On 2015 Mar 16, Donald Forsdyke commented:

NEUTRAL THEORY NOT SUPPORTED. As a reviewer of this paper I recommended acceptance but was unhappy with the conclusion that it supported neutral theory explanations. On the advice of reviewers, my subsequent Letter to the Editor was declined by the Editor (see http://post.queensu.ca/~forsdyke/bioinfor.htm ). The abstract of the letter read:

"Galtier and Lobry compared the optimum growth temperatures of various prokaryotes with the G+C content of their genomic DNA and of various non-mRNA RNA species (e.g. ribosomal RNAs). Since GC bonds confer greater stability on nucleic acid secondary structure than AT bonds, their data strongly suggest that an increase of G+C content is needed for the stabilization at high temperature of rRNA secondary structure (stem-loops), but not of DNA secondary structures.

The authors propose that "any secondary structure that must endure at high temperatures requires a high G+C content", so that "a high proportion" of stem-loop "secondary structures in bacterial genomes is unlikely". Thus, the fact that Chargaff's parity rule (%A=%T, %G=%C) applies to single-stranded DNA (as to single-stranded RNA), is held to be "poorly explained" on the basis of an evolutionary pressure on DNA to form stem-loops (as proposed by Forsdyke 1995; J Mol Evol 41:573-581). Rather the parity rule would be explained by "neutral directional mutational pressure" (Lobry, 1995; J Mol Evol 40:326-330).

However, "any secondary structure" includes the classical duplex DNA secondary structure. This is likely to exist at high temperatures, and presumably requires "other physiological adaptations" than an increase in G+C content. Such adaptations might also apply to DNA stem-loop secondary structure. Thus, in this context selectionist arguments are no less probable than neutralist arguments."

Subsequently the Editor himself (2000; Gene 241: 3-17) came to agree:

"The low GC levels of some thermophilic bacteria do not contradict, as claimed (Galtier and Lobry, 1997), the selectionist interpretation ... . Indeed, different strategies were apparently developed by different organisms to cope with long-term high body temperatures. It is now known that the DNAs of such thermophilic bacteria are very strongly stabilized by particular DNA-binding proteins (Robinson et al., 1998) and that, in turn, their proteins can be stabilized by thermostable chaperoninins (Taguchi et al., 1991)."

For more please see my textbook Evolutionary Bioinformatics (2nd edition 2011, Springer, New York).

On 2017 Nov 30, Donald Forsdyke commented:

CRISPR SPACERS PROVIDE "JUNK" VLA RNAs

A "peculiarity of human thinking" invokes sad head-shaking in some quarters. It is argued, not only that "the vast majority of low abundant transcripts are simply junk," but also that such junk is "simple" (1). Those led to think that junk DNA serves the organism (i.e. can under some conditions be functional and hence selectively advantageous) are labelled "determinists." They can scarcely be distinguished from "ID believers"! There is no mention of the two-decade-old view that very low abundance transcripts (VLA RNAs) represent an intracellular antibody-like repertoire, for which much evidence has since accumulated (2-4).

For microorganisms, the CRISPR system provided a clear example of the functionality of the transcription of their spacer "junk DNA." Ledford notes that the system "adapts to, and remembers, specific genetic invaders in a similar way to how human antibodies provide long-term immunity after an infection" (5). Just as we have germline cascades of V genes that confer immunological specificity on B and T lymphocytes, so microorganisms have their germline spacers that confer a similar specificity on their RNA populations. However, the functionality of an individual spacer "sense" transcript is only tested when a virus with a specific "antisense" sequence enters the cell. Transcription is conditional. The selective advantage can only emerge when the corresponding pathogen attacks.

Thus, the analytical problem is not so "simple" as showing by experimental DNA deletion that the transcript of a specific eukaryotic gene is functional, or as dismissively postulating a requirement for "unacceptably high birth rates." Deletion of a single human V-region gene could show no selective effect if no corresponding pathogens invaded the body. Even if there were such an invasion, other V-regions would likely be able to compensate for the deletion. Similarly, deleting a segment of "junk" DNA is unlikely to impact survival if some of the wide spectrum of alternative "junk" transcripts can compensate for this defect in the RNA antibody-like repertoire.

1.Sverdlov E (2017) Transcribed junk remains junk if it does not acquire a selected function in evolution. BioEssays doi: 10.1002/bies.201700164. Sverdlov E, 2017

2.Cristillo AD, Mortimer JR, Barrette IH, Lillicrap TP, Forsdyke DR (2001) Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load. J Theor Biol 208:475-491. Cristillo AD, 2001

3.Forsdyke DR, Madill CA, Smith SD (2002) Immunity as a function of the unicellular state: implications of emerging genomic data. Trends Immunol 23:575-579. Forsdyke DR, 2002

4.Forsdyke DR (2016) Evolutionary Bioinformatics. 3rd edition. Springer, New York, pp. 279-303.

5.Ledford H (2017) Five big mysteries about CRISPR’s origins. Nature 541:280-282. Ledford H, 2017

On 2017 Oct 02, Donald Forsdyke commented:

VIRAL REPRODUCTIVE ISOLATION WITHIN A COMMON HOST CELL

This otherwise admirable article (Hunter P, 2017) begins with the curious assertion that, "since they depend on their host for replication," then viruses cannot "be categorized as species on the basis of reproductive isolation." The latter prevents recombination between organisms and so forms the most generally accepted definition of species. Virus species whose members share a common host cell, and depend on that cell for their replication, are still able to retain their species individuality. Their members do not mutually destroy each other by recombinational blending of their genomes. They are reproductively isolated from each other.

When we compare two viral species that have a common host cell, with two viral species that, even within a common host, do not share a common cell, we would expect to observe a fundamental difference related to their reproductive isolation mechanisms. If that difference is found to apply to other viral pairs that occupy a common host cell, then a fundamental isolation mechanism has been identified.

Such a difference was first related to the base compositions of insect viruses (1), a then to the base compositions of herpes viruses (2). A more extreme example arose from studies of retroviruses that share a T-lymphocyte host. The AIDS virus (HIV1) and human T cell leukaemia virus (HTLV1), can be assumed to have evolved from a common ancestor. Differentiation of members of that ancestral species within a common host cell into two independent populations would have required some mechanism to prevent their blending. Thus, we see today a wide divergence in base compositions. HIV1 is one of the highest AT-rich species know. HTLV1 is one of the highest GC-rich species known (3). There is high differentiation of chromosomal nucleic acids.

In these viruses there has been no opportunity for other reproductive isolation mechanisms to supersede chromosomal mechanisms. Diffusible cytoplasmic products make the subsequent evolution of genic incompatibilities less likely, and being in a common host cell there is no equivalent of prezygotic isolation as conventionally understood (4).

1. Wyatt GR (1952) The nucleic acids of some insect viruses. J Gen Physiol 36:201-205. WYATT GR, 1952
2. Schachtel GA et al. (1991) Evidence for selective evolution of codon usage in conserved amino acid segments of human alphaherpesvirus proteins. J Mol Evol 33:483-494. Schachtel GA, 1991
3. Bronson EC, Anderson JN (1994) Nucleotide composition as a driving force in the evolution of retroviruses. J Mol Evol 38:506-532. Bronson EC, 1994
4. Forsdyke DR (1996) Different biological species broadcast their DNAs at different (G+C)% wavelengths. J Theoret Biol 178:405-417. Forsdyke DR, 1996% "wavelengths".")

On 2017 Sep 27, Donald Forsdyke commented:

"CLOSE TO SELF" AND "NEAR SELF"

A major conclusion of this elegant modeling study is that "TCR selection against self-peptides has a minimal influence on the recognition of peptides which are 'close' to self." Thus, "TCR negative selection by host peptides has only a weak suppressive effect on detecting peptides which closely resemble self." This agrees with a somewhat less elegant modeling study that invoked lymphocyte clones selected for anti-"near-self" immune reactivity. These would normally have escaped negative selection (i.e. would have been positively selected; 1). The "near-self" viewpoint contrasted with the then prevailing "altered self" viewpoint (2). However, whereas George et al. (2017) regard their study as "empirical," the earlier study (1) arose from consideration of alloreactive phenomena and recognized implications for cancer immunotherapy in keeping with an "overall objective of optimizing CRL therapy" (3, 4). Full historical reviews are available (5, 6).

1. 1.Forsdyke DR (1975) Further implications of a theory of immunity. J Theor Biol 52: l87-l98.Forsdyke DR, 1975

2. 2.Forsdyke DR (2005) "Altered-self" or "near-self" in the positive selection of lymphocyte repertoires? Immunol Lett 100: 103-106.Forsdyke DR, 2005

3. 3.Forsdyke (1977) Grant application

4. 4.Forsdyke DR (1999) Heat shock proteins as mediators of aggregation-induced "danger" signals: implications of the slow evolutionary fine-tuning of sequences for the antigenicity of cancer cel1s. Cell Stress Chaperone 4: 205-210.Forsdyke DR, 1999

5. 5.Forsdyke DR (2012) Immunology (1955-1975): The natural selection theory, the two signal hypothesis and positive repertoire selection. J Hist Biol 45: 139-161.Forsdyke DR, 2012

6. 6.Forsdyke DR (2015) Lymphocyte repertoire selection and intracellular self/not-self discrimination: historical overview. Immun Cell Biol 93: 297-304.Forsdyke DR, 2015

On 2017 Dec 03, Donald Forsdyke commented:

REDESIGNATING SELF AS NOT-SELF MARKERS

A cell's altruistic service to the population of cells that comprise its host organism may be compromised by a foreign pathogen or by a mutated driver cancer gene (both deemed "non-self"). Such intracellular compromising agents can first be addressed by internal sensing and auto-destructive mechanisms. Should one of these fail, then external sensing and destructive mechanisms, involving reactions with specific predatory T cells, may come into play. A compromised cell has the option of displaying peptides as pMHC complexes to see if they are recognized by members of T cell populations that, following thymic surveillance and deletion of nascent strongly self-reacting T cells, are programed to eliminate cells displaying non-self markers.

While such markers may arise from foreign proteins or mutated self proteins, Mishto and Liepe note that the scope of markers ("the antigenic landscape") can be greatly increased by redesignating potential self markers (unspliced peptides in pMHC complexes) as non-self (1). This creation of foreign from self is achieved by splicing and trimming non-contiguous peptides to create novel peptides that would not have passed thymic filters and so would be seen as non-self. Two corollaries of this are that such peptide splicing must not occur in the thymus and that, to militate against autoimmunity, extra-thymic specific splicing of separate protein segments would not occur randomly in uncompromised cells.

Thus, some elements of an internal sensing mechanism within a compromised cell would be needed to foster an extension of the antigenic landscape. The growing evidence for such a mechanism in the antigen presentation pathway (intracellular self/non-self discrimination) is presented elsewhere (2). I agree that "the unexpectedly large frequency and amount of … spliced peptides may … have profound implications for the concept of self/nonself peptide presentation" (3).

1.Mishto M, Liepe J. (2017) Post-translational peptide splicing and T cell responses. Trends in Immunology 38:904-915 Mishto M, 2017

2.Forsdyke DR (2015) Lymphocyte repertoire selection and intracellular self/not-self discrimination: historical overview. Immunology and Cell Biology 93:297-304. Forsdyke DR, 2015

3.Liepe J et al. (2016) A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354:354-358.Liepe J, 2016

On 2018 Jan 20, Donald Forsdyke commented:

PATHOGEN COEVOLUTION AND THE ANTIGENIC UNIVERSE

The distinction between selective and instructive (Lamarckian) systems of immunity (1) – originating with Paul Ehrlich – was clearly set out in 1957 by Talmage (2) who, with Burnet, can be considered a "father of clonal selection theory" (3, 4). Its historical omissions aside, this bold attempt to place the evolution of immune systems in a broad context raises other concerns.

Although mentioning "the complex adaptation of the immune repertoire to the antigenic environment," and the need "continuously to acquire and store open-ended information about the antigenic environment," the coevolution of that antigenic environment (e.g. the coevolution of pathogens) does not seem to have been considered.

While the authors agree with Burnet that "distinguishing tumours from normal self is likely to be the most challenging task for Darwinian immunity," it is not recognized that the most successful pathogens are those that, through mutation, can come close to self. Whereas tumours represent mutations away from self, successful pathogens represent mutations towards self (by means of which they seek to exploit 'holes' in immune repertoires; 5). In both circumstances, this greatly simplifies the evolutionary task of a host. It does not have to depend on "the open-ended nature of the receptor repertoire." It does not have to "constitute a system of 'unlimited heredity' within the immune system." It does not have to "be broad enough to recognize the 'potential universe of antigens'." The scope of its task is greatly reduced.

As long ago proposed (6), and increasingly recognized (7, 8), it would be evolutionarily advantageous for organisms to focus their immune cell receptors on 'near self' antigenic specificities, rather than to attempt to anticipate the entire universe of antigens. Organisms achieve this, not through negative, but through positive selection of their immune repertoires. From the outset, organisms and their pathogens have coevolved and it would seem incorrect to suppose for the immune system that positive selection "could only be added at advanced stages of its evolution" (9). It is fundamental to immune system evolution.

1.Muller V, Boer RJ de, Bonhoeffer S, Szathmary E (2018) Biol Rev 93:505-528. Müller V, 2018

2.Talmage DW (1957) Allergy and immunology. Ann Rev Med 8:239-256 TALMAGE DW, 1957

3.Forsdyke DR (1996) The origins of the clonal selection theory of immunity. FASEB J 9:164-166.Forsdyke DR, 1995

4.Lederberg J (2002) Instructive selection and immunological theory. Immunol Rev 185:50-53.Lederberg J, 2002

5.Calis JJA, de Boer RJ, Kesmir C (2012) Degenerate T-cell recognition of peptides on MHC molecules creates large holes in the T-cell repertoire. PLoS Comput Biol 8:e1002412.Calis JJ, 2012

6.Forsdyke DR (1975) Further implications of a theory of immunity. J Theoret Biol 52:l87-l98.Forsdyke DR, 1975

7.Vrisekoop N, Monteiro JP, Mandl JN, Germain RN (2014) Revisiting thymic positive selection and the mature T cell repertoire for antigen. Immunity 41:181-190.Vrisekoop N, 2014

8.Marrack P. et al. (2017) The somatically generated portion of T cell receptor CDR3alpha contributes to the MHC allele specificity of the T cell receptor. eLife 6:e30918.Marrack P, 2017

9.Forsdyke DR (2016) Evolutionary Bioinformatics. 3rd Edition. Springer, New York.

On 2017 Jul 17, Donald Forsdyke commented:

IF G-QUADRUPLEXES, WHY SO MANY ADENINES?

It is good to see the problem of EBV immune evasion focused, not on the translation product of EBNA1 mRNA (1), but on the mRNA itself (2). However, it is puzzling that the sequence encoding the glycine-alanine repeats is enriched not only in guanines (Gs), but also in adenines (As). In such a GC-rich genome (60% GC), there is a scarcity of As, yet they are concentrated in the glycine-alanine repeat-encoding region. In other words, codons have been selected for their general purine-richness, not just for their G-richness (3). While it is conceivable that the As somehow assist consecutive Gs to form G-quadruplexes, consideration might have been given to the hypothesis that the G-quadruplexes may merely be helpful by-products of the fundamental need to purine-load the mRNA.

EBV is not alone in this respect. EBV and HTLV-1 share common characters. Both are deeply latent, GC-rich viruses. They persist in their human hosts for long periods often with no obvious detrimental effects. Most of their proteins are encoded by pyrimidine-rich mRNAs. The HTLV-1 provirus encodes its pyrimidine-rich mRNAs in its "top" sense strand. But there is a "bottom" strand transcript. This is heavily R-loaded and is translated into a basic zipper protein (HBZ) which is poorly immunogenic and is increasingly seen, like EBNA-1, as playing a major role in immune evasion (4-6).

1. 1.Levitskaya, J. et al. (1995) Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375:685–688. Levitskaya J, 1995
2. 2.Lista MJ et al. (2017) Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA-1 mRNA. Nature Commun 8:16043. Lista MJ, 2017
3. 3.Cristillo AD et al. (2001) Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load. J Theor Biol 208:475–491.Cristillo AD, 2001
4. 4.Cook LB et al. (2013) HTLV-1: Persistence and pathogenesis. Virology 435:131–140. Cook LB, 2013
5. 5.Shiohama et al. (2016) Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status. Retrovirology 13:29 Shiohama Y, 2016
6. 6.Forsdyke DR EBV Webpage

On 2017 May 05, Donald Forsdyke commented:

ORGANIC MEMORY

The view that Richard Semon's work was neglected seems to be based on psychologist Daniel Schacter's 1982 text (1). This was reissued with a new title and a few changes in 2001, without mention of the profound interim account by historian Laura Otis (2). While the authors cite my 2006 text on Samuel Butler and Ewald Hering, later work corroborates and extends Otis’s study and casts a somewhat different light on the authors' prime hero (3, 4).

Even if offering a list of heroes that is "entirely personal," a paper that extolls the "benefits of exploring the history of science" and of acknowledging our "debts … to those scientists who have offered key ideas," could have mentioned the doubts cast on Semon by Freud and Hertzog, and Semon's dismissal of Butler's work as "rather a retrogression than an advance."

1. Schacter DL (1982) Stranger behind the Engram: Theories of Memory and the Psychology of Science. Hillsdale, NJ: Erlbaum.

2. Otis L (1994) Organic Memory. History and the Body in the Late Nineteenth and Early Twentieth Centuries. Lincoln: University of Nebraska Press.

3. Forsdyke DR (2009) Samuel Butler and human long term memory: is the cupboard bare? J Theor Biol 258:156-164.Forsdyke DR, 2009

4. Forsdyke DR (2015) "A vehicle of symbols and nothing more." George Romanes, theory of mind, information, and Samuel Butler. History of Psychiatry 26:270-287. Forsdyke DR, 2015

On 2017 May 19, Donald Forsdyke commented:

THE VIRUS-VIRUS ARMS RACE

For commentary on this paper please see ArXiv preprint (1). For further discussion see commentary on a BioRxiv preprint (2).

(1) Forsdyke DR (2016) Elusive preferred hosts or nucleic acid level selection? ArXiv Preprint (https://arxiv.org/abs/1612.02035).

(2) Shmakov SA, Sitnik V, Makarova KS, Wolf YI, Severinov KV, Koonin EV (2017) The CRISPR spacer space is dominated by sequences from the species-specific mobilome. BioRxiv preprint (http://biorxiv.org/content/early/2017/05/12/137356).

On 2017 Jul 25, Donald Forsdyke commented:

LECTIN PATHWAY STUDIES WITH PLANT MANNOSE-BINDING LECTINS

Papers on the lectin pathway (LP) of complement activation in animal sera generally refer to animal mannose-binding lectins (MBLs), with little reference to work with plant MBLs. For example, citing May and Frank (1973), this fine paper states: "Reports of unconventional complement activation in the absence of C4 and/or C2 predate the discovery of LP." Actually, a case can be made that the discovery of the LP predates May-Frank.

The MASP-binding motif on animal MBL, which is necessary for complement activation, includes the amino acid sequence GKXG (at positions 54-57), where X is often valine. The plant lectin concanavalin-A (Con-A) has this motif at approximately the same position in its sequence (the 237 amino acid subunit of Con-A had the sequence GKVG at positions 45-48). The probability of this being a chance event is very low. Indeed, prior to the discovery of MASP involvement, Milthorp & Forsdyke (1970) reported the dosage-dependent activation of complement by Con-A.

As far as I am aware, it has not been formally shown that MASP is involved in the activation of the complement pathway by this plant MBL. Our studies in the 1970s demonstrated that Con-A activates complement through a cluster-based mechanism, which is consistent with molecular studies of animal MBL showing “juxtaposition- and concentration dependent activation” (Degn et al. 2014). References to our several papers on the topic may be found in a review of innate immunity (Forsdyke 2016).

Degn SE et al. (2014) Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes. Proc Natl Acad Sci USA 111:13445-13450. Degn SE, 2014

Forsdyke DR (2016) Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective. Microb Infect 18: 450-459. Forsdyke DR, 2016

May JE, Frank MM (1973) Hemolysis of sheep erythrocytes in guinea pig serum deficient in the fourth component of complement. I. antibody and serum requirements. J Immunol 111: 1671-1677. May JE, 1973

Milthorp PM, Forsdyke DR (1970) Inhibition of lymphocyte activation at high ratios of concanavalin A to serum depends on complement. Nature 227:1351-1352 Milthorp P, 1970

Yaseem et al. (2017) Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2. FASEBJ 31:2210-2219 Yaseen S, 2017

On 2017 Jan 03, Donald Forsdyke commented:

ASSUME A SPHERICAL COW?

Following a multidisciplinary study of milk production at a dairy farm, a physicist returned to explain the result to the farmer. Drawing a circle she began: "Assume the cow is a sphere … ." (1) This insider math joke may explain Koonin’s puzzlement that "most biologists do not pay much attention to population genetic theory" (2).

The bold statement that "nothing in evolution makes sense except in the light of population genetics," cannot be accepted by biologists when evolution is portrayed in terms of just two variables, "an interplay of selection and random drift," constituting a "core theory." While mathematical biologists might find it "counterintuitive" that "the last common eukaryotic ancestor had an intron density close to that in extant animals," this is not necessarily so for their less mathematical counterparts. They are not so readily inclined to believe that an intron "is apparently there just because it can be" (3).

While expediently adopting "null models" to make the maths easier, population geneticists are not "refuted by a new theoretical development." They have long been refuted by old theoretical developments, as illustrated by the early twentieth century clash between the Mendelians and the Biometricians (4). It is true that by adjusting "selection coefficient values" and accepting that "streamlining is still likely to efficiently purge true functionless sequences," the null models can closer approximate reality. But a host of further variables – obvious to many biologists – still await the acknowledgement of our modern Biometricians.

1.Krauss LM (1994) Fear of Physics: A Guide for the Perplexed. Jonathan Cape, London.

2.Koolin EV (2016) Splendor and misery of adaptation, or the importance of neutral null for understanding evolution. BMC Biology 14:114 Koonin EV, 2016

3.Forsdyke DR (2013) Introns First. Biological Theory 7, 196-203.

4.Cock AG, Forsdyke DR (2008) "Treasure Your Exceptions." The Science and Life of William Bateson. Springer, New York.

On 2016 Nov 21, Donald Forsdyke commented:

THE RNA WORLD AND DARRYL REANNEY

The title of historian Neeraja Sankaran's paper in a "special historical issue" of the Journal of Molecular Evolution implies that the RNA world idea was formulated 30 years ago (i.e. 1986) by a single author, Walter Gilbert (1). Yet the paper traces the story to authors who wrote at earlier times. Missing from the author list is Darryl Reanney who, like Gilbert, documented a "genes in pieces" hypothesis in February 1978 and went on to explore the RNA world idea with the imperative that error-correcting mechanisms must have evolved at a very early stage (2). Much of Reanney's work is now supported (3).

However, Sankaran cites the video of a US National Library of Medicine meeting organized by historian Nathaniel Comfort on 17th March 2016 (4). Here W. F. Doolittle, who had consistently cited Reanney, discusses the evolutionary speculation triggered by the discovery of introns in 1977, declaring that "several things came together at that time," things that "a guy named Darryl Reanney had been articulating before that." Furthermore, "it occurred to several of us simultaneously and to Darryl Reanney a bit before – before me anyway – that you could just recast the whole theory in terms of the RNA world."

Gilbert himself thought that "most molecular biologists did not seriously read the evolution literature; probably still don’t." Indeed, contemporary molecular biologists writing on "the origin of the RNA world," do not mention Reanney (5). Thus, we look to historians to put the record straight.

1.Sankaran N (2016) The RNA world at thirty: a look back with its author. J Mol Evol DOI 10.1007/s00239-016-9767-3 Sankaran N, 2016

2.Reanney DC (1987) Genetic error and genome design. Cold Spring Harb Symp Quant Biol 52:751-757

3.Forsdyke DR (2013) Introns first. Biological Theory 7:196-203 Paper here

4.Comfort N (2016) The origins of the RNA world. Library of Congress Webcast. NLM Webcast

5.Robertson MP, Joyce GF (2012) The origins of the RNA world. Cold Spring Harb Perspect Biol 4:a003608. Robertson MP, 2012

On 2016 Nov 09, Donald Forsdyke commented:

Marketing in science

In it ironic that Vincent Detours insightful analysis of the "managers" who outdo the "competent" comes at a time when the triumph of marketing over ability is so evident on the political scene. For any who might think this could not happen in science, two accounts of the career of Niels Jerne will perhaps provide helpful reading (1, 2).

1.Soderqvist T (2003) Science as Autobiograph: the Troubled Life of Niels Jerne (Yale Univ. Press, New Haven).

2.Eichmann K (2008) The Network Collective: Rise and Fall of a Scientific Paradigm (Birkhauser, Berlin).

On 2016 Nov 03, Donald Forsdyke commented:

CONSIDERATION OF NUCLEIC ACID LEVEL SELECTION?

The authors set out "to investigate the evolutionary factors that affect serine codon set switches" (i.e. between TCN and AGY). Their "findings imply unexpectedly high levels of selection" (1). Indeed, the data strongly support the conclusion that codon mutations "are driven by selection." It is conjectured that the codon mutation "switch would involve as an intermediate either threonine ACN or cysteine TGY, amino acid residues with properties substantially different from those of serine, so that such changes are unlikely to be tolerated at critical functional or structural sites of a protein."

However, it does not follow that the unsuitability of the interim amino acids drove the rapid tandem substitutions. Choice of "coincident codons" has long been seen as influenced by pressures acting at the nucleic acid level (2-4). These pressures evolve in parallel with, and sometimes dominate, protein pressures. One example is purine-loading pressure (3). If this cannot be satisfied by changes at third codon positions, then sometimes the organism must accept a less favorable amino acid. With serine codons, a change from TCN to AGY (i.e. first and second codon positions) can increase purine-loading pressure without compromising the amino acid that is encoded see Ref. 3.

1.Rogozin IB, Belinky F, Pavlenko V, Shabilina SA, Kristensen DM, Koonin EV (2016) Evolutionary switches between two serine codon sets are driven by selection. Proc Natl Acad Sci USA www.pnas.org/cgi/doi/10.1073/pnas.1615832113 Rogozin IB, 2016

2.Bains W. (1987) Codon distribution in vertebrate genes may be used to predict gene length. J Mol Biol 197:379-388. Bains W, 1987

3.Mortimer JR, Forsdyke DR (2003) Comparison of responses by bacteriophage and bacteria to pressures on the base composition of open reading frames. Appl Bioinf 2: 47-62. Mortimer JR, 2003

4.Forsdyke DR (2016) Evolutionary Bioinformatics, 3rd edition (Springer, New York).

On 2016 Dec 05, Donald Forsdyke commented:

SELECTIVE PRESSURE TO CONSERVE VIRUS SPECIES IDENTITY

The authors correctly note that "the most obvious parameter associated with G + C content is the strength of molecular hybridization of polynucleotide duplexes" (1). Such hybridization controls recombination, which is favored when there is close sequence resemblance between different co-infecting viruses ("complete alignment conserved"), and is impeded when there is less sequence resemblance ("complete alignment variable"). The latter anti-recombination activity can be considered in relation to speciation mechanisms that initiate and retain taxonomic differentiations. As recently noted by Meyer et al., allied species of "viruses that infect the same [host] species and cell types are thought to have evolved mechanisms to limit recombination." Without such limitations the genomes would blend and co-infectants would lose their independence as distinct viral species. Mechanisms overcoming this selective disadvantage include "divergences in nucleotide composition and RNA structure that are analogous to pre-zygotic barriers in plants and animals" (2).

Thus, a nucleic acid region may be "conserved," not only because it encodes a protein (i.e. there is "protein pressure" on the sequence), but because it has a specific nucleotide composition (e.g. "GC-pressure"). While protein pressure mainly affects the first and second codon positions, GC-pressure can affect all codon positions. Indeed, at first and second codon positions there may be conflict between pressures, especially when protein pressure is high (i.e. in regions where amino acid conservation is high); then GC-pressure is constrained to vary only at the more flexible third codon position. In contrast, when protein pressure is low (i.e. in regions where amino acid conservation is low), then GC-pressure has greater freedom to affect all codon positions.

If, to avoid recombination, there is selective pressure on one branch of a diverging line to decrease its GC%, then it would be predicted that "the GC% of nucleotides encoding conserved amino acid (AA) residues" would be "consistently higher than that of nucleotides encoding variable AAs," where the pressure to decrease GC% has fuller rein to encompass all three codon positions (1). Conversely, it would be predicted that when there is pressure on a diverging line to increase GC%, then it would be predicted that the GC% corresponding to conserved codons would be consistently lower than that of non-conserved codons (e.g. Ebolavirus).

For flavivirus "the mean G% of the core conserved AA residues is higher (35%) than that of the variable AA residues (28%), but the mean G3% of the core conserved AA residues (28%) is similar to that of the variable AA residues (29%)" (1). While consistent with the above views, there is need for information on C3% and relative frequencies of synonymous codons (e.g. the two cysteine codons correspond either to low or high GC%). More details of selective anti-recombination pressures are presented elsewhere (3, 4). Similar considerations may apply to codon biases and GC% among mycobacteriophages (5).

1.Klitting R, Gould EA & de Lamballerie X (2016) G + C content differs in conserved and variable amino acid residues of flaviviruses and other evolutionary groups. Infection, Genetics and Evolution 45: 332-340.Klitting R, 2016

2.Meyer JR, Dobias DT, Medina SJ, Servilio L, Gupta A, Lenski RE (2016) Ecological speciation of bacteriophage lambda in allopatry and sympatry. Science 354: 1301-1304. Meyer JR, 2016

3.Forsdyke (2014) Implications of HIV RNA structure for recombination, speciation, and the neutralism-selectionism controversy. Microbes & Infect16:96-103. Forsdyke DR, 2014

4.Forsdyke DR (2016) Evolutionary Bioinformatics, 3rd edition. Springer, New York.

5.Esposito LA, Gupta S, Streiter F, Prasad A, Dennehy JJ (2016). Evolutionary interpretations of mycobacteriophage biodiversity and host-range through the analysis of codon usage bias. Microbiol Genomics 2(10), doi: 10.1099/mgen.0.000079. See arXiv preprint

On 2016 Sep 07, Donald Forsdyke commented:

PATERNITY OF INNATE IMMUNITY?

The accolades cast on scientists we admire include that of paternity. Few will dispute that Gregor Mendel was the father of the science we now call genetics. At the outset, this paper (1) hails Metchnikoff (1845-1916) as “the father of innate immunity.” However, an obituary of US immunologist Charles Janeway (1943-2003) hails him similarly (2). Can a science have two fathers? Well, yes. But not if an alternative of Mendelian stature is around. While paternity is not directly ascribed, a review of the pioneering studies on innate immunity of Almroth Wright (1861-1947) will perhaps suggest to some that he is more deserving of that accolade (3).

1.Gordon S (2016) Phagocytosis: the legacy of Metchnikoff. Cell 166:1065-1068 Gordon S, 2016

2.Oransky I (2003) Charles A Janeway Jr. Lancet 362:409.

3.Forsdyke DR (2016) Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective. Microbes & Infection 18:450-459. Forsdyke DR, 2016

On 2016 May 20, Donald Forsdyke commented:

FACTS BEFORE COUNTERFACTS

Counterfactual explorations can provide intriguing insights (1). But we have to be sure that the facts themselves are correct in the first place. In the context of Weldon, reference to "bad-tempered conflict with Mendel’s followers," really means conflict with William Bateson. While it is correct that the doctrinaire "Mendelian ‘genes for’ approach is increasingly seen as out of step with twenty-first-century biology" (1), for Bateson (1861-1926) the approach was also seen as out of step with twentieth-century biology.

Well aware of developmental and environmental factors, Bateson recognized that the biochemical characterization of genes should be high on the twentieth century research agenda. Thus, near the end of his life he declared that “Our knowledge of the nature of unorganized matter must first be increased. For a long time we may have to halt” in getting to grips with the underlying biological principles (2). However, he argued forcefully for looking beyond the visible characters of an organism (its conventional phenotype) to what we now regard as its genome phenotype (3). It was here that the answer to Darwin's fundamental question - the origin of species - was likely to lie.

Weldon had allied himself with Pearson whose brilliant work (later built on by Fisher), was to create modern biostatistics. But those were early days and they made elementary mistakes that Bateson was quick to point out. For example, Bateson would have bridled at the idea that “first year biologists” could serve as a reliable “control” against which to compare “second year humanities undergraduates” (1). Yes, we should “study Mendel, but let him be part of his time”(1). And as related by Meijer (4), Mendel followed the statistics of his time. Indeed, his results have withstood the test of time.

(1) Radick G (2016) Teach students the biology of their time. Nature 533:293 Radick G, 2016

(2) Cock AG, Forsdyke DR (2008) "Treasure Your Exceptions." The Science and Life of William Bateson. Springer, New York.

(3) Forsdyke, D. R. (2010) George Romanes, William Bateson, and Darwin's "Weak Point." Notes Rec R Soc Lond 64:139-154.

(4) Meijer OG (1982) The essence of Mendel’s discovery. In: Gregor Mendel and the Foundations of Genetics. Orel V (ed). The Mendelianum of the Moravian Museaum, Brno, pp. 173-200.

On 2016 Jul 21, Donald Forsdyke commented:

THEORY-DRIVEN RESEARCH

The results of Enard et al. Enard D, 2016 "draw a broader picture where adaptation against viruses involves not only the specialized antiviral response, but also the entire population of host proteins." Indeed, Petrov has remarked: "Organisms have been living with viruses for billions of years" so on theoretical grounds alone "those interactions have affected every part of the cell."

Given recent disparagement of theoretical work Lander ES, 2016, it is nice to see results that are consistent with theory (e.g. Trends Immunol (2002) 23:575-579; Paper with Endnotes). Enard et al. now "conservatively estimate" that "viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals." Such "virus interacting proteins" vastly exceed the known proteins that regularly engage in immune responses to viruses (e.g. protein kinase R).

This is consistent with the 2002 postulate of the existence of intracellular protein "immune receptors" Forsdyke DR, 2002. Thus, over evolutionary time a protein that primarily evolved for a distinct function, but also happened to cross-react with some virus component, would in addition be selected by virtue of the latter function.

On 2016 Apr 28, Donald Forsdyke commented:

RNAS ENCODING HBZ AND EBNA1 PROTEINS ARE BOTH PURINE-LOADED

The sensitive quantification of HBZ protein levels in various clinical conditions (1) is a major advance. Unlike other HTLV1 proteins, the latency-controlling HBZ protein is encoded by the antisense strand. Thus, whereas the main-gene-encoding ‘top’ strand of the latent virus is pyrimidine-rich, the complementary, ‘bottom,’ strand that encodes HBZ is purine-rich. Likewise, most genes in Epstein-Barr virus are encoded by pyrimidine-rich strands, but the latency-controlling EBNA1 protein is encoded by a purine-rich strand.

The speculation that this purine-loading militates against the formation of double-stranded RNA and hence dampens the host immune response (2) is in keeping with:

(a) the “extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection,”

(b) the possibility that “the most important actions of HBZ, which are critical to HTLV-1 persistence, are exerted at the RNA level, and not the protein level,” and

(c) the view that “minimized HBZ protein translation is a sophisticated viral strategy for evasion from the host T cell response.”

(1) Shiohama et al. (2016) Retrovirology 13:29 Shiohama Y, 2016

(2) Forsdyke DR (2014) Microbes and Infection 16, 96-103 Forsdyke DR, 2014

On 2016 Apr 06, Donald Forsdyke commented:

PROTEIN SIZE AND CONCENTRATION DETERMINE DOSAGE-SENSITIVITY?

With the goal of understanding “the evolution of incomplete sex chromosome dosage compensation mechanisms in general,” the authors confirm that, among dosage-sensitive genes, those whose products specifically engage in stoichiometric complexes with other gene products, have a high degree of dosage-compensation. However, most genes are not dosage-limited by stoichiometry and “perplexing questions” remain. It is suggested that “certain loci … simply lack dosage effects” (my italics). In other words, certain loci “simply” contribute more to dosage effects than others.

While far from simple, this proposal is consistent with dosage compensation being more concerned with collective protein functions than with the specific functions of individual proteins (1). In the crowded cytosol the protein collective should exert an entropy-driven aggregation pressure on individual proteins, as part of a process of intracellular self/not-self discrimination (2). It is predicted that small, low concentration, proteins, will hardly influence aggregation pressure, so here there is no necessity for dosage compensation between the sexes. However, large, high concentration, proteins will greatly influence aggregation pressure, so here regulation of dose, on a gene-by-gene basis or otherwise, should be critical. Failure to regulate such dosage in human females would explain their susceptibility to autoimmune diseases (3-5).

1.Forsdyke DR (1994) Relationship of X chromosome dosage compensation to intracellular self/not-self discrimination: a resolution of Muller's paradox? J Theor Biol 167:7-12. Forsdyke DR, 1994

2.Forsdyke DR (2009) X chromosome reactivation perturbs intracellular self/not-self discrimination. Imm Cell Biol (2009) 87:525-528.Forsdyke DR, 2009

3.Dillon SP et al. (2012) Sex chromosome aneuploidies among men with systemic lupus erythematosus. J Autoimmun 38:J129-J134. Dillon SP, 2012

4.Forsdyke DR (2012) Ohno's hypothesis and Muller's paradox: sex chromosome dosage compensation may serve collective gene functions. BioEssays 34:930-933. Forsdyke DR, 2012

5.Wang J et al. (2016) Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proc Natl Acad Sci USA doi/10.1073/pnas.1520113113 Wang J, 2016

On 2016 Apr 04, Donald Forsdyke commented:

COLLECTIVE GENE FUNCTIONS SHOULD BE TAKEN INTO ACCOUNT

Gene products have both individual and collective functions (e.g. the Donnan equilibrium; 1). Wang et al. suggest that female susceptibility to autoimmune diseases may reflect incomplete dosage-compensation, which results in overexpression of certain X chromosome-located, immunity-related, genes (2). However, they refer to studies by Scofield and colleagues [ref. 11] on diseases with changes in numbers of entire X chromosomes (e.g. XXY), indicating biallelic expression of many genes. Scofield has noted that collective, as well as specific, gene functions must be considered (3). This is in keeping the hypothesis that the cytosolic aggregation pressure exerted by the protein collective is immunologically important (4, 5). <br>

1.Loeb J (1921) Donnan equilibrium and the physical properties of proteins. 1. Membrane potentials. J Gen Physiol. 3:667-90. Loeb J, 1921<br>

2.Wang J et al. (2016) Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proc Natl Acad Sci USA doi/10.1073/pnas.1520113113 Wang J, 2016<br>

3.Dillon SP et al. (2012) Sex chromosome aneuploidies among men with systemic lupus erythematosus. J Autoimmun 38:J129-J134.Dillon SP, 2012<br>

4.Forsdyke DR (2009) X chromosome reactivation perturbs intracellular self/not-self discrimination. Imm Cell Biol (2009) 87:525-528. Forsdyke DR, 2009<br>

5.Forsdyke DR (2012) Ohno's hypothesis and Muller's paradox: sex chromosome dosage compensation may serve collective gene functions. BioEssays 34:930-933. Forsdyke DR, 2012

On 2016 Jan 26, Donald Forsdyke commented:

An accurate account of the exciting lead up to the 1977 discovery of split genes and obviously, as indicated by the title, Arnold Berk's fine perspective review does not deal with the alternative hypotheses that then appeared. However, readers are left to conclude that the "original suggestion" of Gilbert is now backed by "considerable evidence," so that perhaps the alternatives are disposed of. We should not forget that, in step with Gilbert, Darryl Reanney in Australia was fostering a viewpoint for which "considerable evidence" has also accumulated [1].

[1] Forsdyke DR (2013) Introns first. Biological Theory 7:196-203 http://post.queensu.ca/~forsdyke/introns3.htm

On 2016 Jan 28, Donald Forsdyke commented:

HYPOTHESIS-DRIVEN RESEARCH

The discoveries of a cytosolic microbial adaptive immune system (CRISPR) and its applications to genome editing are major scientific advances. A review of the history of this magnificent achievement, made mainly by young people close to those with abundant research funds, is welcome. But the implication that this history supports the non-hypothesis-driven approach to research is questionable.

Backed by inexpensive bioinformatic analyses, a hypothesis of cytosolic innate immunity was developed in the 1990s [1-3]. Had this CRISPR-analogous hypothesis been backed by funding, CRISPR and its applications might have been achieved more expeditiously. Thus, there are many roads to Rome. Because the well-equipped army that took route A arrive first, it does not follow that route A is superior to route B. Likewise, this comment could have been written in prose or poetry. Your liking (perhaps) of the present prose rendition, does not disprove the proposition that a poetic version might have been superior.

[1] Forsdyke & Mortimer (2000) Chargaff’s legacy. Gene 261, 127-137.Forsdyke DR, 2000

[2] Cristillo et al. (2001) Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load. J Theor Biol 208, 475-491.Cristillo AD, 2001

[3] Forsdyke, Madill & Smith (2002) Immunity as a function of the unicellular state: implications of emerging genomic data. Trends Immunol 23, 575-579.Forsdyke DR, 2002

On 2016 Jan 26, Donald Forsdyke commented:

None

On 2016 Jun 25, Donald Forsdyke commented:

HISTORY OF LECTIN PATHWAY RESEARCH

The history of this field has recently been reviewed (1,2).

1 Forsdyke DR (2016) Microbes & Infection 18, 450-459. Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective. Forsdyke DR, 2016

2 Sims RB, Schwaeble W, Fujita T (2016) Complement research in the 18th-21st centuries: progress comes with new technology. Immunobiology 221:1037-1045 Sim RB, 2016

On 2015 Nov 16, Donald Forsdyke commented:

COMPLEMENT ACTIVATION BY PLANT AND ANIMAL LECTINS - THE LECTIN PATHWAY

The “proof-of-principle” that “lectins can be made more selective through molecular engineering” was demonstrated with the plant mannose-binding lectin Concanavalin-A in the 1970s (1). The functions then studied were mitogenicity, complement activation, and serum-binding activity. The dimeric form was found to retain mitogenicity without complement activation. The tetrameric form retained both activities (2).

The discovery that plant lectins activate the mammalian complement system (1) was initially attributed to a single antibody-independent “alternative” pathway of complement activation (3). However, subsequently the observation was extended to animal mannose-binding lectins and a second “alternative” pathways was characterized (4). This was named the “lectin pathway” and the other retained the “alternative pathway” name. Thus, there are three complement activation pathways – the classical, the lectin and the alternative.

In this light there are caveats regarding the proposed topical application of lectins to prevent HIV infection.

(i) There is suggestive evidence that lectins can cross mucous membranes, thus accessing body fluids and exerting toxic, perhaps complement-mediated, effects (5). While the elegant studies of Swanson et al. (6) show how mitogenic effects might be avoided, the possibility of toxic effects has not been excluded.

(ii) Reaction of soluble lectins with targets can be extensively buffered by competing lectin-binding activities, both associated with cell surfaces and in body fluids. When HIV buds from infected cells its envelope includes normal cell surface components that bind lectin. Furthermore, when plant lectins activate lymphocytes cultured in serum-containing medium, doubling the serum concentration doubles the lectin requirement (7). Thus, very high lectin concentrations may be needed for in vivo microbicidal effects. Is in vitro BanLec mitogenicity dependent on the BanLec/serum ratio? If so, is molecular engineering able to decrease competitive binding by serum and natural secretions?

(iii) While avoidance of initial HIV infection is important, use of lectins has not been thought promising in this respect. Hopefully the lectin work is not diverting resources from studies of the “shock-and-kill” approach that promises complete HIV eradication (8)?

(1) Milthorp P, Forsdyke DR (1970) Inhibition of lymphocyte activation at high ratios of concanavalin A to serum depends on complement. Nature 227:1351-1352. Milthorp P, 1970

(2) Forsdyke DR (1977) Role of receptor aggregation in complement-dependent inhibition of lymphocytes by high concentrations of concanavalin-A. Nature 267:358-360. Forsdyke DR, 1977

(3) Eidinger D, Gery I, Elleman C (1977) The inhibition of murine lymphocyte mitotic responses by human and mouse sera. 1. Evidence for a role of antibody-independent activation of the alternative complement pathway. Cellular Immunology 30:82-91. Eidinger D, 1977

(4) Degn SE, et al. (2014) Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes. Proc. Natl. Acad. Sci. USA 111:13445-13450. Degn SE, 2014

(5) Forsdyke DR (1978) Role of complement in the toxicity of dietary legumes. Medical Hypothesis 4:97-100. Forsdyke DR, 1978

(6) Swanson MD, et al. (2015) Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity. Cell 163:746-758. Swanson MD, 2015

(7) Forsdyke DR (1967) Quantitative nucleic acid changes during PHA-induced lymphocyte transformation in vitro: dependence of the response on the PHA/serum ratio. Biochemical Journal 105:679-684. Forsdyke DR, 1967

(8) Forsdyke DR (1991) Programmed activation of T-lymphocytes. A theoretical basis for short term treatment of AIDS with azidothymidine. Medical Hypothesis 34:24-27. Forsdyke DR, 1991

On 2015 Oct 11, Donald Forsdyke commented:

GC% – A COLLECTIVE VARIATION THAT FOSTERS SPECIATION

Among members of a species there is generally a mean genomic GC% value with a bell-curve distribution about this mean. The small group to the left of the mean are biased towards low GC%. The small group to the right of the mean are biased towards high GC%. These fringe groups can be said to have collectively varied away from the mean.

In keeping with the proposal that the reproductive isolation needed for speciation would be fostered by a collective variation (1), it has been shown how differences in GC% would impair recombination between a group on the fringe of the bell-curve and the main species group at the centre of the bell-curve (2). Other conditions being propitious (e.g. further rein is given to natural selection), a fringe group could then become a separate species with its own bell-curve GC% distribution.

Thus, if through reproductive isolation the low GC% group departed the main species, the population mean value would move slightly to the right (higher GC%). While Mugal and her colleagues (3) provide a valuable review of the literature on genomic base composition, the implications for speciation are not mentioned. For more please see my speciation text (4) and my bioinformatics textbook (5). There are also recent PubMed comments (6, 7).

(1) Romanes GJ (1886) Physiological selection: An additional suggestion on the origin of species. Journal of the Linnaean Society, Zoology 19, 337-411.

(2) Forsdyke DR (1996) Different biological species "broadcast" their DNAs at different (G+C)% "wavelengths". J. Theoret. Biol. 178, 405-417. Forsdyke DR, 1996% "wavelengths".")

(3) Mugal CF, Weber DD, Ellegren H (2015) GC-biased gene conversion links the recombination landscape and demography to genomic base composition. BioEssays 35: (in press) doi:10.1002/bies.201500058 Mugal CF, 2015

(4) Forsdyke DR (2001) The Origin of Species Revisited. McGill-Queen’s University Press, Montreal.

(5) Forsdyke DR (2011) Evolutionary Bioinformatics. 2nd edition. Springer, New York.

(6) Clément Y, Fustier M-A, Nabholz B, Glémin S. (2014) The bimodal distribution of genic GC content is ancestral to monocot species. Genome Biology and Evolution 7, 336-348. Clément Y, 2014

(7) Turner LM, Harr B (2014) Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions. Elife Dec 9;3 doi: 10.7554/eLife.02504 Turner LM, 2014

On 2015 Dec 09, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION The proteins of thermophiles are generally more heat-stable than the corresponding proteins from mesophiles. This must be reflected in either, or both, of two major amino acid variables – composition and order. In the past the notion that amino acid composition might be reflective of the pressure in thermophiles to retain purine-rich codons (3) has been disparaged by Zeldovich et al. (6). In this elegant new paper (5), Venev and Zeldovich (2015) agree that the “multiple factors” not accounted for in their modelling “include the influence of the genetic code and guanine-cytosine (GC) content of the genomes on amino acid frequencies.” However, there is puzzlement that the “theory and simulations predict a strong increase of leucine content in the thermostable proteins, whereas it is only minimally increased in experimental data.” Perhaps it is of relevance that leucine is on the top left quadrant of the standard presentation of the genic code, its codons being extremely poor in purines.

My response to Zeldovich et al. (6) in 2007, and my follow-up references in 2012 (1, 4), are set out below. One of the coauthors of the 2007 paper has recently further contributed to this topic (2).

2007 Response

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

2012 Response

As a follow up, it can be noted that Dehouck et al. (2008) report that relationship between a protein's thermostability and the optimum growth temperature of the organism containing it, is not so close as previously thought (1). Furthermore, Liu et al. (2012) now conclude from a study of xylanase purine-rich coding sequences that "The codons relating to enzyme thermal property are selected by thermophilic force at [the] nucleotide level," not at the protein level (4).

1.Dehouck Y, Folch B, Rooman M (2008) Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity. Protein Engineering, Design and Selection 21:275-278.Dehouck Y, 2008

2.Goncearenco A, Berezofsky IN (2014) The fundamental tradeoff in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and the physics of nucleic acids and proteins. Biology Direct 9:29 Goncearenco A, 2014

3.Lambros RJ, Mortimer JR, Forsdyke DR (2003) Optimum growth temperature and the base composition of open reading frames in prokaryotes. Extremophiles 7:443–450.Lambros RJ, 2003

4.Liu L, Wang L, Zhang Z, Wang S, Chen H (2012) Effect of codon message on xylanase thermal activity. J. Biol. Chem. 287:27183-27188 Liu L, 2012

5.Venev SV, Zeldovich KB (2015) Massive parallel sampling of lattice proteins reveals foundations of thermal adaptation. J. Chem. Phys. 143: 055101Venev SV, 2015

6.Zeldovich KB, Berezofsky IN, Shakhnovich EI (2007) Protein and DNA sequence determinants of thermophilic adaptation. PLOS Comput. Biol. 3(1), e5.Zeldovich KB, 2007

On 2015 Aug 12, Donald Forsdyke commented:

WINGE PROPOSED HYBRID STERILITY CURED BY WHOLE-GENOME DUPLICATION

That hybrid sterility would be 'cured' by whole genome duplication was suggested by 'the father of yeast genetics' [1], Őjvind Winge [2], and has been extensively discussed in modern texts on speciation [3] and evolutionary biology [4].

He would doubtless have been delighted that his favorite organism (apart from dogs) had formed the basis of the elegant study by Marcet-Houben and Gabaldon that provides a welcome endorsement of his viewpoint.

[1] Szybalski W (2001) My road to Őjvind Winge, the father of yeast genetics. Genetics 158:1–6.

[2] Winge Ő (1917) The chromosomes. Their numbers and general importance. Comptes Rendus des Travaux du Laboratoire Carlsberg. 13:131–275. see Webpage.

[3] Forsdyke DR (2001) The Origin of Species Revisited. Montreal: McGill-Queen’s University Press, pp. 72–79.

[4] Forsdyke DR (2011) Evolutionary Bioinformatics. 2nd edition. New York: Springer, pp. 184–186.

On 2015 Jun 26, Donald Forsdyke commented:

The review covers a field that has occupied geneticist Jianzhi Zhang and colleagues for many years. Their publications are in journals that have usually not permitted direct commenting. The present new PubMed facility allows the release of past comments on their work that previously had only limited circulation (see Yang JR, 2012, Lin F, 2012, Park C, 2013). There are also comments on a paper in PLOS Biology that are accessible at http://www.plosbiology.org/annotation/listThread.action?root=81241.

The review considers the scope of negative selection, which usually associates with low evolutionary rates and, following Hurst and Smith (1999), only briefly alludes to positive selection, which usually associates with high evolutionary rates. In so doing, the review seems to exclude evidence from studies of positive selection that might reflect on its thesis (see comment on Park C, 2013). Indeed, biochemists have long known that some proteins that are deemed “important” for their host organism evolve slowly under negative selection. Other “important” proteins evolve rapidly under positive selection. Within this broad negative-to-positive range are scattered many other “important” or “essential” or “non-dispensable” proteins. Thus, the review correctly concludes that “the functional importance of a protein only has a weak impact on its evolutionary rate,” and “the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance.”

However, the latter statement can be interpreted as implying that, above a certain minimum, expression level and function are not connected. This misinterpretation could be compounded by (i) the narrow range of papers considered the “foundations in the field,” and (ii) frequent allusions to the functional importance (note singular) of a protein, and (iii) focusing too closely on recently acquired genomic datasets (important as they are).

There is extensive literature showing that collective functions of proteins, which are dependent on expression level, can underlie biological phenomena (e.g. the Donnan equilibrium; Donnan FG, 1927). Thus, a protein can have both specific (e.g. enzymic) and general functions (1). The discovery of X chromosome dosage compensation (reviewed by Muller in 1948; see http://post.queensu.ca/~forsdyke/xchromos.htm) gave an early indication of the importance of the general role.

Since many proteins contribute to collective functions, the loss of an individual protein type is more likely to affect its specific function than its contribution to collective functions. Depending on the collective function, some proteins have properties (e.g. size) that would better support that function than other proteins. Thus, there can be degrees of specificity.

And long ago (McConkey EH, 1982) attention was drawn to the importance of functional constraints due to “quinary” interactions between proteins in the crowded intracellular environment. This referred to “macromolecular interactions that are transient in vivo” which should “constitute an important source of constraints on changes in primary structure” (see also Monteith WB, 2015). The E-R anticorrelation, and selection to avoid protein misinteractions, are further considered in a recent review. A cell mutation that may be deemed as imposing a “gain-in-toxicity,” may function to alert an organism’s cytotoxic T cells that the mutant cell should be destroyed before becoming cancerous. We neglect the immunological concomitants of mutation at our peril (Forsdyke DR, 2015).

(1)Forsdyke DR (2012) Functional constraint and molecular evolution. In: Encyclopedia of Life Sciences. Chichester: John Wiley.

On 2015 Aug 26, Donald Forsdyke commented:

DIMINISHED ROLE FOR CONVENTIONAL NATURAL SELECTION

This study decisively demonstrates both that nucleic acid level forces drive amino acid composition, rather than the converse, and that, in this respect, higher oligonucleotide frequencies are more powerful than mononucleotide frequencies (base composition). This is consistent with the case, made on different grounds, that oligonucleotide frequencies drive mononucleotide frequencies (summarized in Forsdyke 2011). Furthermore, there is now better support for Grantham’s “genome hypothesis” that natural selection by way of the conventional environment, may be secondary to some other form of selection that relates to speciation (see comment on Goncearenco A, 2014). Indeed, in some cases, amino acids in a protein may be mere “place holders” - there to serve the needs of the genome (Rayment JH, 2005).

Of course, some adaptation takes place at the protein level, but that the authors’ reading frame-specific analysis provides “contravening evidence” against the power of oligonucleotides is not readily apparent. In thermophiles the low frequency of TpA overlapping successive codons (e.g. NNT,ANN, …), and the depletion of ApT when positioned within a codon (e.g. NAT, NNN, … ), are easily explained by the pressure on thermophiles to purine-load their coding sequences (i.e. there is a nucleic acid level selective pressure). Thermophiles can best achieve this, without imposing excessively on amino acid composition, by incorporating purines in third codon positions. Thus, instead of the classical distribution of purines (R) and pyrimidines (Y) in codons (e.g. RNY, RNY, …), thermophiles tend to follow the RNR rule (e.g. RNR, RNR, … ) (see Lambros RJ, 2003).

Forsdyke DR: Evolutionary Bioinformatics. 2nd edition. New York: Springer, 2011.

On 2015 May 02, Donald Forsdyke commented:

DIABETIC PLASMA IS MORE ROULEAUGENIC THAN NORMAL PLASMA This fine paper reports an impressive in vivo method for evaluating rouleaux formation. The authors correctly state that rouleaux formation is "attributed to some changes in the plasma concentration" of certain proteins, "which modify the interaction between RBC." However, they go on to conclude that "diabetic erythrocytes have a higher propensity to form aggregates." To show this they would have had to study both RBCs from diabetic patients in normal subjects’ plasma and the converse (normal RBCs in diabetic patients’ plasma). But they do not report such experiments.

Following their original premise, it would be predicted that, when suspended in plasma from diabetic patients, normal RBC (of the same blood group) would form rouleaux just as well as the RBC from diabetic patients. That the primary change is in the surrounding plasma has long been known. Indeed, normal plasma can be made rouleaugenic by either heating to generate polymeric albumin, or merely by slightly concentrating. In both circumstances, the plasma will aggregate autologous RBC (1).

It appears that the aggregation is entropy-driven, showing a degree of specificity (like-RBC aggregating with like-RBC) analogous to the homoaggregation of macromolecules that can be induced by increasing the concentrations of surrounding but dissimilar macromolecules (2). The early history and theoretical implications of rouleaux formation are reviewed elsewhere (3), and in my webpages: see Entropy-Driven Protein Self-Aggregation at http://post.queensu.ca/~forsdyke/mhc001.htm

(1) Forsdyke DR, Palfree RGE, Takeda A (1982) Formation of erythrocyte rouleaux in preheated normal serum: roles of albumin polymers and lysophosphatidylcholine, Canadian Journal of Biochemistry 60: 705-711.

(2) Forsdyke DR, Ford PM (1983) Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation. Biochemical Journal 214: 257-260.

(3) Forsdyke DR (1995) Entropy-driven protein self-aggregation as the basis for self/not-self discrimination in the crowded cytosol. Journal of Biological Systems 3: 273-287.

On 2015 Apr 26, Donald Forsdyke commented:

The data in this interesting paper seem not incompatible with the hypothesis that the need to prevent recombination with other organisms drives a organisms GC%. This anti-recombination selective effect (resulting in the reproductive isolation needed for maintaining species integrity) is something the entire organism has to adapt to. Having adapted, it seems not unlikely that, in some cases, an artificial changing of GC% (as in the Kelkar paper) would be deleterious. This would be particularly evident in the case of 'lower' species that had not superimposed other mechanisms for maintaining reproductive isolation. Absence of superimposed mechanisms would prevent GC% values from seeking new equilibrium positions. For more see my text Evolutionary Bioinformatics (Springer, New York, 2011).

On 2015 Mar 10, Donald Forsdyke commented:

ADAPTATION DECOUPLED FROM SPECIATION. This fine new paper presents an impressive synthesis of phylogenetic data aiming to “explore how it bears on evolutionary hypotheses and mechanisms of speciation and diversification.” In keeping with the results of Venditti et al. (2010) that are cited, the major conclusion is that “if adaptation is largely decoupled from speciation, we should not expect it to be a driver of speciation.” Indeed, “Cases where the phenotype has changed little (e.g. cryptic species) … are interpreted here as evidence of uncoupling.” There is reference to geographic isolation as “the major model,” but it is noted that “time constraints should be similar with ecological speciation, and other models exist.”

One of these “other models” is considered by Venditti et al. (2008 Biologist 55, 140-146), who note: “There is a growing appreciation amongst evolutionary biologists that rapid reproductive isolation is more common than previously thought and is often associated with what is known as sympatric speciation, or speciation between populations which share the same geographic range.” The idea of a non-geographic decoupling of adaptation from speciation was advanced by Darwin’s research associate George Romanes in 1886. As with Venditti et al. (2010), the present results nicely support Romanes, whose work is the major focus of my speciation text (The Origin of Species, Revisited, McGill-Queen's University Press, 2001). There is further elaboration both in our biography of the geneticist William Bateson (Treasure Your Exceptions, Springer, New York, 2008) and in my textbook Evolutionary Bioinformatics (Springer, New York, 2011).

On 2015 Dec 12, Donald Forsdyke commented:

LESS BP MEDICATION NEEDED IN HOT WEATHER This paper recommends “More aggressive blood pressure lowering treatment in the cold months … in high risk individuals” (1), which implies less aggressive blood pressure (BP) lowering treatment in hot months. Indeed, a well-documented “J-curve” observation is that, below a certain value, BP lowering is harmful – e.g. acute kidney injury (2). Quite rightly, the cartoon in the accompanying editorial has question marks at both the high and low ends of the temperature scale (3). However, the paper’s focus is on more treatment in winter, not on less treatment in summer, and it only scores the deaths attributed to cardiovascular disease (CVD), a number that declines in hot weather (1). Cases where mortality can be attributed to other causes are excluded.

Regarding CVD it is reported that “The excess risk was similar between people treated with blood pressure lowering agents and those without” (1). But given the widely different mechanisms of action of different BP lowering agents, surely this statement needs backing with more information on agents and their dosages? Large summertime systolic BP declines that mandate treatment adjustment are not uncommon (4). Differential responses in hot weather can depend on type and dosage of antihypertensive medication. Indeed, extrapolation from the supplementary plot (Fig 1) of Yang et al. (2015), the BP would be normal at 40°C and medication could be dispensed with (1). Taking medication at that temperature could be lethal (5). Indeed, a Canadian case study with an angiotensin receptor blocker found that medication could be dispensed with when summer temperatures reached 33°C (6).

It is obvious from Fig. 3 that the statement that “the seasonal variation in blood pressure … was abolished by the use of home central heating” (1), is incorrect. In Harbin province, where winter temperatures are similar to those in Canada, there is less BP increase in cold weather than in other provinces, and this is attributed to central heating. But the seasonal BP increase is clearly not abolished. Despite central heating, in the Canadian case study the BP decline for a 10°C increase in temperature was of the order of 20 mm Hg (6) – a far higher value than the 6 mm Hg reported here (1).

1 Yang L, Li L, Lewington S. Guo Y, Sherliker P, Bian Z, Collins R, et al (2015) Outdoor temperature, blood pressure, and cardiovascular disease mortality among 23000 individuals with diagnosed cardiovascular diseases from China. Eur Heart J 36:1178-1185. Yang L, 2015

2 Tomlinson LA, Abel GA, Chaudhry AN, Tomson CR, Wilkinson IB, Roland MO et al (2013) ACE inhibitor and angiotensin-II receptor antagonist prescribing and hospital admissions with acute kidney injury: a longitudinal ecological study. PLoS One 8:e78465.Tomlinson LA, 2013

3 Bruno RM, Taddei S (2015) ‘Tis bitter cold and I am sick at heart’: establishing the relationship between outdoor temperature, blood pressure, and cardiovascular mortality. Eur Heart J 36:1152-1154.Bruno RM, 2015

4 Stergiou GS, Myrsilidi A, Kollias A, Destounis A, Roussias L, Kalogeropoulos P (2015) Seasonal variation in meteorological parameters and office, ambulatory and home blood pressure: predicting factors and clinical implications. Hypertension Research 38(12):869-875. Stergiou GS, 2015

5 Editorial (2015) Health professionals: be prepared for heatwaves. Lancet 386:219. Anonymous, 2015

6 Forsdyke DR (2015) Summertime dosage-dependent hypersensitivity to an angiotensin II receptor blocker. BMC Res Notes 8:227. Forsdyke DR, 2015

On 2015 Mar 12, Donald Forsdyke commented:

ADAPTATION DECOUPLED FROM SPECIATION. The authors begin by noting that gene flow among populations, rather than assisting speciation, has a “greater impact” when acting “as a homogenizing force, reuniting populations that might otherwise have had separate evolutionary trajectories.” This blending effect, which thwarts speciation, is prevented by some form of reproductive isolation dependent upon an internal or external barrier. The nature of the barrier varies with time. The present work reflects on the order in which barriers arise, an early barrier being superceded by a later.

The two fern types, having diverged 60 million years ago, can still transfer gametes abiotically (so there are no genic incompatibilities conferring prezygotic isolation), and the resulting zygotes can still develop (so there are no genic incompatibilities conferring hybrid inviability). Thus, they are reproductively isolated to a degree sufficient to prevent blending, solely by virtue of hybrid sterility.

Following a line of reasoning that dates back to Romanes (1886), a failure of meiotic pairing, due to the accumulation of base differences in parental DNA sequences (that would not necessarily affect genes), results in sterile hybrids. The line cannot advance, so that in evolutionary terms the parents are reproductively isolated from each other. Over 60 million years, this chromosome pairing barrier would have been elevated by addition of macroscopically observable deletions, duplications or inversions. Accompanying genic changes would have affected phenotypic functions other than those affecting fertilization and development. Thus secondary adaptations would be decoupled from the primary speciation event. This is in keeping with the recent conclusion of Hedges et al. (2015) that “if adaptation is largely decoupled from speciation, we should not expect it to be a driver of speciation” (see: http://1.usa.gov/1ESNM0W where further references may be found).

On 2015 Mar 16, Donald Forsdyke commented:

INTRONS FIRST. The author points out that, for the human genome, Chargaff's second parity rule (PR2) remains significant for oligonucleotides extending to 9 nt and concludes that "the phenomenon must be non-random." Furthermore, he adds:

"At present, what is important is to unravel the origin of strand symmetry, which would contribute greatly to the study of the origin and evolution of genomes. First and foremost, it is necessary, also possible, to figure out whether the phenomenon is a result of convergence of genome evolution (Albrecht-Buehler, 2006; Fickett et al., 1992; Forsdyke and Bell, 2004; Lobry and Lobry, 1999) or, on the contrary, an original trait (vestige) of the primordial genome (Zhang and Huang, 2008, 2010; Zhang et al., 2013). If strand symmetry emerged by means of direct selection, the structural feature would be functional since its appearance. Otherwise, the structural feature would not necessarily be functional, or would be exploited to have a function, if any."

It is incorrect to list Forsdyke and Bell as supporters of the "convergence" hypothesis. As elaborated in 2013 Biological Theory 7: 196-203 ("Introns first"), Forsdyke holds PR2 to be an original trait related to the role of nucleic acid structure in the correction of errors by recombination. Thus, the trait has been functional since its appearance. Indeed, the primordial genome - one long "intron" - could not have existed without it. For more on this see Evolutionary Bioinformatics (2011, Springer, New York).

On 2015 Jan 23, Donald Forsdyke commented:

GC% DIFFERENCES NOT CAUSED BY CONVENTIONAL SELECTION

Its average base composition (GC%) is a characteristic of a biological species. The present work questions the view that GC% differences between species reflect responses to conventional selective pressures on organism function. Thus, the result “challenges the causes and possible functional roles (if any) of GC content variations in grass and Monocot genomes” (1). Likewise, in earlier work, the authors noted that “it is not clear why GC content in introns should also be selected for. Thus, we think that selective hypotheses are not clearly established and are currently insufficient to explain all the data adequately” (2).

To resolve this it would be interesting to examine the GC% values of sympatric, so-called “sibling species” (espèces jumelles, Geschwisterarten). Here phenotypic differences are minimal. Indeed, it has been shown that very small differences in GC% should suffice to spark speciation. These initiating GC% differences could later be obscured by pressures on the phenotype that affect GC%. But when such phenotypic differentiation was minimal, traces of these initiating events might remain (3).

Speciation is still mainly studied in complex organisms. Virus species that infect the same host cell have less scope for developing phenotypic differences and can be construed as sibling species. Indeed, we might recall that the mid-20th century revolution in molecular biology owed much to physicists who studied the simplest living systems – viruses that infect bacteria.

Considering the retroviruses HIV1 and HTLV1 that both infect CD4 T lymphocytes, we find that HIV1 has one of the lowest GC% values known and HTLV1 has one of the highest GC% values known. Large differences are also found when other related viral species share a host cell. Since minute GC% differences can initiate divergence into recombinationally isolated species, it can be assumed that, in the absence of major superceding phenotypic differences, these GC% differences have remained and expanded in HIV1 and HTLV1 (4).

Viewed from a selective perspective, an ancestral retrovirus by virtue of avoiding recombinational blending with its cell-mates, should have been able to develop sufficient functional variation “in sympatry” to achieve full speciation while retaining phenotypic characters needed for the shared intracellular environment. All this harkens back to Romanes who in 1886 proposed that initiation of divergence into species could precede subsequent phenotypic changes (5).

(1) Clément Y, Fustier M-A, Nabholz B, Glémin S. (2015) Genome Biology and Evolution. (In press) doi:10.1093/gbe/evu278

(2) Glémin S, Clément Y, David J, Ressayre A. (2014) GC content evolution in coding regions of angiosperm genomes: a unifying hypothesis. Trends in Genetics 30, 263-270.

(3) Forsdyke DR (2001) The Origin of Species Revisited. McGill-Queen’s University Press, Montreal.

(4) Forsdyke DR (2014) Implications of HIV RNA structure for recombination, speciation, and the neutralism-selectionism controversy. Microbes and Infection 16, 96-103 doi: 10.1016/j.micinf.2013.10.017.

(5) Romanes GJ (1886) Physiological selection: An additional suggestion on the origin of species. Journal of the Linnaean Society, Zoology 19, 337-411.

On 2015 Jan 11, Donald Forsdyke commented:

CONTINUING SUPPORT FOR GRANTHAM'S GENOME HYPOTHESIS

Richard Grantham (1980), after examination of a mere 160 short sequences, proposed his ‘genome hypothesis.’ For nucleic acids he envisaged ‘manifold constraints and adaptations, of both structural and functional natures.’ These ‘could exist, independently of protein coding.’ Thus, there was ‘protein-independent molecular evolution of a non-neutral character.’ The distinctive ‘coding strategy of an organism’ was ‘at the heart of the problem of molecular evolution,’ and was likely to prove of fundamental importance for ‘speciation and systematics in general’ (Grantham et al. 1986).

These observations won extensive support when thousands of much longer sequences became available, even though many were incomplete (Forsdyke and Mortimer 2000; Mortimer and Forsdyke 2003; Lee et al. 2004). The data, consistent with the early base compositional studies of Chargaff, Sueoka and Szybalski, are now further affirmed by this comprehensive new work involving thousands of entirely complete sequences (Goncearenco and Berezovsky 2014).

However, while there is little disagreement on data and the need for evolutionary trade-offs (‘mutual adjustment of the nucleotide and amino acid compositions’), readers should note that there remains disagreement over interpretations (see my comments on a previous paper and my textbook; Zeldovich et al. 2007, Forsdyke 2011). Readers should also note that the present text (p. 3) has proline (P) listed in both the high GC% saturation group and the low GC% saturation group. In error, phenylalanine (F) was replaced by P in the latter. The GC% low and medium groups have amino acids listed in order of increasing codon GC% saturation, but the GC% high group has amino acids listed (p. 3) in decreasing order of codon GC% saturation (see Fig. S3). And it is puzzling that Figures 1a and 1b appear the same, with just axis labels interchanged, yet some points seem incorrectly interchanged. The rectilinear interpretations of obvious curvilinear relationships (Figs. 8, S9) are also problematic, as noted by Reviewer 2.

The notion that aspartate and glutamate (with purine-rich codons GAY and GAR) ‘cannot be used for the efficient tuning of the nucleotide composition,’ does not hold for the tuning of AG%. Furthermore, it should be noted that as GC% increases, the decline of A and T does not affect both bases equally. While G% and T% tend to remain constant, C increases at the expense of A. Likewise, when GC% decreases, A increases at the expense of C (Mortimer and Forsdyke 2003). This A-for-C transversional trading, most evident at extreme GC% values (Fig. S10), should decrease the probabilities of G-quadruplexes and thymine dimers. Finally, noting for example the high AG% in thermophiles, it may be premature to conclude that tradeoffs are a ‘purely compositional phenomenon, linking the realms of nucleic and amino acids in prokaryotes regardless of their life styles, environments, and phylogeny.’ Grantham’s admonition regarding speciation and systematics should not go unheeded.

Forsdyke DR: Evolutionary Bioinformatics. 2nd edition. New York: Springer, 2011.

Forsdyke DR, Mortimer JR: Chargaff’s legacy. Gene 2000, 261:127-137.Forsdyke DR, 2000

Goncearenco A, Berezovsky IN: The fundamental trade-off in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and physics of nucleic acids and proteins. Biology Direct 2014, 9:29.Goncearenco A, 2014

Grantham R: Workings of the genetic code. Trends Biochem Sci 1980, 5:327-331.

Grantham R, Perrin P, Mouchiroud D: Patterns in codon usage of different kinds of species. Oxford Surv Evol Biol 1986, 3:48-81.

Lee S-J, Mortimer JR, Forsdyke DR: Genomic conflict settled in favour of the species rather than of the gene at extreme GC% values. Applied Bioinformatics 2004, 3:219-228.Lee SJ, 2004

Mortimer JR, Forsdyke DR: Comparison of responses by bacteriophage and bacteria to pressures on the base composition of open reading frames. Applied Bioinformatics 2003, 2:47-62.Mortimer JR, 2003

Zeldovich KB, Berezovsky IN, Shakhnovich EI: Protein and DNA sequence determinants of thermophilic adaptation. PLoS Comput Biol 2007, 3(1):e5.Zeldovich KB, 2007

On 2015 Jan 20, Donald Forsdyke commented:

HYPOTHESES OF SPECIATION As hinted at by one of the reviewers, hypotheses on the initiation of speciation are broadly categorized as genic and chromosomal (Nevo, 2012). The possibility that their intriguing observations on putatively sterile hybrids between certain mouse subspecies might be explained in chromosomal (non-genic) terms is not considered by Turner and Harr. Yet, from studies of hybrids of the same subspecies, Bhattacharyya et al. (2013; 2014) infer that “meiotic asynapsis of heterospecific homologous chromosomes is the primary mechanistic basis of hybrid sterility.” This indicates a role for “a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis.” Thus, any observed genic differences would be secondary to this (Page and Orr-Weaver, 1997).

Furthermore, it is incorrectly implied by Turner and Harr that the work of Bateson (1909) supports the genic viewpoint to which the names of Dobzhansky and Muller are attached (“DM incompatibilities”). This is not a minor point, since Bateson consistently favored a non-genic viewpoint that is today best equated with the chromosomal hypothesis (Nei and Nozawa, 2011; Forsdyke, 2011).

Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, Simecek P, Forejt J. 2013. Mechanistic basis of infertility of mouse intersubspecific hybrids. Proceedings of the National Academy of Sciences, U S A 110: E468–477. doi:10.1073/pnas.1219126110.

Bhattacharyya T, Reifova R, Gregorova S, Simecek P, Gergelits V, Mistrik M, Martincova I, Pialek J, Forejt J. 2014. X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids. PLoS Genetics 10: e1004088. doi:10.1371/journal.pgen.1004088

Forsdyke DR. 2011. The ‘B’ in BDM. William Bateson did not advocate a genic speciation theory. Heredity 106:202. doi:10.1038/hdy.2010.15.

Nei M, Nozawa M. 2011. Roles of mutation and selection in speciation: from Hugo de Vries to the modern genomic era. Genome Biology and Evolution 3,812–829. doi:10.1093/gbe/evr028.

Nevo E. 2012. Speciation: chromosomal mechanisms. In: eLS. Chichester: John Wiley & Sons. doi: 10.1002/9780470015902.a0001757.pub3.

Page AW, Orr-Weaver TL. 1997. Stopping and starting the meiotic cycle. Current Opinion in Genetics and Development 7:23–31. doi: 10.1016/S0959-437X(97)80105-0.

On 2015 Sep 28, Donald Forsdyke commented:

General Principle of Juxtaposition- and Concentration-Dependent Activation of Complement

Seeking to understand "immune system … ability to discriminate self from nonself," the authors here describe an elegant “clustering-based mechanism” for complement activation by MBL that is “akin to the mechanism governing signaling through many cellular receptors,” and distinguishes the lectin and classical pathways of complement activation (1). Their demonstration of the general principle of “juxtaposition- and concentration-dependent activation,” is in keeping with studies with the MBL, concanavalin-A, that were reported in 1977 (2).

However, the latter studies were not mentioned (1), and a new review states that “the ‘lectin pathway’ was discovered in the early 1980s” (3). Thus, it is possible that those engaged in this fascinating field are unaware of the earlier work that began to be reported in 1970 (4). Subsequent studies in the 1970s (5-8) culminated in demonstration of “the importance of the sequence of binding events” (9). It is to be hoped that the full relationship of our earlier work to modern studies will be determined in future work.

(1) Degn SE, et al. (2014) Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes. Proc. Natl. Acad. Sci. USA 111:13445-13450.

(2) Forsdyke DR (1977) Role of receptor aggregation in complement-dependent inhibition of lymphocytes by high concentrations of concanavalin-A. Nature 267:358-360. Forsdyke DR, 1977

(3) Thielens NM, Gaborlaud C, Thiel S (2015) Complement: classical and lectin pathways. In: eLS John Wiley & Sons, Chichester.

(4) Milthorp P, Forsdyke DR (1970) Inhibition of lymphocyte activation at high ratios of concanavalin A to serum depends on complement. Nature 227:1351-1352. Milthorp P, 1970

(5) Milthorp P, Forsdyke DR (1973) Serum factors affecting the incorporation of (3H)uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement. _Biochem. J. 132:803-812.Milthorp P, 1973

(6) Milthorp P, Forsdyke DR (1973) A comparison of the activation of thymus and lymph-node cells by concanavalin A and phytohaemagglutinin. Effects of complement. J. Immun. Meth. 2:269-277.Milthorp P, 1973

(7) Forsdyke DR (1978) Role of complement in the toxicity of dietary legumes. Med. Hypoth. 4:97-100 Forsdyke DR, 1978

(8) Forsdyke DR, Milthorp P (1979) Early onset inhibition of lymphocytes in heterologous serum by high concentrations of concanavalin-A. Further studies of the role of complement with suramin and heated serum. Int. J. Immunopharmacol. 1:133-139.Forsdyke DR, 1979

(9) Forsdyke DR (1980) Lectin pulses as determinants of lymphocyte activation and inactivation during the first six hours of culture: sequential action of concanavalin-A and complement cause cell lysis. Can. J. Biochem. 58:1387-1396. Forsdyke DR, 1980

On 2017 Sep 07, Donald Forsdyke commented:

REFERENCE WITHDRAWN

Reference 7 in this paper (de Oliveira MF, 2011) has been retracted by the Editors Human Neuroscience Editorial Office., 2016. The discovery of the problem was made in 2015 by Ondrej Havlicek and announced in the Neurosceptic section of Discover Magazine Here, which was reviewing one of my papers on the topic. In an End-Note (Oct 2012) to an earlier paper on my web-pages Forsdyke DR, 2009 Here, I had juxtaposed the figure of Feuillet et al. (2007) with the one from the paper of Oliviera et al. (2011). With hindsight, it is easy to see that the photographs for the patient of Feuillet et al. are reproduced in Oliviera et al., without appropriate acknowledgement.

.<br> However, the fact that the third repetition of an observation does not withstand scrutiny, in no way implies a similar problem with the first two reports, one by Lorber, and one by Oliviera et al. (2007). While many, like myself, remain sceptical, we await further independent reports (such as that provided by Rosie Raveis in the Neurosceptic blog), and post-mortem verifications of the patients described in the first two reports.

On 2015 Jun 26, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (Feb 6 2013) may be of interest:

'I was delighted to see a new paper from your laboratory in this week’s PNAS Early Edition Park C, 2013 and, as expected, it did not disappoint me. The convergence of our thinking can be seen in the earlier emails and the attached articles on "Functional Constraint" (ELS where I squeezed in a last-minute reference to your work) and on "Collective Gene Functions" (BioEssays). Since I differ from you and your coauthors in some current interpretations, I hope you will not mind my again offering comments, which I hope will be helpful.

1. I think you would agree that your statement "Amino acid substitutions are slower as the mRNA folding strength increases" could be restated as "Amino acid substitutions are faster as the mRNA folding strength decreases". The latter was the main observation of my paper in Molecular Biology and Evolution (1995 12, 1157-1165, http://post.queensu.ca/~forsdyke/introns.htm). In other words, under strong Darwinian positive selection (high dN), amino acid substitutions (codon changes) primarily serve the needs of protein function, rather than of nucleic acid structure. When the protein is conserved (evolving slowly) then the needs of nucleic acid structure are more readily accommodated. This approach can even be used to assay positive Darwinian selection (see Immunogenetics 1996 43, 182-189, http://post.queensu.ca/~forsdyke/introns.htm). The point was recently reiterated (see J. Biol. Systems 2007 15, 95-108, http://post.queensu.ca/~forsdyke/speciat3.htm).

2. While your Introduction implies the possibility of "a selective pressure at any level (DNA, mRNA, or protein)," you assume that there is "a major role of natural selection at the mRNA level in constraining protein evolution." You do not mention the possibility that mRNA structure is by default, because the encoding DNA needs structure (i.e. DNA has the potential to extrude single strands as stem-loops). Since such structure is pervasive in DNA (in exons and introns and in extragenic locations), it appears that DNA needs structure, whether it encodes proteins or not. I have considered the rationale for this elsewhere.

3. You note that "most of the correlations reported in this study are strong" when using enzymic (Fig. 1a) rather than computational (Fig. 1b) RNA structure determination. And you point out that mRNA folding strength may be impacted by "different amino acid, nucleotide, and synonymous codon frequencies." Here it might have been better first to look at the nucleotide component by comparing folding energies with randomly shuffled sequences. Your use of "pseudo-mRNAs" as a basis for comparison was useful for eliminating the possibilities of roles for "specific protein sequences or synonymous codon usages," but would have greatly reduced the statistical significance of your results.

4. You call for "further improvement of the computational prediction." Base composition tends to be a genome-wide, or segment-wide, characteristic; it tends not to be a local characteristic. On the other hand, base order is a local characteristic. Nucleic acid structure depends on both base composition and order, but for many purposes, base order provides a more sensitive measure. If you follow my method for dissecting out the base order-dependent component of the folding energy, I believe you will obtain more satisfactory results with the computational method of structure determination.

5. Compared with low expressed proteins, highly expressed proteins are more likely to have both specific and collective functions. Thus, there are two sources of negative selective pressure on genes with highly expressed protein products, and only one source of negative selection pressure on genes with low expressed protein products. So the former genes tend to evolve more slowly, and are less likely to be affected by deletion mutations since their contributions to collective functions are shared with other highly expressed proteins, which can compensate. Since they evolve slowly, they are better able to accommodate structure at the nucleic acid level. I consider this more extensively elsewhere.

6. Genes under positive Darwinian selection pressure may be of various expression levels, so "amino acid substitution rate is negatively correlated with mRNA folding strength, with or without the control of expression level." Furthermore, there is "a significant [negative] correlation between mRNA folding strength and dN/dS, even when gene expression level is controlled."

All this comes with no guarantee. I may be wrong. If you have any problem with these comments please get back to me. Having crossed this territory in the 1990s I feel some obligation to warn later explorers of possible pitfalls!'

The ELS and BioEssays papers referred to in the above email were:

Nature Encyclopedia of Life Sciences 7, 396-403 (2002). Functional constraint and molecular evolution. This was updated for Wiley Online Library in 2005 and 2012.

BioEssays (2012) 34, 930-933 Forsdyke DR, 2012.

On 2017 Feb 20, Jiri Forejt commented:

I appreciate the historical aspects of speciation theories elaborated in the above comment and other studies of the author (e.g. Forsdyke DR, 2011 or Forsdyke DR, Biol. Theory, 2016, DOI 10.1007/s13752-016-0257-z) since they help to understand the origin of some tacitly agreed dogmas in the field. We interpret the F1 hybrid male sterility described in Bhattacharyya T, 2014 and Bhattacharyya T, 2013 as incompatibility between Prdm9 and Hstx2 genes and diversified genomic sequence of the Mus musculus subspecies. The phenotype is largely chromosomal, representing a failure of meiotic pairing and synapsis of homeologous chromosomes and disruption of MSCI. However, we are hesitant to call it chromosomal sterility to avoid confusion with male sterility associated with large chromosomal rearrangements (see e.g. Forejt J, 1996, Zanders SE, 2014). I agree that non-genic, genomic divergence as a mechanism of reproductive isolation would deserve its own designation or even acronym. But first, it has to be accepted as a reproductive isolation mechanism by the community of evolutionary geneticists.

On 2017 Feb 17, Donald Forsdyke commented:

EPONYMS: "DM" FOR GENIC AND "WCB" FOR CHROMOSOMAL SPECIATION THEORIES

The remarkable observations made in this outstanding paper have been well supported by subsequent studies (e.g. Moehring 2011; Forsdyke 2016). However, encompassing both genic and non-genic incompatibilities under the Dobzhansky-Muller rubric is regrettable. As described elsewhere (Forsdyke 2011), while William Bateson's term "epistasis" is used to describe interactions between genes, he himself espoused a non-genic "chromosomal" model for the initiation of the divergence of one species into two species. Thus, Bhattacharyya et al. (2013) correctly employ the eponymous acronym "DM" rather than the widely employed "BDM."

Yet, they write that "several pieces of indirect evidence are in favor of D-M incompatibilities based on non-genic sequence divergence." This implies that Dobzhansky and Muller had non-genic viewpoints (which they did not). For those who like eponymous acronyms, some new form, relating to the history of the chromosomal viewpoint, would seem appropriate.

Clear articulations of the chromosomal viewpoint trace back to the Danish "father of yeast genetics" Ojvind Winge, and were elaborated in the 1920s by Crowther and Bateson. Thus, it would seem appropriate that, while retaining "DM incompatibilities" for appropriate genic speciation theories, we introduce "WCB incompatibilities" for appropriate chromosomal theories. For further background please see Bateson Webpage.

Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, et al., (2013) Mechanistic basis of infertility of mouse intersubspecific hybrids. Proc Nat Acad Sci USA 110: E468–E477.Bhattacharyya T, 2013

Forsdyke DR (2011) The B in BDM. William Bateson did not advocate a genic speciation theory. Heredity 106: 202.Forsdyke DR, 2011

Forsdyke DR (2016) Evolutionary Bioinformatics, 3rd edn. Springer: New York.

Forsdyke DR (2017) Speciation: Goldschmidt's chromosomal heresy, once supported by Gould and Dawkins, is again reinstated. Biol Theor (in press) doi: 10.1007/s13752-016-0257-z

Moehring AJ (2011). Heterozygosity and its unexpected correlations with hybrid sterility. Evolution 65: 2621–2630.Moehring AJ, 2011

On 2015 Jun 25, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (July 2 2012) may be of interest:

'Your fine new paper on dosage compensation in PNAS Early Edition links up nicely with your previous paper on protein misinteraction; that is, if you care to consider the hypothesis I advanced in 1994 (http://post.queensu.ca/~forsdyke/dominanc.htm), which is updated in my textbook (http://post.queensu.ca/~forsdyke/book05.htm). The basic point is that proteins have collective functions (such as the well-known Donnan equilibrium), as well as specific functions. Over evolutionary time protein concentrations have been fine-tuned to serve such collective functions. My 1994 paper postulated a novel collective function – aggregation pressure – through which an individual cell can initiate self/not-self discrimination. Each protein contributes to, and is acted upon by, this aggregation pressure. The more abundant a protein, the more it contributes and is acted upon.

Subsequent work on X chromosome dosage compensation has nicely supported this view ( http://post.queensu.ca/~forsdyke/theorimm7.htm). If a human female fails to compensate to some degree (both Xs expressed so aggregation pressure is high), then autoimmune disease is more likely. From the point of view of evolution, this would be a short-term effect and individuals would be negatively selected. On the other hand, if she were to excessively compensate (say part of a singly expressed X not expressed, so aggregation pressure is lowered), then she would stand an increased risk of getting infections. But since there are many alternatives for combatting infections, this would tend to be a long-term effect from the point of view of evolution. Thus, in your words “chromosome-wide expression halving has been tolerated” because “Y degeneration is stepwise” so that “expression reduction happened gradually to more and more X-linked genes during evolution.”'

On 2015 Jan 11, Donald Forsdyke commented:

ANOTHER "INTRONS-FIRST" SCENARIO - UPDATE

Punctuation problems garbled my comment published with the paper in Biology Direct in 2012 http://www.biology-direct.com/content/7/1/11/comments#893696. The comment should read:

The statement in the abstract of Rogozin et al. that "the introns-first scenario is not supported by any evidence" seems to refer to the particular version of "introns-first" which was developed from the ideas of Darryl Reanney by Penny and his colleagues [references 47 and 48 of the Rogozin paper]. There is, however, another "introns-first" scenario, which has considerable bioinformatic support and is in at least two textbooks [1, 2]. Since the Rogozin paper does "not attempt a comprehensive coverage" and appears to favor introns invading protein-encoding regions, rather than the converse, readers of Biology Direct might like to review the evidence for the "introns-first" scenario that I began to elaborate in 1981 [3, 4]. Full details may be found in my webpages. A course on introns for High School and College students may be accessed through You Tube (videos 37-54 of Forsdyke Evolution Academy)[5].

Updating this, a full paper was later published [6].

[1] Forsdyke DR: Evolutionary Bioinformatics.: 2nd edition. New York: Springer; 2011: 249-266.

[2] Forsdyke DR: The interrupted gene. In Lewin's Genes X. Edited by Krebs JE, Goldstein ES, Kilpatrick ST. Boston: Jones and Bartlett; 2011:79-97, 172-175.

[3] Forsdyke DR: Are introns in-series error detecting sequences?. J Theoret Biol 1981, 93:861-866.Forsdyke DR, 1981

[4] Barrette IH, McKenna S, Taylor DR, Forsdyke DR: Introns resolve the conflict between base order-dependent stem-loop potential and the encoding of RNA or Protein: Further evidence from overlapping genes. Gene 2001, 270:181-189.Barrette IH, 2001

[5] Evolution Academy http://post.queensu.ca/~forsdyke/videolectures.htm

[6] Forsdyke DR: Introns first. Biological Theory 2013, 7:196-203; DOI 10.1007/s13752-013-0090-6 http://post.queensu.ca/~forsdyke/introns3.htm

On 2015 Jun 25, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (May 27 2012) may be of interest:

'Thank you for a very interesting paper in PNAS Early Edition. The "potentially toxic" effect of protein-protein misinteractions forms the basis of our hypothesis of intracellular self/not-self discrimination, which is now receiving support from studies of the predisposition of females to autoimmune disease (J of Autoimmunity 38, J129-J134). Our earlier studies were influenced by the 1982 paper of E. H. McConkey on the "quinary structure" of proteins (PNAS 79, 3236-40). I have added a reference to your new paper as an "end-note" to the web version of a 2001 paper (see http://post.queensu.ca/~forsdyke/theorimm2.htm). I look forward to your future paper on the effect of interaction avoidance on the usage of synonymous codons.'

On 2017 Sep 13, Donald Forsdyke commented:

WHAT IS THEORETICAL BIOLOGY?

In a 2011 letter to the editor, I wrote in a somewhat too boisterous fashion questioning the Editors-in-Chiefs' statement on the nature of theoretical biology. The advent of PubMed Commons now makes it possible to reproduce my unpublished letter, which quotes from their statement:

With 23 papers in the JTB stretching from 1969 to 2009, I am perhaps qualified to congratulate the various Editors who have built on the foundation Professor Danielli so carefully laid in 1961. Yet the letter of the present Editors-in-Chief [1], while claiming to "reflect on the history of theoretical biology," notes that in 1961 "theoretical biology was largely in its infancy."

This will raise many eyebrows – particularly among those of us who have recently celebrated the Darwin Centenary. Apart from Charles Darwin, the many others set rotating in their graves by this remark will include Gregor Mendel, Francis Galton, George Romanes, William Bateson, Herman Muller, JBS Haldane, Theodosius Dobzhansky, McFarlane Burnet and Francis Crick. At a time when "hundreds of our reviewers are women," the disregarding of these past male contributions hints at political correctness.

That the present Editors also consider this "a field based largely on mathematics" would also have struck many of these giants – even Mendel, Galton and Haldane – as bizarre. Thus to the question: "Whether the focus and content of JTB has changed over these fifty years?" one must suspect increasing tendencies to (1) dismiss the history of our subject, (2) follow political agendas, and (3) depart from verbal analysis to mathematical modeling.

It will be interesting to see how many of the experts in the field who have been commissioned by the present Editors to provide reviews of its past, present and future, will note what I believe to have been an unfortunate departure from Danielli’s original goals.

[1] D. Kirschner, Denise Kirschner, Fifty years of JTB: Past, present and future a letter from the editors-in-chief, J. Theor. Biol., (2011) doi:10.1016/j.jtbi.2010.09.004 Kirschner D, 2011

On 2017 Jan 01, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION (This comment was original posted as a reader response to the original PLOS CompBiol article 20 Feb 2008.)

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

An analogy may help. Passing from winter to summer, you change your outdoor attire. Many people (Imelda Marcos excepted) have a wider range of shirts and sweaters than of footwear. There may be items which provide more reliable indices of outside temperature than others. For example, the weight of your shirt + sweater might more finely correlate with temperature than the weight of your shoes. If you wanted to predict outside temperature from attire, you would choose shirt-sweater weights, rather than shoe weights. But if your clothes (shirt + sweater) weigh more, you might need sturdier shoes to cope with the extra weight. Thus, shoe-weight would depend on clothes-weight. The latter would be primary and the former would be secondary ("derivative"). But showing a better correlation with temperature in the case of shirt + sweater, does not establish a dependence of shoe-weight on shirt-sweater weight. More likely, you wear lighter shoes in warm weather because they keep your feet cooler! Both depend on temperature. It just so happens that the correlation with temperature is coarser and not so finely tuned for shoe weight, as for shirt-sweater weight.

On 2015 Mar 16, Donald Forsdyke commented:

Concluding that this paper showed, if anything, the very opposite of what was claimed, a commentary on this paper was submitted to Bioinformatics in 2005, but was declined for publication. The paper and correspondence with the authors was placed online (2006) at: http://post.queensu.ca/~forsdyke/bioinfo9.htm, and various updates have since been added.

On 2015 Jan 28, Donald Forsdyke commented:

SIMILARITY BETWEEN EPSTEIN-BARR VIRUS AND PLASMODIUM FALCIPARUM. A key observation in this paper (Cristillo et al. 2001) is that the EBNA-1 protein is longer than necessary because of the need to contain a low complexity GLY-ALA repeat region between functional domains. This allows purine-loading at the nucleic acid level. Essentially the same observation was reported by Pizzi and Frontali (Genome Research 2001 11, 218-229). They noted:

"Proteins from Plasmodium falciparum, the etiological agent of the most severe form of human malaria, are often larger than homologous proteins from other organisms. When multiple alignment is possible, the size difference can be seen to be due to the presence of long insertions separating well-conserved blocks that are adjacent in the homologous proteins.... The insertions are characterized by highly recurrent amino acid usage" and correspond to "low complexity regions ... believed to encode non-globular domains of unknown function that are extruded from the protein core and do not impair the functional folding of the protein."... The recurrent amino acids "correlate with A-richness in codons."

This quotation from Pizzi and Frontali is the first of a series of End Notes that were added to the version of the Cristillo paper displayed on one of Forsdyke's webpages http://post.queensu.ca/~forsdyke/EBV.htm

On 2015 Jan 28, Donald Forsdyke commented:

SIMILARITY BETWEEN EPSTEIN-BARR VIRUS AND PLASMODIUM FALCIPARUM. A key observation in this paper (Cristillo et al. 2001) is that the EBNA-1 protein is longer than necessary because of the need to contain a low complexity GLY-ALA repeat region between functional domains. This allows purine-loading at the nucleic acid level. Essentially the same observation was reported by Pizzi and Frontali (Genome Research 2001 11, 218-229). They noted:

"Proteins from Plasmodium falciparum, the etiological agent of the most severe form of human malaria, are often larger than homologous proteins from other organisms. When multiple alignment is possible, the size difference can be seen to be due to the presence of long insertions separating well-conserved blocks that are adjacent in the homologous proteins.... The insertions are characterized by highly recurrent amino acid usage" and correspond to "low complexity regions ... believed to encode non-globular domains of unknown function that are extruded from the protein core and do not impair the functional folding of the protein."... The recurrent amino acids "correlate with A-richness in codons."

This quotation from Pizzi and Frontali is the first of a series of End Notes that were added to the version of the Cristillo paper displayed on one of Forsdyke's webpages http://post.queensu.ca/~forsdyke/EBV.htm

On 2015 Mar 16, Donald Forsdyke commented:

Concluding that this paper showed, if anything, the very opposite of what was claimed, a commentary on this paper was submitted to Bioinformatics in 2005, but was declined for publication. The paper and correspondence with the authors was placed online (2006) at: http://post.queensu.ca/~forsdyke/bioinfo9.htm, and various updates have since been added.

On 2017 Jan 01, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION (This comment was original posted as a reader response to the original PLOS CompBiol article 20 Feb 2008.)

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

An analogy may help. Passing from winter to summer, you change your outdoor attire. Many people (Imelda Marcos excepted) have a wider range of shirts and sweaters than of footwear. There may be items which provide more reliable indices of outside temperature than others. For example, the weight of your shirt + sweater might more finely correlate with temperature than the weight of your shoes. If you wanted to predict outside temperature from attire, you would choose shirt-sweater weights, rather than shoe weights. But if your clothes (shirt + sweater) weigh more, you might need sturdier shoes to cope with the extra weight. Thus, shoe-weight would depend on clothes-weight. The latter would be primary and the former would be secondary ("derivative"). But showing a better correlation with temperature in the case of shirt + sweater, does not establish a dependence of shoe-weight on shirt-sweater weight. More likely, you wear lighter shoes in warm weather because they keep your feet cooler! Both depend on temperature. It just so happens that the correlation with temperature is coarser and not so finely tuned for shoe weight, as for shirt-sweater weight.

On 2017 Sep 13, Donald Forsdyke commented:

WHAT IS THEORETICAL BIOLOGY?

In a 2011 letter to the editor, I wrote in a somewhat too boisterous fashion questioning the Editors-in-Chiefs' statement on the nature of theoretical biology. The advent of PubMed Commons now makes it possible to reproduce my unpublished letter, which quotes from their statement:

With 23 papers in the JTB stretching from 1969 to 2009, I am perhaps qualified to congratulate the various Editors who have built on the foundation Professor Danielli so carefully laid in 1961. Yet the letter of the present Editors-in-Chief [1], while claiming to "reflect on the history of theoretical biology," notes that in 1961 "theoretical biology was largely in its infancy."

This will raise many eyebrows – particularly among those of us who have recently celebrated the Darwin Centenary. Apart from Charles Darwin, the many others set rotating in their graves by this remark will include Gregor Mendel, Francis Galton, George Romanes, William Bateson, Herman Muller, JBS Haldane, Theodosius Dobzhansky, McFarlane Burnet and Francis Crick. At a time when "hundreds of our reviewers are women," the disregarding of these past male contributions hints at political correctness.

That the present Editors also consider this "a field based largely on mathematics" would also have struck many of these giants – even Mendel, Galton and Haldane – as bizarre. Thus to the question: "Whether the focus and content of JTB has changed over these fifty years?" one must suspect increasing tendencies to (1) dismiss the history of our subject, (2) follow political agendas, and (3) depart from verbal analysis to mathematical modeling.

It will be interesting to see how many of the experts in the field who have been commissioned by the present Editors to provide reviews of its past, present and future, will note what I believe to have been an unfortunate departure from Danielli’s original goals.

[1] D. Kirschner, Denise Kirschner, Fifty years of JTB: Past, present and future a letter from the editors-in-chief, J. Theor. Biol., (2011) doi:10.1016/j.jtbi.2010.09.004 Kirschner D, 2011

On 2015 Jun 25, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (May 27 2012) may be of interest:

'Thank you for a very interesting paper in PNAS Early Edition. The "potentially toxic" effect of protein-protein misinteractions forms the basis of our hypothesis of intracellular self/not-self discrimination, which is now receiving support from studies of the predisposition of females to autoimmune disease (J of Autoimmunity 38, J129-J134). Our earlier studies were influenced by the 1982 paper of E. H. McConkey on the "quinary structure" of proteins (PNAS 79, 3236-40). I have added a reference to your new paper as an "end-note" to the web version of a 2001 paper (see http://post.queensu.ca/~forsdyke/theorimm2.htm). I look forward to your future paper on the effect of interaction avoidance on the usage of synonymous codons.'

On 2015 Jan 11, Donald Forsdyke commented:

ANOTHER "INTRONS-FIRST" SCENARIO - UPDATE

Punctuation problems garbled my comment published with the paper in Biology Direct in 2012 http://www.biology-direct.com/content/7/1/11/comments#893696. The comment should read:

The statement in the abstract of Rogozin et al. that "the introns-first scenario is not supported by any evidence" seems to refer to the particular version of "introns-first" which was developed from the ideas of Darryl Reanney by Penny and his colleagues [references 47 and 48 of the Rogozin paper]. There is, however, another "introns-first" scenario, which has considerable bioinformatic support and is in at least two textbooks [1, 2]. Since the Rogozin paper does "not attempt a comprehensive coverage" and appears to favor introns invading protein-encoding regions, rather than the converse, readers of Biology Direct might like to review the evidence for the "introns-first" scenario that I began to elaborate in 1981 [3, 4]. Full details may be found in my webpages. A course on introns for High School and College students may be accessed through You Tube (videos 37-54 of Forsdyke Evolution Academy)[5].

Updating this, a full paper was later published [6].

[1] Forsdyke DR: Evolutionary Bioinformatics.: 2nd edition. New York: Springer; 2011: 249-266.

[2] Forsdyke DR: The interrupted gene. In Lewin's Genes X. Edited by Krebs JE, Goldstein ES, Kilpatrick ST. Boston: Jones and Bartlett; 2011:79-97, 172-175.

[3] Forsdyke DR: Are introns in-series error detecting sequences?. J Theoret Biol 1981, 93:861-866.Forsdyke DR, 1981

[4] Barrette IH, McKenna S, Taylor DR, Forsdyke DR: Introns resolve the conflict between base order-dependent stem-loop potential and the encoding of RNA or Protein: Further evidence from overlapping genes. Gene 2001, 270:181-189.Barrette IH, 2001

[5] Evolution Academy http://post.queensu.ca/~forsdyke/videolectures.htm

[6] Forsdyke DR: Introns first. Biological Theory 2013, 7:196-203; DOI 10.1007/s13752-013-0090-6 http://post.queensu.ca/~forsdyke/introns3.htm

On 2015 Jun 25, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (July 2 2012) may be of interest:

'Your fine new paper on dosage compensation in PNAS Early Edition links up nicely with your previous paper on protein misinteraction; that is, if you care to consider the hypothesis I advanced in 1994 (http://post.queensu.ca/~forsdyke/dominanc.htm), which is updated in my textbook (http://post.queensu.ca/~forsdyke/book05.htm). The basic point is that proteins have collective functions (such as the well-known Donnan equilibrium), as well as specific functions. Over evolutionary time protein concentrations have been fine-tuned to serve such collective functions. My 1994 paper postulated a novel collective function – aggregation pressure – through which an individual cell can initiate self/not-self discrimination. Each protein contributes to, and is acted upon by, this aggregation pressure. The more abundant a protein, the more it contributes and is acted upon.

Subsequent work on X chromosome dosage compensation has nicely supported this view ( http://post.queensu.ca/~forsdyke/theorimm7.htm). If a human female fails to compensate to some degree (both Xs expressed so aggregation pressure is high), then autoimmune disease is more likely. From the point of view of evolution, this would be a short-term effect and individuals would be negatively selected. On the other hand, if she were to excessively compensate (say part of a singly expressed X not expressed, so aggregation pressure is lowered), then she would stand an increased risk of getting infections. But since there are many alternatives for combatting infections, this would tend to be a long-term effect from the point of view of evolution. Thus, in your words “chromosome-wide expression halving has been tolerated” because “Y degeneration is stepwise” so that “expression reduction happened gradually to more and more X-linked genes during evolution.”'

On 2017 Feb 17, Donald Forsdyke commented:

EPONYMS: "DM" FOR GENIC AND "WCB" FOR CHROMOSOMAL SPECIATION THEORIES

The remarkable observations made in this outstanding paper have been well supported by subsequent studies (e.g. Moehring 2011; Forsdyke 2016). However, encompassing both genic and non-genic incompatibilities under the Dobzhansky-Muller rubric is regrettable. As described elsewhere (Forsdyke 2011), while William Bateson's term "epistasis" is used to describe interactions between genes, he himself espoused a non-genic "chromosomal" model for the initiation of the divergence of one species into two species. Thus, Bhattacharyya et al. (2013) correctly employ the eponymous acronym "DM" rather than the widely employed "BDM."

Yet, they write that "several pieces of indirect evidence are in favor of D-M incompatibilities based on non-genic sequence divergence." This implies that Dobzhansky and Muller had non-genic viewpoints (which they did not). For those who like eponymous acronyms, some new form, relating to the history of the chromosomal viewpoint, would seem appropriate.

Clear articulations of the chromosomal viewpoint trace back to the Danish "father of yeast genetics" Ojvind Winge, and were elaborated in the 1920s by Crowther and Bateson. Thus, it would seem appropriate that, while retaining "DM incompatibilities" for appropriate genic speciation theories, we introduce "WCB incompatibilities" for appropriate chromosomal theories. For further background please see Bateson Webpage.

Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, et al., (2013) Mechanistic basis of infertility of mouse intersubspecific hybrids. Proc Nat Acad Sci USA 110: E468–E477.Bhattacharyya T, 2013

Forsdyke DR (2011) The B in BDM. William Bateson did not advocate a genic speciation theory. Heredity 106: 202.Forsdyke DR, 2011

Forsdyke DR (2016) Evolutionary Bioinformatics, 3rd edn. Springer: New York.

Forsdyke DR (2017) Speciation: Goldschmidt's chromosomal heresy, once supported by Gould and Dawkins, is again reinstated. Biol Theor (in press) doi: 10.1007/s13752-016-0257-z

Moehring AJ (2011). Heterozygosity and its unexpected correlations with hybrid sterility. Evolution 65: 2621–2630.Moehring AJ, 2011

On 2015 Jun 26, Donald Forsdyke commented:

In the light of new reviews (Zhang J, 2015 and Forsdyke DR, 2015), the following email to the senior author (Feb 6 2013) may be of interest:

'I was delighted to see a new paper from your laboratory in this week’s PNAS Early Edition Park C, 2013 and, as expected, it did not disappoint me. The convergence of our thinking can be seen in the earlier emails and the attached articles on "Functional Constraint" (ELS where I squeezed in a last-minute reference to your work) and on "Collective Gene Functions" (BioEssays). Since I differ from you and your coauthors in some current interpretations, I hope you will not mind my again offering comments, which I hope will be helpful.

1. I think you would agree that your statement "Amino acid substitutions are slower as the mRNA folding strength increases" could be restated as "Amino acid substitutions are faster as the mRNA folding strength decreases". The latter was the main observation of my paper in Molecular Biology and Evolution (1995 12, 1157-1165, http://post.queensu.ca/~forsdyke/introns.htm). In other words, under strong Darwinian positive selection (high dN), amino acid substitutions (codon changes) primarily serve the needs of protein function, rather than of nucleic acid structure. When the protein is conserved (evolving slowly) then the needs of nucleic acid structure are more readily accommodated. This approach can even be used to assay positive Darwinian selection (see Immunogenetics 1996 43, 182-189, http://post.queensu.ca/~forsdyke/introns.htm). The point was recently reiterated (see J. Biol. Systems 2007 15, 95-108, http://post.queensu.ca/~forsdyke/speciat3.htm).

2. While your Introduction implies the possibility of "a selective pressure at any level (DNA, mRNA, or protein)," you assume that there is "a major role of natural selection at the mRNA level in constraining protein evolution." You do not mention the possibility that mRNA structure is by default, because the encoding DNA needs structure (i.e. DNA has the potential to extrude single strands as stem-loops). Since such structure is pervasive in DNA (in exons and introns and in extragenic locations), it appears that DNA needs structure, whether it encodes proteins or not. I have considered the rationale for this elsewhere.

3. You note that "most of the correlations reported in this study are strong" when using enzymic (Fig. 1a) rather than computational (Fig. 1b) RNA structure determination. And you point out that mRNA folding strength may be impacted by "different amino acid, nucleotide, and synonymous codon frequencies." Here it might have been better first to look at the nucleotide component by comparing folding energies with randomly shuffled sequences. Your use of "pseudo-mRNAs" as a basis for comparison was useful for eliminating the possibilities of roles for "specific protein sequences or synonymous codon usages," but would have greatly reduced the statistical significance of your results.

4. You call for "further improvement of the computational prediction." Base composition tends to be a genome-wide, or segment-wide, characteristic; it tends not to be a local characteristic. On the other hand, base order is a local characteristic. Nucleic acid structure depends on both base composition and order, but for many purposes, base order provides a more sensitive measure. If you follow my method for dissecting out the base order-dependent component of the folding energy, I believe you will obtain more satisfactory results with the computational method of structure determination.

5. Compared with low expressed proteins, highly expressed proteins are more likely to have both specific and collective functions. Thus, there are two sources of negative selective pressure on genes with highly expressed protein products, and only one source of negative selection pressure on genes with low expressed protein products. So the former genes tend to evolve more slowly, and are less likely to be affected by deletion mutations since their contributions to collective functions are shared with other highly expressed proteins, which can compensate. Since they evolve slowly, they are better able to accommodate structure at the nucleic acid level. I consider this more extensively elsewhere.

6. Genes under positive Darwinian selection pressure may be of various expression levels, so "amino acid substitution rate is negatively correlated with mRNA folding strength, with or without the control of expression level." Furthermore, there is "a significant [negative] correlation between mRNA folding strength and dN/dS, even when gene expression level is controlled."

All this comes with no guarantee. I may be wrong. If you have any problem with these comments please get back to me. Having crossed this territory in the 1990s I feel some obligation to warn later explorers of possible pitfalls!'

The ELS and BioEssays papers referred to in the above email were:

Nature Encyclopedia of Life Sciences 7, 396-403 (2002). Functional constraint and molecular evolution. This was updated for Wiley Online Library in 2005 and 2012.

BioEssays (2012) 34, 930-933 Forsdyke DR, 2012.

On 2017 Sep 07, Donald Forsdyke commented:

REFERENCE WITHDRAWN

Reference 7 in this paper (de Oliveira MF, 2011) has been retracted by the Editors Human Neuroscience Editorial Office., 2016. The discovery of the problem was made in 2015 by Ondrej Havlicek and announced in the Neurosceptic section of Discover Magazine Here, which was reviewing one of my papers on the topic. In an End-Note (Oct 2012) to an earlier paper on my web-pages Forsdyke DR, 2009 Here, I had juxtaposed the figure of Feuillet et al. (2007) with the one from the paper of Oliviera et al. (2011). With hindsight, it is easy to see that the photographs for the patient of Feuillet et al. are reproduced in Oliviera et al., without appropriate acknowledgement.

.<br> However, the fact that the third repetition of an observation does not withstand scrutiny, in no way implies a similar problem with the first two reports, one by Lorber, and one by Oliviera et al. (2007). While many, like myself, remain sceptical, we await further independent reports (such as that provided by Rosie Raveis in the Neurosceptic blog), and post-mortem verifications of the patients described in the first two reports.

On 2015 Sep 28, Donald Forsdyke commented:

General Principle of Juxtaposition- and Concentration-Dependent Activation of Complement

Seeking to understand "immune system … ability to discriminate self from nonself," the authors here describe an elegant “clustering-based mechanism” for complement activation by MBL that is “akin to the mechanism governing signaling through many cellular receptors,” and distinguishes the lectin and classical pathways of complement activation (1). Their demonstration of the general principle of “juxtaposition- and concentration-dependent activation,” is in keeping with studies with the MBL, concanavalin-A, that were reported in 1977 (2).

However, the latter studies were not mentioned (1), and a new review states that “the ‘lectin pathway’ was discovered in the early 1980s” (3). Thus, it is possible that those engaged in this fascinating field are unaware of the earlier work that began to be reported in 1970 (4). Subsequent studies in the 1970s (5-8) culminated in demonstration of “the importance of the sequence of binding events” (9). It is to be hoped that the full relationship of our earlier work to modern studies will be determined in future work.

(1) Degn SE, et al. (2014) Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes. Proc. Natl. Acad. Sci. USA 111:13445-13450.

(2) Forsdyke DR (1977) Role of receptor aggregation in complement-dependent inhibition of lymphocytes by high concentrations of concanavalin-A. Nature 267:358-360. Forsdyke DR, 1977

(3) Thielens NM, Gaborlaud C, Thiel S (2015) Complement: classical and lectin pathways. In: eLS John Wiley & Sons, Chichester.

(4) Milthorp P, Forsdyke DR (1970) Inhibition of lymphocyte activation at high ratios of concanavalin A to serum depends on complement. Nature 227:1351-1352. Milthorp P, 1970

(5) Milthorp P, Forsdyke DR (1973) Serum factors affecting the incorporation of (3H)uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement. _Biochem. J. 132:803-812.Milthorp P, 1973

(6) Milthorp P, Forsdyke DR (1973) A comparison of the activation of thymus and lymph-node cells by concanavalin A and phytohaemagglutinin. Effects of complement. J. Immun. Meth. 2:269-277.Milthorp P, 1973

(7) Forsdyke DR (1978) Role of complement in the toxicity of dietary legumes. Med. Hypoth. 4:97-100 Forsdyke DR, 1978

(8) Forsdyke DR, Milthorp P (1979) Early onset inhibition of lymphocytes in heterologous serum by high concentrations of concanavalin-A. Further studies of the role of complement with suramin and heated serum. Int. J. Immunopharmacol. 1:133-139.Forsdyke DR, 1979

(9) Forsdyke DR (1980) Lectin pulses as determinants of lymphocyte activation and inactivation during the first six hours of culture: sequential action of concanavalin-A and complement cause cell lysis. Can. J. Biochem. 58:1387-1396. Forsdyke DR, 1980

On 2015 Jan 20, Donald Forsdyke commented:

HYPOTHESES OF SPECIATION As hinted at by one of the reviewers, hypotheses on the initiation of speciation are broadly categorized as genic and chromosomal (Nevo, 2012). The possibility that their intriguing observations on putatively sterile hybrids between certain mouse subspecies might be explained in chromosomal (non-genic) terms is not considered by Turner and Harr. Yet, from studies of hybrids of the same subspecies, Bhattacharyya et al. (2013; 2014) infer that “meiotic asynapsis of heterospecific homologous chromosomes is the primary mechanistic basis of hybrid sterility.” This indicates a role for “a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis.” Thus, any observed genic differences would be secondary to this (Page and Orr-Weaver, 1997).

Furthermore, it is incorrectly implied by Turner and Harr that the work of Bateson (1909) supports the genic viewpoint to which the names of Dobzhansky and Muller are attached (“DM incompatibilities”). This is not a minor point, since Bateson consistently favored a non-genic viewpoint that is today best equated with the chromosomal hypothesis (Nei and Nozawa, 2011; Forsdyke, 2011).

Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, Simecek P, Forejt J. 2013. Mechanistic basis of infertility of mouse intersubspecific hybrids. Proceedings of the National Academy of Sciences, U S A 110: E468–477. doi:10.1073/pnas.1219126110.

Bhattacharyya T, Reifova R, Gregorova S, Simecek P, Gergelits V, Mistrik M, Martincova I, Pialek J, Forejt J. 2014. X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids. PLoS Genetics 10: e1004088. doi:10.1371/journal.pgen.1004088

Forsdyke DR. 2011. The ‘B’ in BDM. William Bateson did not advocate a genic speciation theory. Heredity 106:202. doi:10.1038/hdy.2010.15.

Nei M, Nozawa M. 2011. Roles of mutation and selection in speciation: from Hugo de Vries to the modern genomic era. Genome Biology and Evolution 3,812–829. doi:10.1093/gbe/evr028.

Nevo E. 2012. Speciation: chromosomal mechanisms. In: eLS. Chichester: John Wiley & Sons. doi: 10.1002/9780470015902.a0001757.pub3.

Page AW, Orr-Weaver TL. 1997. Stopping and starting the meiotic cycle. Current Opinion in Genetics and Development 7:23–31. doi: 10.1016/S0959-437X(97)80105-0.

On 2015 Jan 11, Donald Forsdyke commented:

CONTINUING SUPPORT FOR GRANTHAM'S GENOME HYPOTHESIS

Richard Grantham (1980), after examination of a mere 160 short sequences, proposed his ‘genome hypothesis.’ For nucleic acids he envisaged ‘manifold constraints and adaptations, of both structural and functional natures.’ These ‘could exist, independently of protein coding.’ Thus, there was ‘protein-independent molecular evolution of a non-neutral character.’ The distinctive ‘coding strategy of an organism’ was ‘at the heart of the problem of molecular evolution,’ and was likely to prove of fundamental importance for ‘speciation and systematics in general’ (Grantham et al. 1986).

These observations won extensive support when thousands of much longer sequences became available, even though many were incomplete (Forsdyke and Mortimer 2000; Mortimer and Forsdyke 2003; Lee et al. 2004). The data, consistent with the early base compositional studies of Chargaff, Sueoka and Szybalski, are now further affirmed by this comprehensive new work involving thousands of entirely complete sequences (Goncearenco and Berezovsky 2014).

However, while there is little disagreement on data and the need for evolutionary trade-offs (‘mutual adjustment of the nucleotide and amino acid compositions’), readers should note that there remains disagreement over interpretations (see my comments on a previous paper and my textbook; Zeldovich et al. 2007, Forsdyke 2011). Readers should also note that the present text (p. 3) has proline (P) listed in both the high GC% saturation group and the low GC% saturation group. In error, phenylalanine (F) was replaced by P in the latter. The GC% low and medium groups have amino acids listed in order of increasing codon GC% saturation, but the GC% high group has amino acids listed (p. 3) in decreasing order of codon GC% saturation (see Fig. S3). And it is puzzling that Figures 1a and 1b appear the same, with just axis labels interchanged, yet some points seem incorrectly interchanged. The rectilinear interpretations of obvious curvilinear relationships (Figs. 8, S9) are also problematic, as noted by Reviewer 2.

The notion that aspartate and glutamate (with purine-rich codons GAY and GAR) ‘cannot be used for the efficient tuning of the nucleotide composition,’ does not hold for the tuning of AG%. Furthermore, it should be noted that as GC% increases, the decline of A and T does not affect both bases equally. While G% and T% tend to remain constant, C increases at the expense of A. Likewise, when GC% decreases, A increases at the expense of C (Mortimer and Forsdyke 2003). This A-for-C transversional trading, most evident at extreme GC% values (Fig. S10), should decrease the probabilities of G-quadruplexes and thymine dimers. Finally, noting for example the high AG% in thermophiles, it may be premature to conclude that tradeoffs are a ‘purely compositional phenomenon, linking the realms of nucleic and amino acids in prokaryotes regardless of their life styles, environments, and phylogeny.’ Grantham’s admonition regarding speciation and systematics should not go unheeded.

Forsdyke DR: Evolutionary Bioinformatics. 2nd edition. New York: Springer, 2011.

Forsdyke DR, Mortimer JR: Chargaff’s legacy. Gene 2000, 261:127-137.Forsdyke DR, 2000

Goncearenco A, Berezovsky IN: The fundamental trade-off in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and physics of nucleic acids and proteins. Biology Direct 2014, 9:29.Goncearenco A, 2014

Grantham R: Workings of the genetic code. Trends Biochem Sci 1980, 5:327-331.

Grantham R, Perrin P, Mouchiroud D: Patterns in codon usage of different kinds of species. Oxford Surv Evol Biol 1986, 3:48-81.

Lee S-J, Mortimer JR, Forsdyke DR: Genomic conflict settled in favour of the species rather than of the gene at extreme GC% values. Applied Bioinformatics 2004, 3:219-228.Lee SJ, 2004

Mortimer JR, Forsdyke DR: Comparison of responses by bacteriophage and bacteria to pressures on the base composition of open reading frames. Applied Bioinformatics 2003, 2:47-62.Mortimer JR, 2003

Zeldovich KB, Berezovsky IN, Shakhnovich EI: Protein and DNA sequence determinants of thermophilic adaptation. PLoS Comput Biol 2007, 3(1):e5.Zeldovich KB, 2007

On 2015 Jan 23, Donald Forsdyke commented:

GC% DIFFERENCES NOT CAUSED BY CONVENTIONAL SELECTION

Its average base composition (GC%) is a characteristic of a biological species. The present work questions the view that GC% differences between species reflect responses to conventional selective pressures on organism function. Thus, the result “challenges the causes and possible functional roles (if any) of GC content variations in grass and Monocot genomes” (1). Likewise, in earlier work, the authors noted that “it is not clear why GC content in introns should also be selected for. Thus, we think that selective hypotheses are not clearly established and are currently insufficient to explain all the data adequately” (2).

To resolve this it would be interesting to examine the GC% values of sympatric, so-called “sibling species” (espèces jumelles, Geschwisterarten). Here phenotypic differences are minimal. Indeed, it has been shown that very small differences in GC% should suffice to spark speciation. These initiating GC% differences could later be obscured by pressures on the phenotype that affect GC%. But when such phenotypic differentiation was minimal, traces of these initiating events might remain (3).

Speciation is still mainly studied in complex organisms. Virus species that infect the same host cell have less scope for developing phenotypic differences and can be construed as sibling species. Indeed, we might recall that the mid-20th century revolution in molecular biology owed much to physicists who studied the simplest living systems – viruses that infect bacteria.

Considering the retroviruses HIV1 and HTLV1 that both infect CD4 T lymphocytes, we find that HIV1 has one of the lowest GC% values known and HTLV1 has one of the highest GC% values known. Large differences are also found when other related viral species share a host cell. Since minute GC% differences can initiate divergence into recombinationally isolated species, it can be assumed that, in the absence of major superceding phenotypic differences, these GC% differences have remained and expanded in HIV1 and HTLV1 (4).

Viewed from a selective perspective, an ancestral retrovirus by virtue of avoiding recombinational blending with its cell-mates, should have been able to develop sufficient functional variation “in sympatry” to achieve full speciation while retaining phenotypic characters needed for the shared intracellular environment. All this harkens back to Romanes who in 1886 proposed that initiation of divergence into species could precede subsequent phenotypic changes (5).

(1) Clément Y, Fustier M-A, Nabholz B, Glémin S. (2015) Genome Biology and Evolution. (In press) doi:10.1093/gbe/evu278

(2) Glémin S, Clément Y, David J, Ressayre A. (2014) GC content evolution in coding regions of angiosperm genomes: a unifying hypothesis. Trends in Genetics 30, 263-270.

(3) Forsdyke DR (2001) The Origin of Species Revisited. McGill-Queen’s University Press, Montreal.

(4) Forsdyke DR (2014) Implications of HIV RNA structure for recombination, speciation, and the neutralism-selectionism controversy. Microbes and Infection 16, 96-103 doi: 10.1016/j.micinf.2013.10.017.

(5) Romanes GJ (1886) Physiological selection: An additional suggestion on the origin of species. Journal of the Linnaean Society, Zoology 19, 337-411.

On 2015 Mar 16, Donald Forsdyke commented:

INTRONS FIRST. The author points out that, for the human genome, Chargaff's second parity rule (PR2) remains significant for oligonucleotides extending to 9 nt and concludes that "the phenomenon must be non-random." Furthermore, he adds:

"At present, what is important is to unravel the origin of strand symmetry, which would contribute greatly to the study of the origin and evolution of genomes. First and foremost, it is necessary, also possible, to figure out whether the phenomenon is a result of convergence of genome evolution (Albrecht-Buehler, 2006; Fickett et al., 1992; Forsdyke and Bell, 2004; Lobry and Lobry, 1999) or, on the contrary, an original trait (vestige) of the primordial genome (Zhang and Huang, 2008, 2010; Zhang et al., 2013). If strand symmetry emerged by means of direct selection, the structural feature would be functional since its appearance. Otherwise, the structural feature would not necessarily be functional, or would be exploited to have a function, if any."

It is incorrect to list Forsdyke and Bell as supporters of the "convergence" hypothesis. As elaborated in 2013 Biological Theory 7: 196-203 ("Introns first"), Forsdyke holds PR2 to be an original trait related to the role of nucleic acid structure in the correction of errors by recombination. Thus, the trait has been functional since its appearance. Indeed, the primordial genome - one long "intron" - could not have existed without it. For more on this see Evolutionary Bioinformatics (2011, Springer, New York).

On 2015 Mar 12, Donald Forsdyke commented:

ADAPTATION DECOUPLED FROM SPECIATION. The authors begin by noting that gene flow among populations, rather than assisting speciation, has a “greater impact” when acting “as a homogenizing force, reuniting populations that might otherwise have had separate evolutionary trajectories.” This blending effect, which thwarts speciation, is prevented by some form of reproductive isolation dependent upon an internal or external barrier. The nature of the barrier varies with time. The present work reflects on the order in which barriers arise, an early barrier being superceded by a later.

The two fern types, having diverged 60 million years ago, can still transfer gametes abiotically (so there are no genic incompatibilities conferring prezygotic isolation), and the resulting zygotes can still develop (so there are no genic incompatibilities conferring hybrid inviability). Thus, they are reproductively isolated to a degree sufficient to prevent blending, solely by virtue of hybrid sterility.

Following a line of reasoning that dates back to Romanes (1886), a failure of meiotic pairing, due to the accumulation of base differences in parental DNA sequences (that would not necessarily affect genes), results in sterile hybrids. The line cannot advance, so that in evolutionary terms the parents are reproductively isolated from each other. Over 60 million years, this chromosome pairing barrier would have been elevated by addition of macroscopically observable deletions, duplications or inversions. Accompanying genic changes would have affected phenotypic functions other than those affecting fertilization and development. Thus secondary adaptations would be decoupled from the primary speciation event. This is in keeping with the recent conclusion of Hedges et al. (2015) that “if adaptation is largely decoupled from speciation, we should not expect it to be a driver of speciation” (see: http://1.usa.gov/1ESNM0W where further references may be found).

On 2015 Dec 12, Donald Forsdyke commented:

LESS BP MEDICATION NEEDED IN HOT WEATHER This paper recommends “More aggressive blood pressure lowering treatment in the cold months … in high risk individuals” (1), which implies less aggressive blood pressure (BP) lowering treatment in hot months. Indeed, a well-documented “J-curve” observation is that, below a certain value, BP lowering is harmful – e.g. acute kidney injury (2). Quite rightly, the cartoon in the accompanying editorial has question marks at both the high and low ends of the temperature scale (3). However, the paper’s focus is on more treatment in winter, not on less treatment in summer, and it only scores the deaths attributed to cardiovascular disease (CVD), a number that declines in hot weather (1). Cases where mortality can be attributed to other causes are excluded.

Regarding CVD it is reported that “The excess risk was similar between people treated with blood pressure lowering agents and those without” (1). But given the widely different mechanisms of action of different BP lowering agents, surely this statement needs backing with more information on agents and their dosages? Large summertime systolic BP declines that mandate treatment adjustment are not uncommon (4). Differential responses in hot weather can depend on type and dosage of antihypertensive medication. Indeed, extrapolation from the supplementary plot (Fig 1) of Yang et al. (2015), the BP would be normal at 40°C and medication could be dispensed with (1). Taking medication at that temperature could be lethal (5). Indeed, a Canadian case study with an angiotensin receptor blocker found that medication could be dispensed with when summer temperatures reached 33°C (6).

It is obvious from Fig. 3 that the statement that “the seasonal variation in blood pressure … was abolished by the use of home central heating” (1), is incorrect. In Harbin province, where winter temperatures are similar to those in Canada, there is less BP increase in cold weather than in other provinces, and this is attributed to central heating. But the seasonal BP increase is clearly not abolished. Despite central heating, in the Canadian case study the BP decline for a 10°C increase in temperature was of the order of 20 mm Hg (6) – a far higher value than the 6 mm Hg reported here (1).

1 Yang L, Li L, Lewington S. Guo Y, Sherliker P, Bian Z, Collins R, et al (2015) Outdoor temperature, blood pressure, and cardiovascular disease mortality among 23000 individuals with diagnosed cardiovascular diseases from China. Eur Heart J 36:1178-1185. Yang L, 2015

2 Tomlinson LA, Abel GA, Chaudhry AN, Tomson CR, Wilkinson IB, Roland MO et al (2013) ACE inhibitor and angiotensin-II receptor antagonist prescribing and hospital admissions with acute kidney injury: a longitudinal ecological study. PLoS One 8:e78465.Tomlinson LA, 2013

3 Bruno RM, Taddei S (2015) ‘Tis bitter cold and I am sick at heart’: establishing the relationship between outdoor temperature, blood pressure, and cardiovascular mortality. Eur Heart J 36:1152-1154.Bruno RM, 2015

4 Stergiou GS, Myrsilidi A, Kollias A, Destounis A, Roussias L, Kalogeropoulos P (2015) Seasonal variation in meteorological parameters and office, ambulatory and home blood pressure: predicting factors and clinical implications. Hypertension Research 38(12):869-875. Stergiou GS, 2015

5 Editorial (2015) Health professionals: be prepared for heatwaves. Lancet 386:219. Anonymous, 2015

6 Forsdyke DR (2015) Summertime dosage-dependent hypersensitivity to an angiotensin II receptor blocker. BMC Res Notes 8:227. Forsdyke DR, 2015

On 2015 Mar 10, Donald Forsdyke commented:

ADAPTATION DECOUPLED FROM SPECIATION. This fine new paper presents an impressive synthesis of phylogenetic data aiming to “explore how it bears on evolutionary hypotheses and mechanisms of speciation and diversification.” In keeping with the results of Venditti et al. (2010) that are cited, the major conclusion is that “if adaptation is largely decoupled from speciation, we should not expect it to be a driver of speciation.” Indeed, “Cases where the phenotype has changed little (e.g. cryptic species) … are interpreted here as evidence of uncoupling.” There is reference to geographic isolation as “the major model,” but it is noted that “time constraints should be similar with ecological speciation, and other models exist.”

One of these “other models” is considered by Venditti et al. (2008 Biologist 55, 140-146), who note: “There is a growing appreciation amongst evolutionary biologists that rapid reproductive isolation is more common than previously thought and is often associated with what is known as sympatric speciation, or speciation between populations which share the same geographic range.” The idea of a non-geographic decoupling of adaptation from speciation was advanced by Darwin’s research associate George Romanes in 1886. As with Venditti et al. (2010), the present results nicely support Romanes, whose work is the major focus of my speciation text (The Origin of Species, Revisited, McGill-Queen's University Press, 2001). There is further elaboration both in our biography of the geneticist William Bateson (Treasure Your Exceptions, Springer, New York, 2008) and in my textbook Evolutionary Bioinformatics (Springer, New York, 2011).

On 2015 Apr 26, Donald Forsdyke commented:

The data in this interesting paper seem not incompatible with the hypothesis that the need to prevent recombination with other organisms drives a organisms GC%. This anti-recombination selective effect (resulting in the reproductive isolation needed for maintaining species integrity) is something the entire organism has to adapt to. Having adapted, it seems not unlikely that, in some cases, an artificial changing of GC% (as in the Kelkar paper) would be deleterious. This would be particularly evident in the case of 'lower' species that had not superimposed other mechanisms for maintaining reproductive isolation. Absence of superimposed mechanisms would prevent GC% values from seeking new equilibrium positions. For more see my text Evolutionary Bioinformatics (Springer, New York, 2011).

On 2015 May 02, Donald Forsdyke commented:

DIABETIC PLASMA IS MORE ROULEAUGENIC THAN NORMAL PLASMA This fine paper reports an impressive in vivo method for evaluating rouleaux formation. The authors correctly state that rouleaux formation is "attributed to some changes in the plasma concentration" of certain proteins, "which modify the interaction between RBC." However, they go on to conclude that "diabetic erythrocytes have a higher propensity to form aggregates." To show this they would have had to study both RBCs from diabetic patients in normal subjects’ plasma and the converse (normal RBCs in diabetic patients’ plasma). But they do not report such experiments.

Following their original premise, it would be predicted that, when suspended in plasma from diabetic patients, normal RBC (of the same blood group) would form rouleaux just as well as the RBC from diabetic patients. That the primary change is in the surrounding plasma has long been known. Indeed, normal plasma can be made rouleaugenic by either heating to generate polymeric albumin, or merely by slightly concentrating. In both circumstances, the plasma will aggregate autologous RBC (1).

It appears that the aggregation is entropy-driven, showing a degree of specificity (like-RBC aggregating with like-RBC) analogous to the homoaggregation of macromolecules that can be induced by increasing the concentrations of surrounding but dissimilar macromolecules (2). The early history and theoretical implications of rouleaux formation are reviewed elsewhere (3), and in my webpages: see Entropy-Driven Protein Self-Aggregation at http://post.queensu.ca/~forsdyke/mhc001.htm

(1) Forsdyke DR, Palfree RGE, Takeda A (1982) Formation of erythrocyte rouleaux in preheated normal serum: roles of albumin polymers and lysophosphatidylcholine, Canadian Journal of Biochemistry 60: 705-711.

(2) Forsdyke DR, Ford PM (1983) Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation. Biochemical Journal 214: 257-260.

(3) Forsdyke DR (1995) Entropy-driven protein self-aggregation as the basis for self/not-self discrimination in the crowded cytosol. Journal of Biological Systems 3: 273-287.

On 2015 Aug 26, Donald Forsdyke commented:

DIMINISHED ROLE FOR CONVENTIONAL NATURAL SELECTION

This study decisively demonstrates both that nucleic acid level forces drive amino acid composition, rather than the converse, and that, in this respect, higher oligonucleotide frequencies are more powerful than mononucleotide frequencies (base composition). This is consistent with the case, made on different grounds, that oligonucleotide frequencies drive mononucleotide frequencies (summarized in Forsdyke 2011). Furthermore, there is now better support for Grantham’s “genome hypothesis” that natural selection by way of the conventional environment, may be secondary to some other form of selection that relates to speciation (see comment on Goncearenco A, 2014). Indeed, in some cases, amino acids in a protein may be mere “place holders” - there to serve the needs of the genome (Rayment JH, 2005).

Of course, some adaptation takes place at the protein level, but that the authors’ reading frame-specific analysis provides “contravening evidence” against the power of oligonucleotides is not readily apparent. In thermophiles the low frequency of TpA overlapping successive codons (e.g. NNT,ANN, …), and the depletion of ApT when positioned within a codon (e.g. NAT, NNN, … ), are easily explained by the pressure on thermophiles to purine-load their coding sequences (i.e. there is a nucleic acid level selective pressure). Thermophiles can best achieve this, without imposing excessively on amino acid composition, by incorporating purines in third codon positions. Thus, instead of the classical distribution of purines (R) and pyrimidines (Y) in codons (e.g. RNY, RNY, …), thermophiles tend to follow the RNR rule (e.g. RNR, RNR, … ) (see Lambros RJ, 2003).

Forsdyke DR: Evolutionary Bioinformatics. 2nd edition. New York: Springer, 2011.

On 2015 Jun 26, Donald Forsdyke commented:

The review covers a field that has occupied geneticist Jianzhi Zhang and colleagues for many years. Their publications are in journals that have usually not permitted direct commenting. The present new PubMed facility allows the release of past comments on their work that previously had only limited circulation (see Yang JR, 2012, Lin F, 2012, Park C, 2013). There are also comments on a paper in PLOS Biology that are accessible at http://www.plosbiology.org/annotation/listThread.action?root=81241.

The review considers the scope of negative selection, which usually associates with low evolutionary rates and, following Hurst and Smith (1999), only briefly alludes to positive selection, which usually associates with high evolutionary rates. In so doing, the review seems to exclude evidence from studies of positive selection that might reflect on its thesis (see comment on Park C, 2013). Indeed, biochemists have long known that some proteins that are deemed “important” for their host organism evolve slowly under negative selection. Other “important” proteins evolve rapidly under positive selection. Within this broad negative-to-positive range are scattered many other “important” or “essential” or “non-dispensable” proteins. Thus, the review correctly concludes that “the functional importance of a protein only has a weak impact on its evolutionary rate,” and “the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance.”

However, the latter statement can be interpreted as implying that, above a certain minimum, expression level and function are not connected. This misinterpretation could be compounded by (i) the narrow range of papers considered the “foundations in the field,” and (ii) frequent allusions to the functional importance (note singular) of a protein, and (iii) focusing too closely on recently acquired genomic datasets (important as they are).

There is extensive literature showing that collective functions of proteins, which are dependent on expression level, can underlie biological phenomena (e.g. the Donnan equilibrium; Donnan FG, 1927). Thus, a protein can have both specific (e.g. enzymic) and general functions (1). The discovery of X chromosome dosage compensation (reviewed by Muller in 1948; see http://post.queensu.ca/~forsdyke/xchromos.htm) gave an early indication of the importance of the general role.

Since many proteins contribute to collective functions, the loss of an individual protein type is more likely to affect its specific function than its contribution to collective functions. Depending on the collective function, some proteins have properties (e.g. size) that would better support that function than other proteins. Thus, there can be degrees of specificity.

And long ago (McConkey EH, 1982) attention was drawn to the importance of functional constraints due to “quinary” interactions between proteins in the crowded intracellular environment. This referred to “macromolecular interactions that are transient in vivo” which should “constitute an important source of constraints on changes in primary structure” (see also Monteith WB, 2015). The E-R anticorrelation, and selection to avoid protein misinteractions, are further considered in a recent review. A cell mutation that may be deemed as imposing a “gain-in-toxicity,” may function to alert an organism’s cytotoxic T cells that the mutant cell should be destroyed before becoming cancerous. We neglect the immunological concomitants of mutation at our peril (Forsdyke DR, 2015).

(1)Forsdyke DR (2012) Functional constraint and molecular evolution. In: Encyclopedia of Life Sciences. Chichester: John Wiley.

On 2015 Aug 12, Donald Forsdyke commented:

WINGE PROPOSED HYBRID STERILITY CURED BY WHOLE-GENOME DUPLICATION

That hybrid sterility would be 'cured' by whole genome duplication was suggested by 'the father of yeast genetics' [1], Őjvind Winge [2], and has been extensively discussed in modern texts on speciation [3] and evolutionary biology [4].

He would doubtless have been delighted that his favorite organism (apart from dogs) had formed the basis of the elegant study by Marcet-Houben and Gabaldon that provides a welcome endorsement of his viewpoint.

[1] Szybalski W (2001) My road to Őjvind Winge, the father of yeast genetics. Genetics 158:1–6.

[2] Winge Ő (1917) The chromosomes. Their numbers and general importance. Comptes Rendus des Travaux du Laboratoire Carlsberg. 13:131–275. see Webpage.

[3] Forsdyke DR (2001) The Origin of Species Revisited. Montreal: McGill-Queen’s University Press, pp. 72–79.

[4] Forsdyke DR (2011) Evolutionary Bioinformatics. 2nd edition. New York: Springer, pp. 184–186.

On 2015 Dec 09, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION The proteins of thermophiles are generally more heat-stable than the corresponding proteins from mesophiles. This must be reflected in either, or both, of two major amino acid variables – composition and order. In the past the notion that amino acid composition might be reflective of the pressure in thermophiles to retain purine-rich codons (3) has been disparaged by Zeldovich et al. (6). In this elegant new paper (5), Venev and Zeldovich (2015) agree that the “multiple factors” not accounted for in their modelling “include the influence of the genetic code and guanine-cytosine (GC) content of the genomes on amino acid frequencies.” However, there is puzzlement that the “theory and simulations predict a strong increase of leucine content in the thermostable proteins, whereas it is only minimally increased in experimental data.” Perhaps it is of relevance that leucine is on the top left quadrant of the standard presentation of the genic code, its codons being extremely poor in purines.

My response to Zeldovich et al. (6) in 2007, and my follow-up references in 2012 (1, 4), are set out below. One of the coauthors of the 2007 paper has recently further contributed to this topic (2).

2007 Response

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

2012 Response

As a follow up, it can be noted that Dehouck et al. (2008) report that relationship between a protein's thermostability and the optimum growth temperature of the organism containing it, is not so close as previously thought (1). Furthermore, Liu et al. (2012) now conclude from a study of xylanase purine-rich coding sequences that "The codons relating to enzyme thermal property are selected by thermophilic force at [the] nucleotide level," not at the protein level (4).

1.Dehouck Y, Folch B, Rooman M (2008) Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity. Protein Engineering, Design and Selection 21:275-278.Dehouck Y, 2008

2.Goncearenco A, Berezofsky IN (2014) The fundamental tradeoff in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and the physics of nucleic acids and proteins. Biology Direct 9:29 Goncearenco A, 2014

3.Lambros RJ, Mortimer JR, Forsdyke DR (2003) Optimum growth temperature and the base composition of open reading frames in prokaryotes. Extremophiles 7:443–450.Lambros RJ, 2003

4.Liu L, Wang L, Zhang Z, Wang S, Chen H (2012) Effect of codon message on xylanase thermal activity. J. Biol. Chem. 287:27183-27188 Liu L, 2012

5.Venev SV, Zeldovich KB (2015) Massive parallel sampling of lattice proteins reveals foundations of thermal adaptation. J. Chem. Phys. 143: 055101Venev SV, 2015

6.Zeldovich KB, Berezofsky IN, Shakhnovich EI (2007) Protein and DNA sequence determinants of thermophilic adaptation. PLOS Comput. Biol. 3(1), e5.Zeldovich KB, 2007

On 2015 Oct 11, Donald Forsdyke commented:

GC% – A COLLECTIVE VARIATION THAT FOSTERS SPECIATION

Among members of a species there is generally a mean genomic GC% value with a bell-curve distribution about this mean. The small group to the left of the mean are biased towards low GC%. The small group to the right of the mean are biased towards high GC%. These fringe groups can be said to have collectively varied away from the mean.

In keeping with the proposal that the reproductive isolation needed for speciation would be fostered by a collective variation (1), it has been shown how differences in GC% would impair recombination between a group on the fringe of the bell-curve and the main species group at the centre of the bell-curve (2). Other conditions being propitious (e.g. further rein is given to natural selection), a fringe group could then become a separate species with its own bell-curve GC% distribution.

Thus, if through reproductive isolation the low GC% group departed the main species, the population mean value would move slightly to the right (higher GC%). While Mugal and her colleagues (3) provide a valuable review of the literature on genomic base composition, the implications for speciation are not mentioned. For more please see my speciation text (4) and my bioinformatics textbook (5). There are also recent PubMed comments (6, 7).

(1) Romanes GJ (1886) Physiological selection: An additional suggestion on the origin of species. Journal of the Linnaean Society, Zoology 19, 337-411.

(2) Forsdyke DR (1996) Different biological species "broadcast" their DNAs at different (G+C)% "wavelengths". J. Theoret. Biol. 178, 405-417. Forsdyke DR, 1996% "wavelengths".")

(3) Mugal CF, Weber DD, Ellegren H (2015) GC-biased gene conversion links the recombination landscape and demography to genomic base composition. BioEssays 35: (in press) doi:10.1002/bies.201500058 Mugal CF, 2015

(4) Forsdyke DR (2001) The Origin of Species Revisited. McGill-Queen’s University Press, Montreal.

(5) Forsdyke DR (2011) Evolutionary Bioinformatics. 2nd edition. Springer, New York.

(6) Clément Y, Fustier M-A, Nabholz B, Glémin S. (2014) The bimodal distribution of genic GC content is ancestral to monocot species. Genome Biology and Evolution 7, 336-348. Clément Y, 2014

(7) Turner LM, Harr B (2014) Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions. Elife Dec 9;3 doi: 10.7554/eLife.02504 Turner LM, 2014

On 2015 Nov 16, Donald Forsdyke commented:

COMPLEMENT ACTIVATION BY PLANT AND ANIMAL LECTINS - THE LECTIN PATHWAY

The “proof-of-principle” that “lectins can be made more selective through molecular engineering” was demonstrated with the plant mannose-binding lectin Concanavalin-A in the 1970s (1). The functions then studied were mitogenicity, complement activation, and serum-binding activity. The dimeric form was found to retain mitogenicity without complement activation. The tetrameric form retained both activities (2).

The discovery that plant lectins activate the mammalian complement system (1) was initially attributed to a single antibody-independent “alternative” pathway of complement activation (3). However, subsequently the observation was extended to animal mannose-binding lectins and a second “alternative” pathways was characterized (4). This was named the “lectin pathway” and the other retained the “alternative pathway” name. Thus, there are three complement activation pathways – the classical, the lectin and the alternative.

In this light there are caveats regarding the proposed topical application of lectins to prevent HIV infection.

(i) There is suggestive evidence that lectins can cross mucous membranes, thus accessing body fluids and exerting toxic, perhaps complement-mediated, effects (5). While the elegant studies of Swanson et al. (6) show how mitogenic effects might be avoided, the possibility of toxic effects has not been excluded.

(ii) Reaction of soluble lectins with targets can be extensively buffered by competing lectin-binding activities, both associated with cell surfaces and in body fluids. When HIV buds from infected cells its envelope includes normal cell surface components that bind lectin. Furthermore, when plant lectins activate lymphocytes cultured in serum-containing medium, doubling the serum concentration doubles the lectin requirement (7). Thus, very high lectin concentrations may be needed for in vivo microbicidal effects. Is in vitro BanLec mitogenicity dependent on the BanLec/serum ratio? If so, is molecular engineering able to decrease competitive binding by serum and natural secretions?

(iii) While avoidance of initial HIV infection is important, use of lectins has not been thought promising in this respect. Hopefully the lectin work is not diverting resources from studies of the “shock-and-kill” approach that promises complete HIV eradication (8)?

(1) Milthorp P, Forsdyke DR (1970) Inhibition of lymphocyte activation at high ratios of concanavalin A to serum depends on complement. Nature 227:1351-1352. Milthorp P, 1970

(2) Forsdyke DR (1977) Role of receptor aggregation in complement-dependent inhibition of lymphocytes by high concentrations of concanavalin-A. Nature 267:358-360. Forsdyke DR, 1977

(3) Eidinger D, Gery I, Elleman C (1977) The inhibition of murine lymphocyte mitotic responses by human and mouse sera. 1. Evidence for a role of antibody-independent activation of the alternative complement pathway. Cellular Immunology 30:82-91. Eidinger D, 1977

(4) Degn SE, et al. (2014) Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes. Proc. Natl. Acad. Sci. USA 111:13445-13450. Degn SE, 2014

(5) Forsdyke DR (1978) Role of complement in the toxicity of dietary legumes. Medical Hypothesis 4:97-100. Forsdyke DR, 1978

(6) Swanson MD, et al. (2015) Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity. Cell 163:746-758. Swanson MD, 2015

(7) Forsdyke DR (1967) Quantitative nucleic acid changes during PHA-induced lymphocyte transformation in vitro: dependence of the response on the PHA/serum ratio. Biochemical Journal 105:679-684. Forsdyke DR, 1967

(8) Forsdyke DR (1991) Programmed activation of T-lymphocytes. A theoretical basis for short term treatment of AIDS with azidothymidine. Medical Hypothesis 34:24-27. Forsdyke DR, 1991

On 2016 Jun 25, Donald Forsdyke commented:

HISTORY OF LECTIN PATHWAY RESEARCH

The history of this field has recently been reviewed (1,2).

1 Forsdyke DR (2016) Microbes & Infection 18, 450-459. Almroth Wright, opsonins, innate immunity and the lectin pathway of complement activation: a historical perspective. Forsdyke DR, 2016

2 Sims RB, Schwaeble W, Fujita T (2016) Complement research in the 18th-21st centuries: progress comes with new technology. Immunobiology 221:1037-1045 Sim RB, 2016

On 2016 Jan 26, Donald Forsdyke commented:

None

On 2016 Jan 28, Donald Forsdyke commented:

HYPOTHESIS-DRIVEN RESEARCH

The discoveries of a cytosolic microbial adaptive immune system (CRISPR) and its applications to genome editing are major scientific advances. A review of the history of this magnificent achievement, made mainly by young people close to those with abundant research funds, is welcome. But the implication that this history supports the non-hypothesis-driven approach to research is questionable.

Backed by inexpensive bioinformatic analyses, a hypothesis of cytosolic innate immunity was developed in the 1990s [1-3]. Had this CRISPR-analogous hypothesis been backed by funding, CRISPR and its applications might have been achieved more expeditiously. Thus, there are many roads to Rome. Because the well-equipped army that took route A arrive first, it does not follow that route A is superior to route B. Likewise, this comment could have been written in prose or poetry. Your liking (perhaps) of the present prose rendition, does not disprove the proposition that a poetic version might have been superior.

[1] Forsdyke & Mortimer (2000) Chargaff’s legacy. Gene 261, 127-137.Forsdyke DR, 2000

[2] Cristillo et al. (2001) Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load. J Theor Biol 208, 475-491.Cristillo AD, 2001

[3] Forsdyke, Madill & Smith (2002) Immunity as a function of the unicellular state: implications of emerging genomic data. Trends Immunol 23, 575-579.Forsdyke DR, 2002

On 2016 Jan 26, Donald Forsdyke commented:

An accurate account of the exciting lead up to the 1977 discovery of split genes and obviously, as indicated by the title, Arnold Berk's fine perspective review does not deal with the alternative hypotheses that then appeared. However, readers are left to conclude that the "original suggestion" of Gilbert is now backed by "considerable evidence," so that perhaps the alternatives are disposed of. We should not forget that, in step with Gilbert, Darryl Reanney in Australia was fostering a viewpoint for which "considerable evidence" has also accumulated [1].

[1] Forsdyke DR (2013) Introns first. Biological Theory 7:196-203 http://post.queensu.ca/~forsdyke/introns3.htm

On 2016 Apr 04, Donald Forsdyke commented:

COLLECTIVE GENE FUNCTIONS SHOULD BE TAKEN INTO ACCOUNT

Gene products have both individual and collective functions (e.g. the Donnan equilibrium; 1). Wang et al. suggest that female susceptibility to autoimmune diseases may reflect incomplete dosage-compensation, which results in overexpression of certain X chromosome-located, immunity-related, genes (2). However, they refer to studies by Scofield and colleagues [ref. 11] on diseases with changes in numbers of entire X chromosomes (e.g. XXY), indicating biallelic expression of many genes. Scofield has noted that collective, as well as specific, gene functions must be considered (3). This is in keeping the hypothesis that the cytosolic aggregation pressure exerted by the protein collective is immunologically important (4, 5). <br>

1.Loeb J (1921) Donnan equilibrium and the physical properties of proteins. 1. Membrane potentials. J Gen Physiol. 3:667-90. Loeb J, 1921<br>

2.Wang J et al. (2016) Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proc Natl Acad Sci USA doi/10.1073/pnas.1520113113 Wang J, 2016<br>

3.Dillon SP et al. (2012) Sex chromosome aneuploidies among men with systemic lupus erythematosus. J Autoimmun 38:J129-J134.Dillon SP, 2012<br>

4.Forsdyke DR (2009) X chromosome reactivation perturbs intracellular self/not-self discrimination. Imm Cell Biol (2009) 87:525-528. Forsdyke DR, 2009<br>

5.Forsdyke DR (2012) Ohno's hypothesis and Muller's paradox: sex chromosome dosage compensation may serve collective gene functions. BioEssays 34:930-933. Forsdyke DR, 2012

On 2016 Apr 06, Donald Forsdyke commented:

PROTEIN SIZE AND CONCENTRATION DETERMINE DOSAGE-SENSITIVITY?

With the goal of understanding “the evolution of incomplete sex chromosome dosage compensation mechanisms in general,” the authors confirm that, among dosage-sensitive genes, those whose products specifically engage in stoichiometric complexes with other gene products, have a high degree of dosage-compensation. However, most genes are not dosage-limited by stoichiometry and “perplexing questions” remain. It is suggested that “certain loci … simply lack dosage effects” (my italics). In other words, certain loci “simply” contribute more to dosage effects than others.

While far from simple, this proposal is consistent with dosage compensation being more concerned with collective protein functions than with the specific functions of individual proteins (1). In the crowded cytosol the protein collective should exert an entropy-driven aggregation pressure on individual proteins, as part of a process of intracellular self/not-self discrimination (2). It is predicted that small, low concentration, proteins, will hardly influence aggregation pressure, so here there is no necessity for dosage compensation between the sexes. However, large, high concentration, proteins will greatly influence aggregation pressure, so here regulation of dose, on a gene-by-gene basis or otherwise, should be critical. Failure to regulate such dosage in human females would explain their susceptibility to autoimmune diseases (3-5).

1.Forsdyke DR (1994) Relationship of X chromosome dosage compensation to intracellular self/not-self discrimination: a resolution of Muller's paradox? J Theor Biol 167:7-12. Forsdyke DR, 1994

2.Forsdyke DR (2009) X chromosome reactivation perturbs intracellular self/not-self discrimination. Imm Cell Biol (2009) 87:525-528.Forsdyke DR, 2009

3.Dillon SP et al. (2012) Sex chromosome aneuploidies among men with systemic lupus erythematosus. J Autoimmun 38:J129-J134. Dillon SP, 2012

4.Forsdyke DR (2012) Ohno's hypothesis and Muller's paradox: sex chromosome dosage compensation may serve collective gene functions. BioEssays 34:930-933. Forsdyke DR, 2012

5.Wang J et al. (2016) Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proc Natl Acad Sci USA doi/10.1073/pnas.1520113113 Wang J, 2016

On 2016 Apr 28, Donald Forsdyke commented:

RNAS ENCODING HBZ AND EBNA1 PROTEINS ARE BOTH PURINE-LOADED

The sensitive quantification of HBZ protein levels in various clinical conditions (1) is a major advance. Unlike other HTLV1 proteins, the latency-controlling HBZ protein is encoded by the antisense strand. Thus, whereas the main-gene-encoding ‘top’ strand of the latent virus is pyrimidine-rich, the complementary, ‘bottom,’ strand that encodes HBZ is purine-rich. Likewise, most genes in Epstein-Barr virus are encoded by pyrimidine-rich strands, but the latency-controlling EBNA1 protein is encoded by a purine-rich strand.

The speculation that this purine-loading militates against the formation of double-stranded RNA and hence dampens the host immune response (2) is in keeping with:

(a) the “extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection,”

(b) the possibility that “the most important actions of HBZ, which are critical to HTLV-1 persistence, are exerted at the RNA level, and not the protein level,” and

(c) the view that “minimized HBZ protein translation is a sophisticated viral strategy for evasion from the host T cell response.”

(1) Shiohama et al. (2016) Retrovirology 13:29 Shiohama Y, 2016

(2) Forsdyke DR (2014) Microbes and Infection 16, 96-103 Forsdyke DR, 2014

On 2016 Jul 21, Donald Forsdyke commented:

THEORY-DRIVEN RESEARCH

The results of Enard et al. Enard D, 2016 "draw a broader picture where adaptation against viruses involves not only the specialized antiviral response, but also the entire population of host proteins." Indeed, Petrov has remarked: "Organisms have been living with viruses for billions of years" so on theoretical grounds alone "those interactions have affected every part of the cell."

Given recent disparagement of theoretical work Lander ES, 2016, it is nice to see results that are consistent with theory (e.g. Trends Immunol (2002) 23:575-579; Paper with Endnotes). Enard et al. now "conservatively estimate" that "viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals." Such "virus interacting proteins" vastly exceed the known proteins that regularly engage in immune responses to viruses (e.g. protein kinase R).

This is consistent with the 2002 postulate of the existence of intracellular protein "immune receptors" Forsdyke DR, 2002. Thus, over evolutionary time a protein that primarily evolved for a distinct function, but also happened to cross-react with some virus component, would in addition be selected by virtue of the latter function.

On 2016 May 20, Donald Forsdyke commented:

FACTS BEFORE COUNTERFACTS

Counterfactual explorations can provide intriguing insights (1). But we have to be sure that the facts themselves are correct in the first place. In the context of Weldon, reference to "bad-tempered conflict with Mendel’s followers," really means conflict with William Bateson. While it is correct that the doctrinaire "Mendelian ‘genes for’ approach is increasingly seen as out of step with twenty-first-century biology" (1), for Bateson (1861-1926) the approach was also seen as out of step with twentieth-century biology.

Well aware of developmental and environmental factors, Bateson recognized that the biochemical characterization of genes should be high on the twentieth century research agenda. Thus, near the end of his life he declared that “Our knowledge of the nature of unorganized matter must first be increased. For a long time we may have to halt” in getting to grips with the underlying biological principles (2). However, he argued forcefully for looking beyond the visible characters of an organism (its conventional phenotype) to what we now regard as its genome phenotype (3). It was here that the answer to Darwin's fundamental question - the origin of species - was likely to lie.

Weldon had allied himself with Pearson whose brilliant work (later built on by Fisher), was to create modern biostatistics. But those were early days and they made elementary mistakes that Bateson was quick to point out. For example, Bateson would have bridled at the idea that “first year biologists” could serve as a reliable “control” against which to compare “second year humanities undergraduates” (1). Yes, we should “study Mendel, but let him be part of his time”(1). And as related by Meijer (4), Mendel followed the statistics of his time. Indeed, his results have withstood the test of time.

(1) Radick G (2016) Teach students the biology of their time. Nature 533:293 Radick G, 2016

(2) Cock AG, Forsdyke DR (2008) "Treasure Your Exceptions." The Science and Life of William Bateson. Springer, New York.

(3) Forsdyke, D. R. (2010) George Romanes, William Bateson, and Darwin's "Weak Point." Notes Rec R Soc Lond 64:139-154.

(4) Meijer OG (1982) The essence of Mendel’s discovery. In: Gregor Mendel and the Foundations of Genetics. Orel V (ed). The Mendelianum of the Moravian Museaum, Brno, pp. 173-200.

On 2016 Jul 03, Donald Forsdyke commented:

GENIC OR NON-GENIC ORIGIN OF SPECIES?

Beginning by citing William Bateson’s magnificent 1909 paper, this elegant study focuses on the role of gene products in the hybrid sterility that can lead to speciation. However, there is an “inherent difficulty of distinguishing when, during the course of speciation, [genic] incompatibilities [resulting in hybrid sterility] may have arisen.” Indeed, the authors’ calculations “imply that most [genic] hybrid incompatibility factors identified in well-established, reproductively isolated species, are likely to have arisen since speciation.”

Thus there is a distinction between initiation and maintenance. While the genes studied are held to play a role in “contributing to genetic conflicts resulting in reproductive isolation,” this role is one of “contributing to the maintenance of postzygotic reproductive isolation,” not contributing to its initiation. Under the heading “temporal origins of reproductive incompatibility,” it is held “unlikely that either Taf1 or agt was involved in the origin of reproductive isolation between D. simulans and D. mauritiana.”

On the other hand Bateson had argued that the initial origin of species could often be non-genic (1). There are a large number of sequence differences in the HMS1 region that are non-genic, or intronic, or change codons synonymously (Table 1), and the authors note “considerable opportunity for interactions among sites.” This could affect DNA structure. When such interactions change, the secondary structure of the extruded DNA segments involved in meiotic pairing between D. simulans and D. mauritiana chromosomes could also change.

The hybrid sterility resulting from such “internal genomic conflicts” could have initiated the divergence, and the observed genic changes would have arisen subsequently. So it can be concluded that “any [genic] incompatibility factor between D. simulans and D. mauritiana is very likely to have evolved since their divergence.” Yet, the authors do not mention the growing evidence for a non-genic basis for initiation of divergence (2-4).

1.Forsdyke DR (1999) Relationship of Romanes' "intrinsic" variability of the reproductive system, and Bateson's "residue", to the species-dependent component of the base composition, (C+G)%. J Theor Biol 201:47-61 Forsdyke DR, 1999%.")

2.Forsdyke DR (2001) The Origin of Species Revisited. McGill-Queen’s University Press, Montreal.

3.Forsdyke DR (2010) George Romanes, William Bateson, and Darwin's "Weak Point." Notes and Records of the Royal Society 64:139-154 (doi:10.1098/rsnr.2009.0045)

4.Forsdyke DR (2016) Evolutionary Bioinformatics, 3rd edition, (Springer, New York).