-specific, and made it difficult to incorporate specific genes. A newer technique called shuttle mutagenesis uses specific cloned genes from the host species to incorporate genetic elements. [ 2 ]  Another effective approach is to deliver transposons through viral  capsids . This facilitates integration into the chromosome and long-term  transgene  expression. [ 5 ]


The Tn5 transposon system is a model system for the study of transposition and for the application of transposon mutagenesis. Tn5 is a bacterial composite transposon in which genes (the original system containing  antibiotic resistance  genes) are flanked by two nearly identical  insertion sequences , named IS50R and IS50L corresponding to the right and left sides of the transposon respectively. [ 6 ]  The IS50R sequence codes for two proteins, Tnp and Inh. These two proteins are identical in sequence, save for the fact that Inh is lacking the 55  N-terminal   amino acids . Tnp codes for a transposase for the entire system, and Inh encodes an  inhibitor  of transposase. The DNA-binding  domain  of Tnp resides in the 55 N-terminal amino acids, and so these residues are essential for function. [ 6 ]  The IS50R and IS50L sequences are both flanked by 19- base pair  elements on the inside and outside ends of the transposon, labelled IE and OE respectively. Mutation of these regions results in an inability of transposase genes to bind to the sequences. The binding interactions between transposase and these sequences is very complicated, and is affected by  DNA methylation  and other  epigenetic  marks. [ 6 ]  In addition, other proteins seem to be able to bind with and affect the transposition of the IS50 elements, such as DnaA.


The most likely pathway of Tn 5 transposition is the common pathway for all transposon systems. It begins with Tnp binding the OE and IE sequences of each IS50 sequence. The two ends are brought together, and through  oligomerization  of DNA, the sequence is cut out of the chromosome. After introducing 9-base pair  5' ends  in target DNA, the transposon and its incorporated genes are inserted into the target DNA, duplicating the regions on either end of the transposon. [ 6 ]  Genes of interest can be  genetically engineered  into the transposon system between the IS50 sequences. By placing the transposon under the control of a host  promoter , the genes will be expressed. Incorporated genes usually include, in addition to the gene of interest, a  selectable marker  to identify transformants, a  eukaryotic  promoter/ terminator  (if expressing in a eukaryote), and 3'  UTR  sequences to separate genes in a  polycistronic  stretch of sequence.


The  Sleeping Beauty transposon system  (SBTS) is the first successful non-viral  vector  for incorporation of a  gene cassette  into a  vertebrate  genome. Up until the development of this system, the major problems with non-viral gene therapy have been the intracellular breakdown of the transgene due to it being recognized as  Prokaryotes  and the inefficient delivery of the transgene into organ systems. The SBTS revolutionized these issues by combining the advantages of viruses and naked DNA. It consists of a transposon containing the cassette of genes to be expressed, as well as its own transposase enzyme. By transposing the cassette directly into the genome of the organism from the plasmid, sustained expression of the transgene can be attained. [ 2 ]  This can be further refined by enhancing the transposon sequences and the transposase enzymes used. SB100X is a hyperactive mammalian transposase which is roughly 100x more efficient than the typical first-generation transposase. Incorporation of this enzyme into the cassette results in even more sustained transgene expression (over one year). Additionally, transgenesis frequencies can be as high as 45% when using  pronuclear injection  into mouse  zygotes . [ 7 ]


The mechanism of the SBTS is similar to the Tn5 transposon system, however the enzyme and gene sequences are eukaryotic in nature as opposed to prokaryotic. The system's tranposase can act in  trans  as well as in  cis , allowing a diverse collection of transposon structures. The transposon itself is flanked by  inverted repeat sequences , which are each repeated twice in a direct fashion, designated IR/DR sequences. The internal region consists of the gene or sequence to be transposed, and could also contain the transposase gene. Alternatively, the transposase can be encoded on a separate plasmid or injected in its protein form. Yet another approach is to incorporate both the transposon and the transposase genes into a viral vector, which can target a cell or tissue of choice. The transposase protein is extremely specific in the sequences that it binds, and is able to discern its IR/DR sequences from a similar sequence by three base pairs. Once the enzyme is bound to both ends of the transposon, the IR/DR sequences are brought together and held by the transposase in a Synaptic Complex Formation (SCF). The formation of the SCF is a checkpoint ensuring proper cleavage.  HMGB1  is a non- histone  protein from the host which is associated with eukaryotic chromatin. It enhances the preferential binding of the transposase to the IR/DR sequences and is likely essential for SCF complex formation/stability. Transposase cleaves the DNA at the target sites, generating  3' overhangs . The enzyme then targets TA  dinucleotides  in the host genome as target sites for integration. The same enzymatic catalytic site which cleaved the DNA is responsible for integrating the DNA into the genome, duplicating the region of the genome in the process. Although transposase is specific for TA dinucleotides, the high frequency of these pairs in the genome indicates that the transposon undergoes fairly random integration. [ 8 ]


As a result of the capacity of transposon mutagenesis to incorporate genes into most areas of target chromosomes, there are a number of functions associated with the process.


In 1999, the virulence genes associated with  Mycobacterium tuberculosis  were identified through transposon mutagenesis-mediated  gene knockout . A plasmid named pCG113 containing  kanamycin  resistance genes and the IS 1096  insertion sequence was engineered to contain variable 80-base pair tags. The plasmids were then transformed into  M. tuberculosis  cells by  electroporation . Colonies were plated on kanamycin to select for resistant cells. Colonies that underwent random transposition events were identified by  Bam HI   digestion  and  Southern blotting  using an internal IS 1096  DNA probe. Colonies were screened for  attenuated  multiplication to identify colonies with mutations in candidate virulence genes. Mutations leading to an attenuated phenotype were  mapped  by  amplification  of adjacent regions to the IS 1096  sequences and compared with the published  M. tuberculosis  genome. In this instance transposon mutagenesis identified 13  pathogenic   loci  in the  M. tuberculosis  genome which were not previously associated with disease. [ 10 ]  This is essential information in understanding the infectious cycle of the bacterium.


The  PiggyBac  (PB) transposon from the  cabbage looper moth  Trichoplusia   ni  was engineered to be highly active in mammalian cells, and is capable of genome-wide mutagenesis. Transposons contained both  PB  and  Sleeping Beauty  inverted repeats, in order to be recognized by both transposases and increase the frequency of transposition. In addition, the transposon contained promoter and  enhancer  elements, a  splice donor and acceptors  to allow gain- or loss-of-function mutations depending on the transposon's orientation, and bidirectional  polyadenylation  signals. The transposons were transformed into mouse cells  in vitro  and mutants containing tumours were analyzed. The mechanism of the mutation leading to tumour formation determined if the gene was classified as an oncogene or a tumour-suppressor gene. Oncogenes tended to be characterized by  insertions  in regions leading to overexpression of a gene, whereas tumour-suppressor genes were classified as such based on loss-of-function mutations. Since the mouse is a model organism for the study of human physiology and disease, this research will help lead to an increased understanding of cancer-causing genes and potential therapeutic targets. [ 9 ]


Transposon insertion sequencing  ( Tn-seq ) combines  transposon insertional mutagenesis  with  massively parallel sequencing  (MPS) of the transposon insertion sites to identify genes contributing to a function of interest in  bacteria . The method was originally established by concurrent work in four laboratories under the acronyms HITS, [ 1 ]  INSeq, [ 2 ]  TraDIS, [ 3 ]  and Tn-Seq. [ 4 ]  Numerous variations have been subsequently developed and applied to diverse biological systems. Collectively, the methods are often termed Tn-Seq as they all involve monitoring the fitness of transposon insertion mutants via DNA sequencing approaches. [ 5 ]


Transposons are highly regulated, discrete  DNA  segments that can relocate within the genome. They are universal and are found in  Eubacteria ,  Archaea , and  Eukarya , including humans. Transposons have a large influence on  gene expression  and can be used to determine gene function.  In fact, when a transposon inserts itself in a gene, the gene's function will be disrupted. [ 6 ]  Because of that property, transposons have been manipulated for use in insertional mutagenesis. [ 7 ]  The development of microbial genome sequencing was a major advance for the use of transposon mutagenesis. [ 8 ] [ 9 ]  The function affected by a transposon insertion could be linked to the disrupted gene by sequencing the genome to locate the transposon insertion site. Massively parallel sequencing allows simultaneous sequencing of transposon insertion sites in large mixtures of different mutants.  Therefore, genome-wide analysis is feasible if transposons are positioned throughout the genome in a mutant collection. [ 5 ]


Transposon sequencing requires the creation of a transposon insertion library, which will contain a group of mutants that collectively have transposon insertions in all non-essential genes.  The library is grown under an experimental condition of interest.  Mutants with transposons inserted in genes required for growth under the test condition will diminish in frequency from the population. To identify mutants being lost, genomic sequences adjacent to the transposon ends are amplified by  PCR  and sequenced by MPS to determine the location and abundance of each insertion mutation.  The importance of each gene for growth under the test condition is determined by comparing the abundance of each mutant before and after growth under the condition being examined. Tn-seq is useful for both the study of a single gene's fitness as well as gene interactions  [ 10 ]


Signature–tagged mutagenesis (STM) is an older technique that also involves pooling transposon insertion mutants to determine the importance of the disrupted genes under selective growth conditions. [ 11 ]  High-throughput versions of STM use genomic microarrays, which are less accurate and have a lower dynamic range than massively-parallel sequencing. [ 5 ]  With the invention of next generation sequencing, genomic data became increasingly available.  However, despite the increase in genomic data, our knowledge of gene function remains the limiting factor in our understanding of the role genes play. [ 12 ] [ 13 ]  Therefore, a need for a high throughput approach to study genotype–phenotype relationships like Tn-seq was necessary.


Transposon sequencing begins by transducing [ clarification needed ]  bacterial populations with transposable elements [ clarification needed ]  using  bacteriophages . Tn-seq [ clarification needed ]  uses the Himar I Mariner transposon, a common and stable [ clarification needed ]  transposon.   After transduction, the DNA is cleaved [ clarification needed ]  and the inserted sequence amplified through  PCR .  The recognition sites [ clarification needed ]  for MmeI, a type IIS restriction endonuclease [ clarification needed ] , can be introduced by a single nucleotide change in the terminal repeats [ clarification needed ]  of Mariner [ clarification needed ] . [ 14 ]  It [ clarification needed ]  is located 4 base pairs before the end of the terminal repeat.


MmeI makes a 2 base pair staggered cut [ clarification needed ]  20 bases downstream [ clarification needed ]  of the recognition site [ clarification needed ] . [ 15 ]


When MmeI digests DNA from a library [ clarification needed ]  of transposon insertion mutants [ clarification needed ] , fragmented DNA including the left and right transposon and 16 base pair of surrounding genomic DNA is produced. The 16 base pair fragment is enough to determine the location of the transposon insertion in the bacterial genome.  The ligation [ clarification needed ]  of the adaptor [ clarification needed ]  is facilitated by the 2 base overhang [ clarification needed ] . A primer [ clarification needed ]  specific to the adaptor and a primer specific to the transposon are used to amplify the sequence via PCR. The 120 base pair product [ clarification needed ]  is then isolated using agarose gel [ clarification needed ]  or PAGE [ clarification needed ]  purification.  Massively parallel sequencing  is then used to determine the sequences of the flanking 16 base pairs [ clarification needed ] . [ 10 ]


Gene function is inferred after looking at the effects of the insertion on gene function under certain conditions [ clarification needed ] .


Unlike high-throughput insertion track by deep sequencing (HITS) and transposon-directed insertion site sequencing (TraDIS) [ clarification needed ] , Tn-seq is specific to the  Himar I Mariner transposon,  and cannot be applied to other transposons or insertional elements. [ 10 ]  However, the protocol for Tn-seq [ clarification needed ]  is less time intensive [ citation needed ] . HITS and TraDIS [ clarification needed ]  use a DNA shearing [ clarification needed ]  technique that produce a range of  PCR  product sizes that could cause shorter DNA templates being preferentially amplified over longer templates. Tn-seq produces a product that is uniform in size, therefore reducing the possibility of PCR bias. [ 10 ]


Tn-seq can be used to identify both the fitness of single genes and to map gene interactions in microorganisms. Existing methods for these types of study are dependent on preexisting genomic microarrays or gene knockout arrays, whereas Tn-seq is not. Tn-seq's utilization of  massively parallel sequencing  makes this technique easily reproducible, sensitive, and robust. [ 10 ] [ clarification needed ]


Tn-seq has proven to be a useful technique for identifying new gene functions. [ clarification needed ]  The highly sensitive nature of Tn-seq [ citation needed ]  can be used to determine phenotype-genotype relationships that may have been deemed insignificant by less sensitive methods. Tn-seq identified essential genes and pathways that are important for the utilization of cholesterol in  Mycobacterium tuberculosis . [ 16 ]


Tn-seq has been used to study higher order genome organization using gene interactions. [ citation needed ]  Genes function as a highly linked network [ citation needed ] . Therefore, in order to study a gene's impact on  phenotype , gene interactions must also be considered [ citation needed ] . These gene networks can be studied by screening for synthetic lethality and gene interactions where a double mutant shows an unexpected fitness value compared to each individual mutant [ clarification needed ] [ citation needed ] .  Tn-seq was used to determine genetic interactions between five query genes and the rest of the genome in Streptococcus pneumoniae, which revealed both aggravating and alleviating genetic interactions. [ 4 ] [ clarification needed ] [ 10 ]


Tn-seq used in combination with  RNA-seq  can be utilized to examine the role of  non-coding DNA  regions. [ 17 ]


Transposons  are semi- parasitic   DNA  sequences which can replicate and spread through the host's  genome . They can be harnessed as a  genetic  tool for analysis of  gene  and  protein  function. The use of transposons is well-developed in  Drosophila  (in which  P elements  are most commonly used) and in Thale cress ( Arabidopsis thaliana ) and bacteria such as  Escherichia coli  ( E. coli  ). [ 1 ] [ 2 ]


Currently  transposons  can be used in genetic research and recombinant  genetic engineering  for  insertional mutagenesis . Insertional mutagenesis is when transposons function as  vectors  to help remove and integrate genetic sequences. Given their relatively simple design and inherent ability to move DNA sequences, transposons are highly compatible at transducing genetic material, making them ideal genetic tools.


Signature-tagging mutagenesis  (also known as STM) is a technique focused on using transposable element insertion to determine the phenotype of a locus in an organism's genome. While genetic sequencing techniques can determine the genotype of a genome, they cannot determine the function or phenotypic expression of gene sequences. [ 3 ] [ 4 ]  STM can bypass this issue by mutating a locus, causing it form a new phenotype; by comparing the observed phenotypic expressions of the mutated and unaltered locus, one can deduce the phenotypic expression of the locus.


In STM, specially tagged transposons are inserted into an organism, such as a bacterium, and randomly integrated into the host genome. In theory, the modified mutant organism should express the altered gene, thus altering the phenotype. If a new phenotype is observed, the genome is sequenced and searched for tagged transposons. [ 3 ]  If the site of transposon integration is found, then the locus may be responsible for expressing the phenotypes. [ 5 ] [ 6 ]


There have been many studies conducted transposon based STM, most notably with the  P elements [ 4 ]  in  Drosophila .  P elements  are transposons originally described in  Drosophila melanogaster  genome capable of being artificially synthesized or spread to other  Drosophila  species through horizontal transfer. [ 4 ]  In experimental trials, artificially created P elements and transposase genes are inserted into the genomes of  Drosophila  embryos. Subsequently, embryos that exhibit mutations have their genomes sequenced and compared, thus revealing the loci that have been affected by insertion and the roles of the loci., [ 4 ] [ 6 ]


Insertional inactivation  focuses on suppressing the expression of a gene by disrupting its sequence with an insertion. When additional nucleotides are inserted near or into a locus, the locus can suffer a  frameshift mutation  that could prevent it from being properly expressed into  polypeptide chain . Transposon-based Insertional inactivation is considered for medical research from suppression of  antibiotic resistance  in bacteria to the treatment of  genetic diseases . [ 7 ]  In the treatment of genetic diseases, the insertion of a transposon into deleterious gene locus of organism's genome would misalign the locus sequence, truncating any harmful proteins formed and rendering them non-functional. Alternatively insertional inactivation could be used to suppress genes that express antibiotic-resistance in bacteria., [ 7 ] [ 8 ]


While transposons have been used successfully in plants and invertebrate subjects through insertional mutagenesis and insertional activation, the usage of transposons in vertebrates has been limited due to a lack of transposons specific to vertebrates. Nearly all transposons compatible to and present within vertebrate genomes are inactive and are often relegated to "junk" DNA. [ 6 ]  However it is possible to identify dormant transposons and artificially recreate them as active agents. [ 6 ]  Genetic researchers Zsuzsanna Izsvák and Zoltán Ivics discovered a fish transposon sequence that, despite being dormant for 15 million years, could be resurrected as a vector for introducing foreign genes into the vertebrate genomes, including those of humans. This transposon, called  Sleeping Beauty  was described in 1997, and could be artificially reactivated into a functioning transposon. [ 6 ]


Sleeping Beauty can also be viable in gene therapy procedures by helping introduce beneficial transgenes into host genomes. Belcher et al. tested this notion by using Sleeping Beauty transposons to help insert sequences into mice with sickle cell anemia so they can produce the enzymes need to counteract their anemia. [ 6 ]  Belcher et al. began their experiment by constructing a genetic sequence consisting of the Hmox-1 transposable element and transposase from Sleeping Beauty. This sequence was then added inserted into a plasmid and introduced into the cells of the mice. The transposase from Sleeping Beauty helped insert the Hmox-1 transposon into the mice genome, allowing the production of enzyme heme oxygenase-1 (HO-1). The mice that receive the insertion showed a fivefold increase in the expression of HO-1, which in turn reduced blood vessel blockage from sickle-cell anemia. The publication of the experiment in 2010 showed that transposons can be useful in gene therapy. [ 6 ]


Naturally occurring  P elements  contain:


Transposase is an enzyme which regulates and catalyzes the excision of a P element from the host DNA, cutting at two recognition sites, and then reinserts the P element randomly. It is the random-insertion process, that can interfere with existing genes, or carry an additional gene, that can be used as a process for genetic research.


To use this process as a useful and controllable genetic tool, the two parts of the P element must be separated to prevent uncontrolled transposition. The normal genetic tools are therefore:


P Plasmids always contain:


and may contain:


(Forward genetics methods)
There are two main ways to utilise these tools: Fly Transformation, and Insertional Mutagenesis, each described below.


(hoping for insertion in non-coding regions)


It is important to note that the inserted gene may have damaged the function of one of the host's genes. Several lines of flies are required so comparison can take place and ensure that no additional genes have been knocked out.


(hoping for insertion in coding region)


Possible mutations:


The hijack of an enhancer from another gene allows the analysis of the function of that enhancer. This, especially if the reporter gene is for a fluorescent protein, can be used to help map expression of the mutated gene through the organism, and is a very powerful tool.


(Reverse genetics method)


If there is an old P element near the gene of interest (with a broken transposase) you can remobilise by microinjection of the embryo with coding for transposase or transposase itself. The P element will often transpose within a few kilobases of the original location, hopefully affecting your gene of interest as for 'Insertional Mutagenisis'.


Once the function of the mutated protein has been determined it is possible to sequence/purify/clone the regions flanking the insertion by the following methods:


The process of cutting, self ligation and re cutting allows the amplification of the flanking regions of DNA without knowing the sequence. The point at which the ligation occurred can be seen by identifying the cut site of [enzyme 1].


7. The gene can be cloned for further analysis.


The genomes of other organisms can be analysed in a similar way, although with different transposable elements. The recent discovery of the ' mariner transposon ' (from the reconstruction of the original sequence from many 'dead' versions in the human genome) has allowed many new experiments, mariner has well conserved homologues across a wide range of species and is a very versatile tool.


This article incorporates material from the  Citizendium  article " Transposons as a genetic tool ", which is licensed under the  Creative Commons Attribution-ShareAlike 3.0 Unported License  but not under the  GFDL .


A  transpoviron  is a  plasmid -like  genetic element  found in the  genomes  of  giant DNA viruses . [ 1 ] [ 2 ]


They are linear  DNA  elements of approximately 7 kilobases that encompass six to eight  protein  encoding  genes . Two of these genes are homologous to  virophage  genes. Transpovirons encode a superfamily 1  helicase , which encompasses an inactivated family B DNA polymerase domain. [ 3 ]  Homologs of this unique polymerase-helicase fusion protein are widespread in  Polinton -Like Viruses (PLV). [ 4 ]  Based on the phylogenetic analysis of the helicase domain the discoverers of PLVs concluded that transpovirons evolved from PLV via the loss of several genes including those encoding the morphogenetic module proteins. [ 3 ]


The  Ty5  is a type of  retrotransposon  native to the  Saccharomyces cerevisiae  organism.


Ty5  is one of five  [ 1 ]  endogenous retrotransposons native to the model organism  Saccharomyces cerevisiae , all of which target integration to gene poor regions. Endogenous retrotransposons are hypothesized to target gene poor chromosomal targets in order to reduce the chance of inactivating host genes. [ 2 ]  Ty1-Ty4 integrate upstream of Pol III promoters, while Ty5 targets integration to loci bound in  heterochromatin . [ 3 ]  In the case of Ty5, this likely occurs by means of an interaction between the  C-terminus  of  integrase  and a target protein. [ 4 ]  The tight targeting patterns seen for the Ty elements are thought to be a means to limit damage to its host, which has a very gene dense genome. [ 5 ]  Ty5 was discovered in the mid 1990s in the laboratory of  Daniel Voytas  at  Iowa State University . [ 6 ]  Ty5 is used as a model system by which to understand the biology of the  telomere  and heterochromatin. The Ty5 retrotransposon is used as a genetic model to study the architecture and dynamics of the telomeres and heterochromatin. [ 7 ]


Heterochromatin in  S. cerevisiae  is composed of a wide array of proteins and plays several roles. The first stage of heterochromatin formation requires DNA binding proteins, which interact with specific cis DNA sequences at the telomeres, rDNA and HM loci. These proteins, including Rap1p and the  origin recognition complex  (ORC), serve as a platform for other proteins to bind, condense the DNA, and modify neighboring  histones . Some of these proteins, notably Rap1p, also play other roles, including initiation of transcription. The first known step in the formation of dedicated heterochromatin is the binding of Sir4p to Rap1p (Luo, Vega-Palas et al. 2002). Sir4p is one of four ‘Silent Information Regulator’ proteins that also include Sir1p, Sir2p and Sir3p. Of these, Sir2p, Sir3p and Sir4p form the core of heterochromatin. [ 8 ]  Sir4p serves as a binding site for Sir2p, which is the next to bind. Sir2p deacetylates adjacent histones, which is thought to further condense the chromatin and prevent the binding of other transcription promoting histone modification enzymes. [ 9 ]  Sir3p binding follows, further condensing the heterochromatin. Sir1p plays a role in the initiation of silencing at the HM loci. A large number of other proteins act in both a synergistic and antagonistic manner. [ 10 ]


Early work characterizing Ty5 targeted transposition focused on two fronts: identifying the component of Ty5 responsible for targeting and identifying the factor with which it interacted. Due to the central role of the Sir proteins in heterochromatin formation, they were initially considered as potential targeting signals. Because  integration  is mediated by the retrotransposon integrase enzyme, it was speculated to contain a component that would recognize heterochromatin.
	The C-terminus of the Ty retrotransposon’s integrase contains an extension not seen in the retroviruses. This region is also not conserved between Ty1 and Ty5, whereas the rest of the integrase is, suggesting that this divergence could be responsible for the different targeting of the yeast Ty elements. A mutation was identified in the integrase C-terminus that randomized Ty5 integration, suggesting that this region of integrase was in fact involved in targeted transposition. [ 4 ]


Ty5 is a relative of the  Retroviridae  family of  retroviruses , which includes the human pathogen  HIV . Ty5 is a tractable system in which to study the biology of retrovirus integration.


UniVec  is a database that can be used to remove  vector  contamination from DNA sequences. [ 1 ]


VectorDB  was a database of  sequence information for  common  vectors  used in  molecular biology [ 1 ]


In  biology ,  polymorphism [ 1 ]  is the occurrence of two or more clearly different  morphs  or  forms , also referred to as alternative  phenotypes , in the  population  of a species. To be classified as such, morphs must occupy the same habitat at the same time and belong to a  panmictic  population (one with random mating). [ 2 ]


Put simply, polymorphism is when there are two or more possibilities of a trait on a gene. For example, there is more than one possible trait in terms of a jaguar's skin colouring; they can be light morph or dark morph. Due to having more than one possible variation for this gene, it is termed 'polymorphism'. However, if the jaguar has only one possible trait for that gene, it would be termed "monomorphic". For example, if there was only one possible skin colour that a jaguar could have, it would be termed monomorphic.


The term  polyphenism  can be used to clarify that the different forms arise from the same  genotype .  Genetic polymorphism  is a term used somewhat differently by  geneticists  and  molecular biologists  to describe certain  mutations  in the genotype, such as  single nucleotide polymorphisms  that may not always correspond to a phenotype, but always corresponds to a branch in the genetic tree. See  below .


Polymorphism is common in nature; it is related to  biodiversity ,  genetic variation , and  adaptation . Polymorphism usually functions to retain a variety of forms in a population living in a varied environment. [ 3 ] : 126   The most common example is  sexual dimorphism , which occurs in many organisms. Other examples are mimetic forms of butterflies (see  mimicry ), and human  hemoglobin  and  blood types .


According to the theory of evolution, polymorphism results from evolutionary processes, as does any aspect of a species. It is  heritable  and is modified by  natural selection . In polyphenism, an individual's genetic makeup allows for different morphs, and the switch mechanism that determines which morph is shown is environmental. In genetic polymorphism, the genetic makeup determines the morph.


The term polymorphism also refers to the occurrence of structurally and functionally more than two different types of individuals, called  zooids , within the same organism. It is a characteristic feature of  cnidarians . [ 2 ]
For example,   Obelia  has feeding individuals, the  gastrozooids ; the individuals capable of asexual reproduction only, the gonozooids, blastostyles; and free-living or sexually reproducing individuals, the  medusae .


Balanced polymorphism refers to the maintenance of different phenotypes in population.


Monomorphism means having only one form. Dimorphism means having two forms.


Polymorphism crosses several discipline boundaries, including ecology, genetics, evolution theory, taxonomy, cytology, and biochemistry. Different disciplines may give the same concept different names, and different concepts may be given the same name. For example, there are the terms established in ecological genetics by  E.B. Ford  (1975), [ 4 ]  and for classical genetics by  John Maynard Smith  (1998). [ 7 ]  The shorter term morphism was preferred by the evolutionary biologist  Julian Huxley  (1955). [ 8 ]


Various synonymous terms exist for the various polymorphic forms of an organism. The most common are morph and morpha, while a more formal term is morphotype.  Form and  phase  are sometimes used, but are easily confused in zoology with, respectively,  "form"  in a population of animals, and "phase" as a color or other change in an organism due to environmental conditions (temperature, humidity, etc.).  Phenotypic traits  and characteristics are also possible descriptions, though that would imply just a limited aspect of the body. [ citation needed ]


In the  taxonomic nomenclature  of  zoology , the word "morpha" plus a Latin name for the morph can be added to a  binomial  or  trinomial  name. However, this invites confusion with geographically variant  ring species  or  subspecies , especially if polytypic. Morphs have no formal standing in the  ICZN . In  botanical taxonomy , the concept of morphs is represented with the terms " variety ", " subvariety " and " form ", which are formally regulated by the  ICN . Horticulturists sometimes confuse this usage of "variety" both with  cultivar  ("variety" in  viticultural  usage, rice agriculture jargon, and informal  gardening  lingo) and with the legal concept " plant variety " (protection of a cultivar as a form of  intellectual property ). [ citation needed ]


Three mechanisms may cause polymorphism: [ 9 ]


Endler's survey of natural selection gave an indication of the relative importance of polymorphisms among studies showing natural selection. [ 10 ]  The results, in summary: Number of species demonstrating natural selection: 141. Number showing quantitative traits: 56. Number showing polymorphic traits: 62. Number showing both Q and P traits: 23. This shows that polymorphisms are found to be at least as common as continuous variation in studies of natural selection, and hence just as likely to be part of the evolutionary process. [ citation needed ]


Since all polymorphism has a genetic basis,  genetic polymorphism  has a particular meaning:


The definition has three parts: a)  sympatry : one interbreeding population; b) discrete forms; and c) not maintained just by mutation. [ citation needed ]


In simple words, the term polymorphism was originally used to describe variations in shape and form that distinguish normal individuals within a species from each other. Presently, geneticists use the term genetic polymorphism to describe the functionally silent differences in  DNA  sequence between individuals that make each human genome unique. [ 14 ]


Genetic polymorphism is actively and steadily maintained in populations by natural selection, in contrast to  transient polymorphisms  where a form is progressively replaced by another. [ 15 ] : 6–7   By definition, genetic polymorphism relates to a balance or equilibrium between morphs. The mechanisms that conserve it are types of  balancing selection . [ citation needed ]


Most genes have more than one effect on the  phenotype  of an organism ( pleiotropism ). Some of these effects may be visible, and others cryptic, so it is often important to look beyond the most obvious effects of a gene to identify other effects. Cases occur where a gene affects an unimportant visible characteristic, yet a change in fitness is recorded. In such cases, the gene's subsurface effects may be responsible for the change in fitness. Pleiotropism is posing continual challenges for many clinical dysmorphologists in their attempt to explain birth defects which affect one or more organ system, with only a single underlying causative agent. For many pleiotropic disorders, the connection between the genetic abnormality and its manifestations is neither apparent nor understood. [ 16 ]


Epistasis  occurs when the expression of one gene is modified by another gene. For example, gene A only shows its effect when allele B1 (at another  locus ) is present, but not if it is absent. This is one of the ways in which two or more genes may combine to produce a coordinated change in more than one characteristic (for instance, in mimicry). Unlike the supergene, epistatic genes do not need to be closely  linked  or even on the same  chromosome . [ citation needed ]


Both pleiotropism and epistasis show that a gene need not relate to a character in the simple manner that was once supposed. [ citation needed ]


Although a polymorphism can be controlled by  alleles  at a single  locus  (e.g. human  ABO  blood groups), the more complex forms are controlled by  supergenes  consisting of several tightly  linked genes  on a single  chromosome .  Batesian mimicry  in butterflies and  heterostyly  in angiosperms are good examples. There is a long-standing debate as to how this situation could have arisen, and the question is not yet resolved.


Whereas a  gene family  (several tightly linked genes performing similar or identical functions) arises by duplication of a single original gene, this is usually not the case with supergenes. In a supergene some of the constituent genes have quite distinct functions, so they must have come together under selection. This process might involve suppression of crossing-over, translocation of chromosome fragments and possibly occasional cistron duplication. That crossing-over can be suppressed by selection has been known for many years. [ 18 ] [ 19 ]


Debate has centered round the question of whether the component genes in a super-gene could have started off on separate chromosomes, with subsequent reorganization, or if it is necessary for them to start on the same chromosome. Originally, it was held that chromosome rearrangement would play an important role. [ 20 ]  This explanation was accepted by E. B. Ford and incorporated into his accounts of ecological genetics. [ 4 ] : ch. 6  [ 11 ] : 17–25


However, many believe it more likely that the genes start on the same chromosome. [ 21 ]  They argue that supergenes arose  in situ . This is known as Turner's sieve hypothesis. [ 22 ]  John Maynard Smith agreed with this view in his authoritative textbook, [ 7 ]  but the question is still not definitively settled.


Selection, whether natural or artificial, changes the frequency of morphs within a population; this occurs when morphs reproduce with different degrees of success. A genetic (or balanced) polymorphism usually persists over many generations, maintained by two or more opposed and powerful selection pressures. [ 6 ]  Diver (1929) found banding morphs in  Cepaea nemoralis  could be seen in  prefossil shells  going back to the  Mesolithic   Holocene . [ 23 ] [ 24 ]  Non-human apes have similar blood groups to humans; this strongly suggests that this kind of polymorphism is ancient, at least as far back as the last common ancestor of the apes and man, and possibly even further.


The relative proportions of the morphs may vary; the actual values are determined by the  effective fitness  of the morphs at a particular time and place. The mechanism of  heterozygote advantage  assures the population of some alternative  alleles  at the  locus  or loci involved. Only if competing selection disappears will an allele disappear. However, heterozygote advantage is not the only way a polymorphism can be maintained.  Apostatic selection , whereby a predator consumes a common morph whilst overlooking rarer morphs is possible and does occur. This would tend to preserve rarer morphs from extinction.


Polymorphism is strongly tied to the adaptation of a species to its environment, which may vary in colour, food supply, and predation and in many other ways including sexual harassment avoidance. Polymorphism is one good way the opportunities [ vague ]  get to be used; it has survival value, and the selection of modifier genes may reinforce the polymorphism. In addition, polymorphism seems to be associated with a higher rate of  speciation .


G. Evelyn Hutchinson , a founder of niche research, commented "It is very likely from an ecological point of view that all species, or at least all common species, consist of populations adapted to more than one niche". [ 26 ]  He gave as examples sexual size dimorphism and mimicry. In many cases where the male is short-lived and smaller than the female, he does not compete with her during her late pre-adult and adult life. Size difference may permit both sexes to exploit different niches. In elaborate cases of  mimicry , such as the African butterfly  Papilio dardanus , female morphs mimic a range of distasteful models called Batesian mimicry, [ 27 ]  often in the same region. The fitness of each type of mimic decreases as it becomes more common, so the polymorphism is maintained by frequency-dependent selection. Thus the efficiency of the mimicry is maintained in a much increased total population. However it can exist within one gender. [ 4 ] : ch. 13


Female-limited polymorphism and sexual assault avoidance


Female-limited polymorphism in  Papilio dardanus  can be described as an outcome of sexual conflict. Cook et al. (1994) [ 28 ]  argued that the male-like phenotype in some females in  P. dardanus  population on Pemba Island, Tanzania functions to avoid detection from a mate-searching male. The researchers found that male mate preference is controlled by frequency-dependent selection, which means that the rare morph suffers less from mating attempt than the common morph. The reasons why females try to avoid male sexual harassment are that male mating attempt can reduce female fitness in many ways such as fecundity and longevity. [ 29 ] [ 30 ]


The mechanism which decides which of several morphs an individual displays is called the  switch . This switch may be genetic, or it may be environmental. Taking sex determination as the example, in humans the determination is genetic, by the  XY sex-determination system . In  Hymenoptera  ( ants ,  bees  and  wasps ), sex determination is by haplo-diploidy: the females are all  diploid , the males are  haploid . However, in some animals an environmental trigger determines the sex:  alligators  are a famous case in point. In ants the distinction between workers and guards is environmental, by the feeding of the grubs. Polymorphism with an environmental trigger is called  polyphenism .


The polyphenic system does have a degree of environmental flexibility not present in the genetic polymorphism. However, such environmental triggers are the less common of the two methods.


Investigation of polymorphism requires use of both field and laboratory techniques. In the field:


And in the laboratory:


Without proper field-work, the significance of the polymorphism to the species is uncertain and without laboratory breeding the genetic basis is obscure. Even with insects, the work may take many years; examples of  Batesian mimicry  noted in the nineteenth century are still being researched.


Polymorphism was crucial to research in  ecological genetics  by E. B. Ford and his co-workers from the mid-1920s to the 1970s (similar work continues today, especially on  mimicry ). The results had a considerable effect on the mid-century  evolutionary synthesis , and on present  evolutionary theory . The work started at a time when  natural selection  was largely discounted as the leading mechanism for evolution, [ 31 ] [ 32 ]  continued through the middle period when  Sewall Wright 's ideas on  drift  were prominent, to the last quarter of the 20th century when ideas such as  Kimura 's  neutral theory of molecular evolution  was given much attention. The significance of the work on ecological genetics is that it has shown how important selection is in the evolution of natural populations, and that selection is a much stronger force than was envisaged even by those population geneticists who believed in its importance, such as  Haldane  and  Fisher . [ 33 ]


In just a couple of decades the work of Fisher, Ford,  Arthur Cain ,  Philip Sheppard  and  Cyril Clarke  promoted natural selection as the primary explanation of variation in natural populations, instead of genetic drift. Evidence can be seen in Mayr's famous book  Animal Species and Evolution , [ 34 ]  and Ford's  Ecological Genetics . [ 4 ]  Similar shifts in emphasis can be seen in most of the other participants in the evolutionary synthesis, such as  Stebbins  and  Dobzhansky , though the latter was slow to change. [ 3 ] [ 35 ] [ 36 ] [ 37 ]


Kimura  drew a distinction between  molecular evolution , which he saw as dominated by selectively neutral mutations, and phenotypic characters, probably dominated by natural selection rather than drift. [ 38 ]


Aggressive mimicry  is a form of  mimicry  in which  predators ,  parasites , or  parasitoids  share similar  signals , using a harmless model, allowing them to avoid being correctly identified by their prey or  host . Zoologists have repeatedly compared this strategy to a  wolf in sheep's clothing . [ 2 ] [ 3 ] [ 4 ]  In its broadest sense, aggressive mimicry could include various types of exploitation, as when an orchid exploits a male insect by mimicking a sexually receptive female (see  pseudocopulation ), [ 5 ]  but will here be restricted to forms of exploitation involving feeding.  For example,  indigenous Australians  who dress up as and imitate  kangaroos  when  hunting  would not be considered aggressive mimics, nor would a human  angler , though they are undoubtedly practising  self-decoration camouflage . Treated separately is  molecular mimicry , which shares some similarity; for instance a  virus  may mimic the molecular properties of its host, allowing it access to its cells. An alternative term,  Peckhamian mimicry , has been suggested (after  George and Elizabeth Peckham ), [ 6 ] [ 7 ] [ 8 ]  but it is seldom used. [ a ]


Aggressive mimicry is opposite in principle to  defensive mimicry , where the mimic generally benefits from being treated as harmful. The mimic may resemble its own prey, or some other organism which is beneficial or at least not harmful to the prey. The model, i.e. the organism being 'imitated', may experience increased or reduced  fitness , or may not be affected at all by the relationship. On the other hand, the signal receiver inevitably suffers from being tricked, as is the case in most mimicry complexes.


Aggressive mimicry often involves the predator employing signals which draw its potential prey towards it, a strategy which allows predators to simply sit and wait for prey to come to them. The promise of food or  sex  are most commonly used as lures. However, this need not be the case; as long as the predator's true identity is concealed, it may be able to approach prey more easily than would otherwise be the case. In terms of species involved, systems may be composed of two or three species; in two-species systems the signal receiver, or "dupe", is the model.


In terms of the  visual  dimension, the distinction between aggressive mimicry and  camouflage  is not always clear. Authors such as Wickler [ 8 ]  have emphasized the significance of the signal to its receiver as delineating mimicry from camouflage. However, it is not easy to assess how 'significant' a signal may be for the dupe, and the distinction between the two can thus be rather fuzzy. Mixed signals may be employed: aggressive mimics often have a specific part of the body sending a deceptive signal, with the rest being hidden or camouflaged.


Aggressive mimicry stands in semantic contrast with  defensive mimicry , where it is the  prey  that acts as a mimic, with  predators  being duped. Defensive mimicry includes the well-known  Batesian  and  Müllerian  forms of mimicry, where the mimic shares outward characteristics with an  aposematic  or harmful model. In Batesian mimicry, the mimic is modeled on a dangerous (usually unpalatable) species, while in Müllerian mimicry both species are harmful, and act as comimics, converging on a common set of signals and sharing the burden of 'educating' their predators. Included in defensive mimicry is the lesser known  Mertensian mimicry , where the mimic is  more  harmful than the model, and  Vavilovian mimicry , where  weeds  come to mimic crops through unintentional artificial selection. In defensive mimicry, the mimic benefits by avoiding a harmful interaction with another organism that would be more likely to take place without the deceptive signals employed. Harmful interactions might involve being eaten, or pulled out of the ground as a weed. In contrast, the aggressive mimic benefits from an interaction that would be less likely to take place without the deception, at the expense of its target. [ 9 ]


In some cases the dupe is lured toward the mimic. This involves mimicry of a resource that is often vital to the prey's survival (or more precisely, the survival of its  genes ) such as nutrition or a mate. If the bait offered is of little value to prey they would not be expected to take such a risk. For example, in all known cases of sexual signal mimicry it is always the male  sex  that is deceived (in fact, it has been suggested that females of some species have evolved mimicry as a strategy to avoid  unwanted matings ). [ 10 ]  In these cases the predator need not move about  foraging  for prey, but may simply stay still and allow prey to come to it.
Some studies suggest that the  northern shrike  ( Lanius excubitor ) sings in winter often imitating small passerines that may be preyed upon when lured within reach. [ 11 ]
There has been one report of a  margay  using mimicry of the cry of an infant  pied tamarin  to try to lure an adult tamarin within striking distance. [ 12 ]


Many aggressive mimics use the promise of nourishment as a way of attracting prey.  The  alligator snapping turtle  ( Macrochelys temminckii ) is a well-camouflaged  ambush predator . Its tongue bears a conspicuous pink extension that resembles a  worm  and can be wriggled around; [ 13 ]  fish that try to eat the "worm" are themselves eaten by the turtle. Similarly, some snakes employ  caudal luring  (using the tail) [ 14 ] [ 15 ]  or  lingual luring  (using the tongue) to entice small vertebrates into striking range. [ 16 ] [ 17 ]


Aggressive mimicry is common amongst  spiders , both in luring prey and stealthily approaching predators. [ 18 ]  One case is the  golden orb weaver  ( Nephila clavipes ), which spins a conspicuous golden coloured web in well-lit areas. Experiments show that bees are able to associate the webs with danger when the yellow pigment is not present, as occurs in less well-lit areas where the web is much harder to see. Other colours too were learned and avoided, but bees seemed least able to effectively associate yellow pigmented webs with danger. Yellow is the colour of many nectar bearing flowers, however, so perhaps avoiding yellow is not worthwhile. Another form of mimicry is based not on colour but pattern. Species such as  Argiope argentata  employ  prominent patterns  in the middle of their webs, such as zigzags. These may reflect  ultraviolet  light, and mimic the pattern seen in many flowers known as  nectar guides . Spiders change their web day to day, which can be explained by bees' ability to remember web patterns. Bees are able to associate a certain pattern with a spatial location, meaning the spider must spin a new pattern regularly or suffer diminishing prey capture. [ 19 ]


Spiders can be the prey of aggressive mimics. The  assassin bug   Stenolemus bituberus  preys on spiders, entering their web and plucking its silk threads until the spider approaches. This vibrational aggressive mimicry matches a general pattern of vibrations which spiders treat as prey, having a similar temporal structure and amplitude to leg and body movements of typical prey caught in the web. [ 20 ]


Larvae of the  ground beetle   Epomis  move their mandibles one after another to lure amphibians toward them and then prey on them. Their body structure allows them to bite and feed on the amphibians even when they are ingested by larger prey such as frogs. [ 21 ]


Although  plants  are better known for defensive mimicry, there are exceptions. For example, many flowers use mimicry to attract pollinators, while others may trick insects into dispersing their seeds. Nonetheless, most mimicry in plants [ b ]  would not be classified as aggressive, as although  luring pollinators  is similar to cases above, they are certainly not eaten by the plant. However some  carnivorous plants  may be able to increase their rate of capture through mimicry. [ 24 ]  For example, some have patterns in the ultraviolet region of the  electromagnetic spectrum , much like the spider webs described above. [ 25 ]


Mimicry systems involving only two species are known as  bipolar . [ 9 ]  Only one bipolar arrangement is possible here, namely where the dupe is itself the model. [ c ]  There are two such variants on this arrangement of mimic imitating its target. In one case, Kirbyan mimicry, the model is the host of a  brood parasite . [ 26 ]  In the other case, termed  Batesian-Wallacian mimicry [ 9 ]  after  Henry Walter Bates [ 27 ]  and  Alfred Russel Wallace , [ 28 ]  the model is the prey species.


Host-parasite mimicry  is a situation where a parasite mimics its own host. As with mimicry of the female sex outlined previously, only two species are involved, the model and mimic being of the same species.  Brood parasitism , a form of  kleptoparasitism  where the mother has its offspring raised by another unwitting organism, is one such situation where host-parasite mimicry has evolved. Georges Pasteur [ 9 ]  terms this form of aggressive-reproductive mimicry  Kirbyan mimicry , after the English  entomologist   William Kirby , who noticed that the young of  syrphid  hoverflies are raised by  bumblebees . [ 26 ]


In Batesian-Wallacian mimicry, the model is a sexually receptive female, which provides a strong attractive effect on males. Some spiders use chemical rather than visual means to ensnare prey. Female  bolas spiders  of the genus  Mastophora  lure male moth-flies ( Diptera , true flies, but resembling moths) by producing analogues of the fly species'  sex pheromones . Each species of spider appears to  specialize  in a particular species of prey in the family  Psychodidae . Juveniles use their front pair of legs to capture prey, such as  flies . Older spiders use a different strategy however, swinging a sticky ball known as a  bolas  suspended by a silk thread at moths. But both old and juvenile are able to lure prey via this olfactory signal; even young spiderlings have been shown to attract prey species. [ 29 ]


The listroscelidine katydid  Chlorobalius leucoviridis  of inland Australia is capable of attracting male cicadas of the Tribe Cicadettini by imitating the species-specific reply clicks of sexually receptive female cicadas. This example of acoustic aggressive mimicry is similar to the  Photuris  firefly case in that the predator's mimicry is remarkably versatile – playback experiments show that  C. leucoviridis  is able to attract males of many cicada species, including Cicadettine cicadas from other continents, even though cicada mating signals are species-specific. The evolution of versatile mimicry in  C. leucoviridis  may have been facilitated by constraints on song evolution in duetting communication systems in which reply signals are recognizable only by their precise timing in relation to the male song (<< 100 ms reply latency). [ 30 ] [ 31 ]


Female  fireflies  of the genus  Photuris  emit the same light signals that females of the genus  Photinus  use as a mating signal. [ 32 ]  Male fireflies from several different  genera  are attracted to these mimics, and are subsequently captured and eaten. Female signals are based on that received from the male, each female having a repertoire of signals matching the delay and duration of the female of the corresponding species. This mimicry may have evolved from non-mating signals that have become modified for predation. [ 33 ]


The prey does not have to be attracted towards the predator for the predator to benefit: it is sufficient for the predator simply not to be identified as a threat. Wicklerian-Eisnerian mimics may resemble a  mutualistic  ally, or a species of little significance to the prey such as a  commensal . [ 9 ]  For example, the  hemipteran   Arachnocoris berytoides  resembles  Faiditus caudatus , a spider commensal of ants. [ 34 ]


In  cryptic aggressive mimicry , the predator mimics an organism that its prey is indifferent to. This allows the predator to avoid detection until the prey are close enough for the predator to strike, effectively a form of  camouflage . The  zone-tailed hawk  ( Buteo albonotatus ), which resembles the  turkey vulture  ( Cathartes aura ), may provide one such example. It flies amongst them, suddenly breaking from the formation and ambushing its prey. [ 35 ]  There is some controversy over whether this is a true case of mimicry. [ 36 ]


Mimicry of mutualistic species is seen in coral reef fish, where the models, certain  cleaner fish , are greatly disadvantaged by the presence of the mimic. Cleaner fish are  mutually beneficial  to many other species, which allows them to eat their parasites and dead skin. Some allow the cleaner to venture inside their mouths and gill cavities to hunt these parasites. However, one species of cleaner, the  bluestreak cleaner wrasse  ( Labroides dimidiatus ), is the model of a mimic, the  sabre-toothed blenny  ( Aspidontus taeniatus ). The  cleaner wrasse , shown in the image cleaning a  grouper  of the genus  Epinephelus , resides in  coral reefs  in the Indian and the Pacific Oceans, and is recognised by other fishes who allow it to clean them. The  blenny  lives in the  Indian Ocean  and not only looks like the cleaner wrasse in terms of size and  coloration , but even mimics the cleaner wrasse's 'dance'. Having fooled its prey into letting its guard down, the sabre-toothed blenny bites it,  tearing off scales  or pieces of fin. Fish grazed upon in this fashion learn to distinguish mimic from model, but because of the similarity between the two, they become much more cautious of the model as well, such that both are affected. Due to victims' ability to discriminate between foe and helper, the blennies have evolved close similarity, down to the regional level. [ 37 ]
Another aggressive mimic of the cleaner wrasse, the  bluestriped fangblenny , has evolved an  opioid -containing venom which dulls pain and lowers blood pressure, confusing the bitten host and giving the mimic time to escape. [ 38 ]


Just as predators such as  angler fish  have a structure that lures prey, so some parasites mimic their host's natural prey, but with roles reversed; the parasite gets eaten by the host. This  deception  provides the parasite easy entry into the host, which they can then feed upon, allowing them to continue their life cycle. [ 39 ]


One such case is a genus of mussel,  Lampsilis , which feeds on the  gills  of fish in the larval stage of their development. Once they mature, they leave the fish as adult mollusc. Gaining entry into the host is not an easy task though, despite the fact that several hundred thousand larvae are released at once. This is especially the case in flowing water bodies such as streams, where they cannot lie on the substrate and wait to be taken up in the course of foraging. Female  Lampsilis  have evolved a special technique for delivering their offspring into a suitable host, however. Structures on the edge of the  mantle  are able to capture the interest of fish. Some resemble small fish themselves, with eye spots, a "tail" and horizontal stripes, and may even move in a similar fashion, as if facing the current ( rheotaxis ). When overshadowed by a fish, the larvae are forcefully expelled, becoming ecto-parasites on their unsuspecting host. [ 8 ]  Some species of  Lampsilis , notably  Lampsilis ovata , attract fish in the genus  Micropterus ,  Villosa  has fish-like mantle lures that attract predatory fish  Percina . [ 40 ]


Cercaria mirabilis , a  trematode , has an especially large larval stage, a  cercaria , which looks much like a small crustacean or mosquito larva. It mimics the  locomotory  behavior of such animals, allowing it to be eaten by predaceous fish. [ 39 ]


Another parasitic trematode example is seen in a terrestrial setting.  Leucochloridium  is a genus of  flatworm  (phylum Platyhelminthes) which matures in the intestine of  songbirds . Their eggs pass out of the bird in the  feces  and are then taken in by  Succinea , a terrestrial snail that lives in moist environments. The eggs develop into larvae inside this  intermediate host , and then must find their way into the digestive system of a suitable bird. The problem here is that these birds do not eat snails, so the sporocyst must find some way of manipulating its future host into eating it. Unlike related species, these parasites are brightly colored and able to move in a pulsating manner. A sporocyst sac forces its way into the snail's eye stalks, and pulsates at high speed, enlarging the tentacle in the process. [ 41 ]  It affects the host's behavior: the snail moves towards light, which it usually avoids. These combined factors make the sporocysts highly conspicuous, such that they are soon eaten by a hungry songbird. The snail then regenerates its tentacles, and  Leucochloridium  carries on with its life cycle. [ 8 ]


Zoologists have repeatedly compared aggressive mimicry to the  wolf in sheep's clothing  strategy of  fable , [ 42 ]  including when describing  jumping spiders , [ 2 ] [ 3 ]   lacewings , [ 43 ]   ant-mimicking   aphids , [ 44 ]   hemipteran  bugs mimicking  chrysomelid beetles , [ 45 ]   bird-dropping spiders , [ 4 ]   orchid mantises , [ 4 ]   cichlid fish , [ 46 ] [ 47 ]  and the zone-tailed hawk which flies with  vultures , enabling it to approach terrestrial prey. [ 48 ] [ 49 ]  These animals have  evolved  to  deceive their prey  by appearing as other prey, or like  angler fish [ 48 ]  and  snapping turtles [ 48 ]  lure the prey by appearing as the prey's prey.


Cepaea  is a  genus  of large air-breathing  land snails ,  terrestrial   pulmonate   gastropod   molluscs  in the family  Helicidae . The shells are often brightly coloured and patterned with brown stripes. The two species in this genus,  C. nemoralis  and  C. hortensis , are widespread and common in Western and Central Europe and have been introduced to North America. Both have been influential  model species  for ongoing studies of genetics and natural selection. Like many Helicidae, these snails use  love darts  during mating. [ 2 ]


For a long time, four species were classified in the genus  Cepaea . However,  molecular phylogenetic studies  suggested that two of them should be placed in the genera  Macularia  and  Caucasotachea , which are not immediate relatives of either  Cepaea  or each other: [ 3 ] [ 4 ]


The range of  C. hortensis  extends further north than that of  C. nemoralis  in Scotland and Scandinavia and it is the only one of the two species in Iceland. [ 5 ] [ 6 ]  Likewise in the Swiss Alps  C. hortensis  is found as high as 2050 m, but  C. nemoralis  only up to 1600 m. [ 7 ]  Conversely, the southern edge of the range lies further north in  C. hortensis ; unlike  C. nemoralis  it does not occur in Italy, and in Spain it has a more restricted distribution (in the north-east corner). [ 8 ] [ 9 ]


Where the ranges overlap  C. hortensis  prefers cooler sites with longer and damper vegetation. But the two species often co-occur at a site, in which situation the densities of both affect each other's growth, fecundity and mortality. However, they differ somewhat in their behaviour:  C. hortensis  is more active at lower temperatures, aestivates higher on the vegetation and is more  diurnal , although this appears to be independent of whether the other species is present or not. [ 10 ] [ 11 ]


When given no choice of partner in the laboratory, the two  Cepaea  species can form hybrids, which will  backcross  with the parental species, but the fertility is very low. [ 12 ]


The two  Cepaea  species share a  genetic polymorphism  for the colour and banding pattern of the shell.


The background colour of the shell ranges from dark brown, through pink to yellow or even approaching white. This variation is continuous, but there are peaks in the distribution corresponding to brown, pink and yellow morphs. [ 13 ]  The colour is mainly determined by  alleles  at a single  locus  with brown  dominant  to pink, which is dominant to yellow. [ 11 ]


Up to five bands (very rarely more) run spirally around the shell, numbered 1 to 5 with the larger numbers further from the shell apex. The conventional scoring annotation is to write 12345 if all bands are present and separated, but to replace a number with 0 if a band is absent from its usual position and to enclose numbers in parentheses if bands are fused with their neighbours. Thus 003(45) would mean that the top two bands are absent and the lower two fused. [ 14 ]


A dominant allele at one locus causes the absence of all bands, a dominant allele at another locus causes the loss of all bands except band 3, and a dominant allele at a third locus causes the loss of just bands 1 and 2. The first of these three loci is closely  linked  to the locus determining shell colour, to another influencing the spread of the band pigment, and to one determining the colour of the lip and bands. This collection of linked loci are part of a  supergene . A consequence of this arrangement is that the shells of different background colours within a population often exhibit different ratios of banded to unbanded shells: this is an example of  linkage disequilibrium . [ 11 ]


The bands are usually dark brown, but this is affected by genes influencing intensity and colouration (e.g. black or orange). Another locus (part of the supergene) determines whether the band is continuous or forms a sequence of spots. The genetics underlying the fusion of adjacent bands is not well understood. [ 11 ]


In both species, most populations exhibit polymorphism in one or more of these shell characters. Nevertheless, statistically we can detect systematic variation at continental scales, and also between habitats, and at various scales down to a few tens of metres. There is also statistical evidence of change with time, based both on comparisons between sub-fossil and modern shells, [ 15 ]  and on resampling the same sites some decades apart, although the latter has more often found little change over the period ( stasis ). [ 16 ]  Very much research in  ecological genetics  has addressed the reasons for both the variation and the systematic trends. [ 11 ] [ 17 ]


The two selection pressures that might most feasibly act on the appearance of shells are  climatic selection  and predation. Darker shells heat up more quickly in the sun, which might well be advantageous for cold-blooded animals in shaded woodland but risks causing overheating and death in open habitats. [ 18 ] [ 19 ]  This trade-off is also presumed to be responsible for the greater proportion of yellow  C. nemoralis  to the south, but it is curious why the trend is not present in  C. hortensis . [ 11 ]  Contrary to predictions, recent global warming has not led to a detectable increase in yellow morphs on a continental scale. [ 20 ]  The use of photosensitive paint has shown that paler morphs spend more time exposed to the sun, which may imply that the shell polymorphism allows different morphs to coexist at a site by occupying different microhabitats. [ 21 ]


Both temperature regulation and predation make the same prediction of pale shells in open habitats and dark shells in woodland, so—although the prediction has often been confirmed [ 22 ] —it is difficult to test which is the more important explanation. However,  song thrushes  ( Turdus philomelos ) break open  Cepaea  shells on stones ("anvils"), allowing a comparison of those they predate with those present in the local environment. Besides the directional selection favouring camouflaged individuals, [ 23 ]  visually searching predators might cause  apostatic selection . The hypothesis is that they form a search image for the commonest morphs, favouring whichever morphs are locally rare, thus promoting diversity. [ 24 ]  As well as its visual effect, the shell pigments are associated with differences in shell strength, so may affect predation by predators searching non-visually, for instance at night. [ 25 ]


Several studies have demonstrated a predicted evolutionary response of shell appearance to a change of habitat. [ 26 ]  However, the association of shell appearance and habitat is not always consistent, especially in more disturbed environments, [ 22 ]  so it is believed that random effects are also influential, particularly  founder effects . The two  Cepaea  species colonised much of Europe only within the last 4000 generations, [ 17 ]  so the time available for selection to act has been limited, and local anthropogenetic disturbances must often have reversed which morphs are optimal. Moreover, snails disperse more slowly than many other animals, so the most suitable genes may be locally absent.


For instance, biologists were at one time puzzled by the phenomenon of " area effects "; the same morph of  Cepaea  may be found consistently over a wide area but in adjacent areas of similar habitat a different set of morphs predominate instead, with a sharp transition between. The explanation accepted nowadays is that relatively recently a change of habitat allowed the rapid colonisation of vacant areas by descendants of a few founder individuals until the colony had expanded out to areas occupied by other populations; subsequently intraspecific competition slowed the dispersal of genes into the neighbouring, occupied areas. [ 27 ] [ 28 ] [ 29 ]  Nevertheless, occasional transfer of genes between areas of different habitat is proposed to be important in maintaining the local diversity of phenotypes. [ 17 ]


Darwin's finches  (also known as the  Galápagos finches ) are a group of about 18  species  of  passerine  birds. [ 1 ] [ 2 ] [ 3 ] [ 4 ]  They are well known for being a classic example of adaptive radiation and for their remarkable diversity in beak form and function. [ 5 ]   They are often classified as the  subfamily   Geospizinae  or  tribe   Geospizini . They belong to the  tanager   family  and are not closely related to the  true finches . The closest known relative of the Galápagos finches is the South American  dull-coloured grassquit  ( Asemospiza obscura ). [ 6 ]  They were first collected when  the second voyage of the  Beagle  visited the   Galápagos Islands , with  Charles Darwin  on board as a gentleman naturalist. Apart from the  Cocos finch , which is from  Cocos Island , the others are found only on the Galápagos Islands.


The term "Darwin's finches" was first applied by  Percy Lowe  in 1936, and popularised in 1947 by  David Lack  in his book  Darwin's Finches . [ 7 ] [ 8 ]  Lack based his analysis on the large collection of museum specimens collected by the 1905–06 Galápagos expedition of the California Academy of Sciences, to whom Lack dedicated his 1947 book. The birds vary in size from 10 to 20 cm (4 to 8 in) and weigh between 8 and 38 grams (0.3 and 1.3 oz). The smallest are the  warbler-finches  and the largest is the  vegetarian finch . The most important differences between species are in the size and shape of their beaks, which are highly  adapted  to different food sources. Food availability was different among the islands of the Galapagos and could also change dramatically due to natural events such as droughts. The birds are all dull-coloured. They are thought to have evolved from a single finch species that came to the islands more than a million years ago. [ 9 ]


During  the survey voyage  of  HMS  Beagle , Darwin was unaware of the significance of the birds of the Galápagos. He had learned how to preserve bird specimens from  John Edmonstone  while at the  University of Edinburgh  and had been keen on shooting, but he had no expertise in  ornithology  and by this stage of the voyage concentrated mainly on geology. [ 10 ]  In Galápagos he mostly left bird shooting to his servant  Syms Covington . [ 11 ]  Nonetheless, these birds were to play an important part in the  inception of Darwin's theory  of  evolution  by  natural selection .


On the  Galápagos Islands  and afterward, Darwin thought in terms of "centres of creation" and rejected ideas concerning the  transmutation of species . [ 12 ]  From  Henslow 's teaching, he was interested in the geographical distribution of species, particularly links between species on oceanic islands and on nearby continents. On  Chatham Island , he recorded that a  mockingbird  was similar to those he had seen in  Chile , and after finding a different one on  Charles Island  he carefully noted where mockingbirds had been caught. [ 10 ]  In contrast, he paid little attention to the finches. When examining his specimens on the way to  Tahiti , Darwin noted that all of the mockingbirds on Charles Island were of one species, those from  Albemarle  of another, and those from  James  and Chatham Islands of a third. As they sailed home about nine months later, this, together with other facts, including what he had heard about  Galápagos tortoises , made him wonder about the stability of species. [ 13 ] [ 14 ]


Following his return from the voyage Darwin presented the finches to the  Zoological Society of London  on 4 January 1837, along with other mammal and bird specimens that he had collected. The bird specimens, including the finches, were given to  John Gould , the famous English  ornithologist , for identification. Gould set aside his paying work and at the next meeting, on 10 January, reported that the birds from the  Galápagos Islands  that Darwin had thought were  blackbirds , " gross-beaks " and  finches  were actually "a series of ground Finches which are so peculiar [as to form] an entirely new group, containing 12 species." This story made the newspapers. [ 15 ] [ 16 ]


Darwin had been in  Cambridge  at that time. In early March, he met Gould again and for the first time to get a full report on the findings, including the point that his Galápagos " wren " was another closely allied species of finch. The  mockingbirds  that Darwin had labelled by island were separate species rather than just varieties. Gould found more species than Darwin had expected, [ 17 ]  and concluded that 25 of the 26 land birds were new and distinct forms, found nowhere else in the world but closely allied to those found on the  South American  continent. [ 16 ]  Darwin now saw that, if the finch species were confined to individual islands, like the mockingbirds, this would help to account for the number of species on the islands, and he sought information from others on the expedition. Specimens had also been collected by Captain  Robert FitzRoy , FitzRoy's steward Harry Fuller, and Darwin's servant  Covington , who had labelled them by island. [ 18 ]  From these, Darwin tried to reconstruct the locations from where he had collected his own specimens. The conclusions supported his idea of the transmutation of species. [ 16 ]


At the time that he rewrote his diary for publication as  Journal and Remarks  (later  The Voyage of the Beagle ), he described Gould's findings on the number of birds, noting that "Although the species are thus peculiar to the archipelago, yet nearly all in their general structure, habits, colour of feathers, and even tone of voice, are strictly American". [ 19 ]  In the first edition of  The Voyage of the Beagle , Darwin said that


It is very remarkable that a nearly perfect gradation of structure in this one group can be traced in the form of the beak, from one exceeding in dimensions that of the largest gros-beak, to another differing but little from that of a warbler". [ 20 ]


By the time the first edition was published, the  development of Darwin's theory  of  natural selection  was in progress. For the 1845 second edition of  The Voyage  (now titled  Journal of Researches ), Darwin added more detail about the beaks of the birds, and two closing sentences which reflected his changed ideas:


Seeing this gradation and diversity of structure in one small, intimately related group of birds, one might really fancy that from an original paucity of birds in this archipelago, one species had been taken and modified for different ends." [ 21 ] [ 22 ]


The remaining land-birds form a most singular group of finches, related to each other in the structure of their beaks, short tails, form of body and plumage: There are thirteen species, which Mr. Gould has divided into four subgroups. All these species are peculiar to this archipelago; and so is the whole group, with the exception of one species of the sub-group  Cactornis , lately brought from Bow Island, in the Low Archipelago. Of  Cactornis , the two species may be often seen climbing about the flowers of the great cactus-trees; but all the other species of this group of finches, mingled together in flocks, feed on the dry and sterile ground of the lower districts. The males of all, or certainly of the greater number, are jet black; and the females (with perhaps one or two exceptions) are brown. The most curious fact is the perfect gradation in the size of the beaks in the different species of  Geospiza , from one as large as that of a hawfinch to that of a chaffinch, and (if Mr. Gould is right in including his sub-group,  Certhidea , in the main group) even to that of a warbler. The largest beak in the genus  Geospiza  is shown in Fig. 1, and the smallest in Fig. 3; but instead of there being only one intermediate species, with a beak of the size shown in Fig. 2, there are no less than six species with insensibly graduated beaks. The beak of the sub-group  Certhidea , is shown in Fig. 4. The beak of  Cactornis  is somewhat like that of a starling, and that of the fourth subgroup,  Camarhynchus , is slightly parrot-shaped. Seeing this gradation and diversity of structure in one small, intimately related group of birds, one might really fancy that from an original paucity of birds in this archipelago, one species had been taken and modified for different ends. In a like manner it might be fancied that  a bird originally a buzzard , had been induced here to undertake the office of the carrion-feeding  Polybori  of the American continent. [ 23 ]


Darwin discussed the divergence of various species of birds in the Galápagos more explicitly in his chapter on geographical distribution in  On the Origin of Species ; however, he does not single out the finches:


The most striking and important fact for us in regard to the inhabitants of islands, is their affinity to those of the nearest mainland, without being actually the same species. [In] the Galapagos Archipelago ... almost every product of the land and water bears the unmistakable stamp of the American continent. There are twenty-six land birds, and twenty-five of these are ranked by Mr. Gould as distinct species, supposed to have been created here; yet the close affinity of most of these birds to American species in every character, in their habits, gestures, and tones of voice, was manifest. ... The naturalist, looking at the inhabitants of these volcanic islands in the Pacific, distant several hundred miles from the continent, yet feels that he is standing on American land. Why should this be so? Why should the species which are supposed to have been created in the Galapagos Archipelago, and nowhere else, bear so plain a stamp of affinity to those created in America? There is nothing in the conditions of life, in the geological nature of the islands, in their height or climate, or in the proportions in which the several classes are associated together, which resembles closely the conditions of the South American coast: In fact there is a considerable dissimilarity in all these respects. On the other hand, there is a considerable degree of resemblance in the volcanic nature of the soil, in climate, height, and size of the islands, between the Galapagos and Cape de Verde Archipelagos: But what an entire and absolute difference in their inhabitants! The inhabitants of the Cape de Verde Islands are related to those of Africa, like those of the Galapagos to America. I believe this grand fact can receive no sort of explanation on the ordinary view of independent creation; whereas on the view here maintained, it is obvious that the Galapagos Islands would be likely to receive colonists, whether by occasional means of transport or by formerly continuous land, from America; and the Cape de Verde Islands from Africa; and that such colonists would be liable to modification — the principle of inheritance still betraying their original birthplace. [ 24 ]


Whereas Darwin spent just five weeks in the Galápagos, and  David Lack  spent three months,  Peter and Rosemary Grant  and their colleagues have made research trips to the Galápagos for about 30 years, particularly studying Darwin's finches.


Females are dimorphic in song type: songs A and B are quite distinct. Also, males with song A have shorter bills than B males, another clear difference. With these beaks, males are able to feed differently on their favourite cactus, the prickly pear  Opuntia . Those with long beaks are able to punch holes in the cactus fruit and eat the fleshy  aril  pulp, which surrounds the seeds, whereas those with shorter beaks tear apart the cactus base and eat the pulp and any insect larvae and pupae (both groups eat flowers and buds). This dimorphism clearly maximises their feeding opportunities during the non-breeding season when food is scarce.


If the population is  panmictic , [ 25 ] [ 26 ]  then  Geospiza conirostris  exhibits a balanced genetic polymorphism and not, as originally supposed, a case of nascent  sympatric speciation . The selection maintaining the polymorphism maximises the species'  niche  by expanding its feeding opportunity. The genetics of this situation cannot be clarified in the absence of a detailed breeding program, but two loci with  linkage disequilibrium [ 27 ]  is a possibility.


Another interesting dimorphism is for the bills of young finches, which are either 'pink' or 'yellow'. All species of Darwin's finches exhibit this morphism, which lasts for two months. No interpretation of this phenomenon is known. [ 28 ]


For some decades, taxonomists have placed these birds in the family  Emberizidae  along with the New World sparrows and Old World buntings. [ 29 ]  However, the  Sibley–Ahlquist taxonomy  puts Darwin's finches with the  tanagers  (Monroe and Sibley 1993), and at least one recent work follows that example (Burns and Skutch 2003). The  American Ornithologists' Union , in its  North American  checklist, places the Cocos finch in the Emberizidae, but with an asterisk indicating that the placement is probably wrong (AOU 1998–2006); in its tentative  South American  check-list, the Galápagos species are  incertae sedis , of uncertain place (Remsen et al. 2007).


A long-term study carried out for more than 40 years by the Princeton University researchers  Peter and Rosemary Grant  has documented evolutionary changes in beak size affected by  El Niño/La Niña  cycles in the Pacific. [ 37 ]


Developmental research in 2004 found that  bone morphogenetic protein 4  (BMP4), and its differential expression during development, resulted in variation of beak size and shape among finches. BMP4 acts in the developing embryo to lay down skeletal features, including making the beak stronger. [ 38 ]  The same group showed that the development of the different beak shapes in Darwin's finches are also influenced by slightly  different timing  and  spatial expressions  of a gene called calmodulin (CaM). [ 39 ]   Calmodulin  acts in a similar way to BMP4, affecting some of the features of beak growth like making them long and pointy. The authors suggest that changes in the temporal and spatial expression of these two factors are possible developmental controls of beak morphology. In a recent study genome sequencing revealed a 240 kilobase haplotype encompassing the  ALX1  gene that encodes a transcription factor affecting craniofacial development is strongly associated with beak shape diversity. [ 40 ] [ 41 ]  Moreover, these changes in the beak size have also altered vocalizations in Darwin's finches. [ 5 ]


Further research from 2016, in which genomes from each of the Darwin's finch species were sequenced, established that a single nucleotide polymorphism in the high mobility AT-hook 2 gene ( HMGA2 ) locus is significantly associated with variation in beak size (Lamichhaney et al. 2016).  HMGA2  codes for a transcription factor which in humans has been associated with variation in height, craniofacial distances, and primary tooth eruption.


In an analysis of the genomes of individuals from three  Geospiza  ground finch species found in sympatry ( G. fortis ,  G. fuliginosa ,  G. magnirostris ), 11 out of 32,569 SNPs were identified as representing four independent groups of statistically linked SNPs that together explained 83.6% of the variance in beak size (Chaves 2016). What this means is that only a small fraction of the genome in Darwin's finches is responsible for variation in beak morphology which is consistent with the rapid changes in beak form in response to the varying environments on the Galapagos Islands.


Ecophenotypic variation  ("ecophenotype") refers to  phenotypical variation  as a function of life station. In wide-ranging species, the contributions of  heredity  and environment are not always certain, but their interplay can sometimes be determined by experiment.


Plants display the most obvious examples of ecophenotypic variation. One example are trees growing in the woods developing long straight trunks, with branching crowns high in the canopy, while the same species growing alone in the open develops a spreading form, branching much lower to the ground.  Genotypes  often have much flexibility in the modification and expression of  phenotypes ; in many organisms these phenotypes are very different under varying environmental conditions. The plant  Hieracium umbellatum  is found growing in two different habitats in Sweden. One habitat is rocky sea-side cliffs, where the plants are bushy with broad leaves and expanded  inflorescences ; the other is among sand dunes where the plants grow prostrate with narrow leaves and compact inflorescences. These habitats alternate along the coast of Sweden and the habitat that the seeds of  H. umbellatum  land in determines the phenotype that grows. [ 1 ]   Invasive plants  such as the  honeysuckle  can thrive by altering their  morphology  in response to changes in the environment, [ 2 ]  which gives them a competitive advantage. Another example of a plants phenotypic reaction and adaptation with its environment is how  Thlaspi caerulescens  can absorb the metals in the soil to use to its advantage in defending against harmful microbes and bacteria in its leaves. [ 3 ]  The more immediate responses shown by vascular plants to their environment, for instance a vine's ability to conform to the wall or tree upon which it grows, are not usually considered ecophenotypic, even though the mechanisms may be related. [ 4 ]


Since animals are far less plastic than plants, ecophenotypic variation is noteworthy. When encountered, it can cause confusion in identification if it is not anticipated. The most obvious examples are again common observations, as the dwarfing of aquarium fish living in a restricted environment. [ 5 ]  In asexual reproduction, the parent passes on the entire genome to the next generation. Mutations to the genes are the only source of genetic variation. In sexual reproduction, each parent contributes half of his or her genome to the offspring; thus the offspring contain a mixture of genetic material. Adaptations are traits that increase fitness, the driving force for natural selection. The level of fitness associated with an allele can only be ascertained by comparison with alternate alleles. Traits that increase the survival rate of a species contribute to an animal's fitness, but selection will only favor such traits insofar as survival improves the reproductive success of the organism. More interesting are examples where causation is less clear. Among  mollusks , examples include the  muricid  snail species  Nucella lamellosa , which in rough, shallow waters is generally less spiny than in deeper, quiet waters. [ 6 ]  In  unionid   freshwater bivalves , there are lake, small river, and large river forms of several species. [ 7 ]  In  vertebrates , experiments on mice show reduced length of ears and tails in response to being reared in a lower temperature, a phenomenon known as  Allen's rule . [ 8 ]


In humans, environmental differences due to  lifestyle choices  are a consideration, for instance the differences between someone who spends much time on the sofa before the television, beer in hand, and an individual who spends his time in the gym or the soccer field can be pronounced.  Franz Boas  found that  cephalic index  was to some degree dependent on where a child was born, independent of the child's genetic or cultural heritage. [ 9 ]
Another way in which environmental differences can cause physical and/or behavioral alterations is in being put under great levels of  stress , causing a wide range of effects. Chronic Stress has been proven to cause health issues in many individuals. "Early childhood attempts to cope with fear or rejection ... set up psychological patterns of behavior for the person's later life. Those behaviors in turn affect the biochemical imbalances in the brain's neuronal systems. Those altered imbalances in turn reinforce the behaviors, and the cycle feeds upon itself." "The body reacts biochemically to excessive stress as it attempts to regain its healthy dynamic balance"; "In psychologically stressful situations, hormones may be brought into play to remedy the imbalance the body finds itself in." [ 10 ]


In the General Adaption Syndrome, which is the biological response to stress, there are three stages.
1.) The "Alarm Action" - heart rate increases, blood sugar levels rise, pupils dilate, and digestion slows.
2.) The "Resistance" or "Adaptive" stage - The body attempts to repair the damage which caused the emergency arousal
3.) The "Exhaustion Stage" - The body grows ill; Mentally, possibly by neurosis or even psychotic disturbances, or physically, having the possibility to trigger several kinds of cardiovascular and kidney diseases, and Quite commonly, certain forms of  asthma . [ 10 ]


The  genotype  of an organism is its complete set of genetic material. [ 1 ]  Genotype can also be used to refer to the  alleles  or variants an individual carries in a particular gene or genetic location. [ 2 ]  The number of alleles an individual can have in a specific gene depends on the number of copies of each  chromosome  found in that species, also referred to as  ploidy . In diploid species like humans, two full sets of chromosomes are present, meaning each individual has two alleles for any given gene. If both alleles are the same, the genotype is referred to as  homozygous . If the alleles are different, the genotype is referred to as heterozygous.


Genotype contributes to  phenotype , the observable traits and characteristics in an individual or organism. [ 3 ]  The degree to which genotype affects phenotype depends on the trait. For example, the  petal color  in a  pea plant  is exclusively determined by genotype. The petals can be purple or white depending on the alleles present in the pea plant. [ 4 ]  However, other traits are only partially influenced by genotype. These traits are often called  complex traits  because they are influenced by additional factors, such as environmental and  epigenetic  factors. Not all individuals with the same genotype look or act the same way because appearance and behavior are modified by environmental and growing conditions. Likewise, not all organisms that look alike necessarily have the same genotype.


The term  genotype  was coined by the  Danish   botanist   Wilhelm Johannsen  in 1903. [ 5 ]


Any given gene will usually cause an observable change in an organism, known as the phenotype.  The terms  genotype and phenotype  are distinct for at least two reasons:


A simple example to illustrate genotype as distinct from phenotype is the flower colour in pea plants (see  Gregor Mendel ). There are three available genotypes, PP ( homozygous dominant ), Pp (heterozygous), and pp (homozygous recessive). All three have different genotypes but the first two have the same phenotype (purple) as distinct from the third (white).