The Cecal AppendixOne More Immune Component With a Function Disturbed By Post-Industrial Culture.код для вставкиСкачать
THE ANATOMICAL RECORD 294:567–579 (2011) The Cecal Appendix: One More Immune Component With a Function Disturbed By Post-Industrial Culture 1 MICHEL LAURIN,1 MARY LOU EVERETT,2 AND WILLIAM PARKER2* UMR 7207, CNRS/MNHN/UPMC, Centre de Recherches sur la Paléobiodiversité et les Paléoenvironnements, Muséum National d’Histoire Naturelle, Paris, France 2 Department of Surgery, Duke University Medical Center, Durham, North Carolina ABSTRACT This review assesses the current state of knowledge regarding the cecal appendix, its apparent function, and its evolution. The association of the cecal appendix with substantial amounts of immune tissue has long been taken as an indicator that the appendix may have some immune function. Recently, an improved understanding of the interactions between the normal gut ﬂora and the immune system has led to the identiﬁcation of the appendix as an apparent safe-house for normal gut bacteria. Further, a variety of observations related to the evolution and morphology of the appendix, including the identiﬁcation of the structure as a ‘‘recurrent trait’’ in some clades, the presence of appendix-like structures in monotremes and some non-mammalian species, and consistent features of the cecal appendix such as its narrow diameter, provide direct support for an important function of the appendix. This bacterial safehouse, which is likely important in the event of diarrheal illness, is presumably of minimal importance to humans living with abundant nutritional resources, modern medicine and modern hygiene practices that include clean drinking water. Consistent with this idea, epidemiologic studies demonstrate that diarrheal illness is indeed a major source of selection pressure in developing countries but not in developed countries, whereas appendicitis shows the opposite trend, being associated with modern hygiene and medicine. The cecal appendix may thus be viewed as a part of the immune system that, like those immune compartments that cause allergy, is vital to life in a ‘‘natural’’ environment, but which is poorly suited to post-industrialized societies. Anat Rec, 294:567–579, C 2011 Wiley-Liss, Inc. 2011. V Key words: cecum; appendix; evolution THE CECAL APPENDAGE AND MICROBIAL ECOLOGY IN THE GUT The cecal appendix in a variety of primates, rodents, and diprotodont marsupials is a relatively narrow, closed-end, tube-like extension from the terminal end of the cecum, characterized by a sharp and distinct change in the diameter of the bowel at the junction between the cecum and appendix. In addition, small ‘‘appendix-like structures,’’ which resemble the cecal appendix in shape but which are present in the absence of a cecum, appear at the junction between the small bowel and large bowel in all extant monotremes as well as in some birds and some actinopterygians (Smith et al., 2009). It is the purC 2011 WILEY-LISS, INC. V pose of this review to summarize recent changes in the understanding of the function and evolution of the appendix. *Correspondence to: William Parker, Ph.D., Department of Surgery, Duke University Medical Center, Box 2605, Durham, NC 27710. Fax: 919-681-7263. E-mail: email@example.com Received 7 April 2010; Accepted 6 January 2011 DOI 10.1002/ar.21357 Published online 2 March 2011 in Wiley Online Library (wileyonlinelibrary.com). 568 LAURIN ET AL. Since its identiﬁcation in humans more than 400 years ago and until the 21st century, the function of the appendix was not known, with substantial evidence from the ﬁeld of medicine pointing toward the idea that no function existed. On the other hand, a wide range of biological evidence from histological and phylogenetic studies has persistently kept the idea alive that some as yet unknown function must exist. However, only recently was an apparent function of the human appendix identiﬁed for which its tube-like structure is apparently well adapted, when Bollinger and colleagues (Bollinger et al., 2007) deduced that the cecal appendix is well adapted to facilitate maintenance of bioﬁlms containing the mutualistic intestinal ﬂora. The appendix apparently plays an important role in the microbial ecology within the gut (Bollinger et al., 2007). It is now well established that a wide range of animals exist in a co-dependent manner with a large complement of micro-organisms in their gut and on their skin (Backhed et al., 2005; Pennisi, 2008; Hattori and Taylor, 2009). These microorganisms depend on the host for food and a habitat, and the animal depends on these commensal microbes to repel infectious organisms, to drive the normal development of the immune system, and to aid in digestion. Thus is seems reasonable that, like other complex organisms such as angiosperms (Campbell and Greaves, 1990; Weller and Thomashow, 1994; Gupta et al., 2000; Fraysse et al., 2003) and corals (Reshef et al., 2006; Ritchie, 2006; Rosenberg et al., 2007), mammals expend substantial energy in the maintenance of the commensal bacterial ﬂora (Bollinger et al., 2003; Sonnenburg et al., 2004). A wide range of experimental evidence points toward the idea that secretory IgA and mucin, two of the most abundantly produced macromolecules in the body, both support the growth of bioﬁlms (adherent colonies of bacteria growing in an extracellular matrix) on the gut epithelium (Bollinger et al., 2003; Orndorff et al., 2004; Palestrant et al., 2004; Sonnenburg et al., 2004; Bollinger et al., 2005). This production of bioﬁlms is apparently beneﬁcial to both the host and the microbes, and it provides a protected habitat for the microbes, while simultaneously providing a barrier to infection for the host. The deduction that the cecal appendix is well adapted to maintain bioﬁlms containing the mutualistic intestinal ﬂora (Bollinger et al., 2007) was based on a number of observations: First, as described above, the immune system maintains microbial bioﬁlms in the mammalian gut as a key component of the mutualistic relationship between mammals and microbes (Bollinger et al., 2003; Orndorff et al., 2004; Palestrant et al., 2004; Sonnenburg et al., 2004; Bollinger et al., 2005). Second, bioﬁlms are widely known to be ‘‘safe’’ zones for bacteria, where they are protected from assault by a variety of factors including other microbial species, and where they form a corporative community (Costerton et al., 1987; LappinScott and Costerton, 1989; Costerton, 1995; Costerton et al., 1995; Kolenbrander, 2000; Xu et al., 2000; Gilbert and McBain, 2001; Lewis, 2001; Davies, 2003; Dykes et al., 2003). Third, immune tissue (gut-associated lymphoid tissue; GALT) is known to be concentrated in the appendix of several species, and thus the appendix is considered to be an ‘‘immune organ’’ (Berry, 1900). The presence of this immune tissue, along with the fact Fig. 1. The apparent function of the appendix. The cycle of recovery from diarrheal illness as supported by the vermiform appendix is illustrated. In this scheme, beneﬁcial bacteria (green) are rapidly eliminated in a diarrheal response along with pathogenic bacteria (red), leaving a digestive tract with far less bacteria than would normally be present (indicated by the white areas). Following this purge, bacteria shed from the cecal appendix inoculate the colon, initiating re-growth of the colonic ﬂora. The location in the digestive tract, narrow lumen, and extensive bioﬁlms of the cecal appendix all serve to protect bacteria within the appendix from contamination with pathogenic bacteria. that the immune system supports mutualistic bioﬁlms in the gut (point #1, above), points toward the appendix as a potential site of maintenance for the bioﬁlms. Fourth, bioﬁlms were found to be most concentrated on the epithelium of the appendix, with less coverage of the epithelium by bioﬁlms in the more distal parts of the large bowel (Bollinger et al., 2007). Fifth, bioﬁlms in a variety of settings are known to be in a continual state of shedding and regeneration, and it was expected that the relatively rapid turnover of the gut epithelium in general, regardless of location in the gut, would dictate a rapid turnover of any bioﬁlms that happen to be adherent in the gut. Thus, it is expected that any epithelial surface, including that of the appendix, will regularly shed or release fragments of any adherent bioﬁlms, thus serving as an inoculum for any downstream niches that might potentially be available. Sixth, the location of the appendix at the terminal end of the cecum is well suited to avoid infection by a pathogen invading the host by passive ﬂow with the fecal stream. Finally, the long, narrow lumen of the appendix is well shaped to retard infection by pathogens invading the large bowel via the oral route. These factors, when combined, paint a picture of the appendix as well adapted to facilitate re-inoculation of the intestinal ﬂora in the event that the bowel should be infected by a pathogen (Bollinger et al., 2007). The cycle of infection, purging of the gut by diarrhea, and reinoculation of the gut by commensal organisms from the appendix is shown in Fig. 1. Given the tremendous disease burden imposed by untreated drinking water in FUNCTION AND EVOLUTION OF THE CECAL APPENDIX human populations without access to modern water treatment and sanitation facilities (see discussion below), it seems highly likely that this proposed function of the appendix might be advantageous for survival, at least in humans. A HISTORY OF THE FUNCTION OF THE APPENDIX Leanardo da Vinci’s failure to publish his now wellknown drawings of the human appendix made in the 1490’s had little to do with the length of time required to ﬁnd the apparent function of the appendix. Although Berengario da Carpi published the ﬁrst description of the human appendix in 1521, Antony van Leeuwenhoek did not identify bacteria until more than 150 years later, in 1676 (Van Leeuwenhoek, 1684). After the discovery of bacteria, almost another 200 years elapsed before a rudimentary understanding of mutualistic relationships was proposed (Schwendener, 1868; De Bary, 1879; Honnegger, 2000), and it was not until the late 1800’s that Louis Pasteur and other microbiologists pioneered the idea that the bacteria in the gut were vital to the life of the host (Schottelius, 1902; Gordon and Pesti, 1971). Further, it was not until the early 1900’s that an increased understanding of the immune system and the observation that the appendix is associated with substantial amounts of immune tissue (GALT) led to the conclusion by Berry that the appendix had some speciﬁc yet unidentiﬁed immune function (Berry, 1900). Studies throughout the 1900’s based on phylogeny and immunology (e.g., Keith, 1912; Neiburger et al., 1976; Gorgollon, 1978; Scott, 1980; Spencer et al., 1985; Zahid, 2004) continued to support the conclusion that the cecal appendix possesses some speciﬁc yet unknown function for which it is adapted. However, well-known but misleading observations made in the ﬁeld of medicine, discussed below, in conjunction with a failure to identify the function of the appendix, left most people comfortable with the idea that the appendix had no function at all. Because of the intense focus on pathogenic microbiology during the 1900’s, the immune system was considered to be strictly anti-microbial in nature. This view was extended even to the relationship between the immune system and the mutualistic microbes of the gut (Williams and Gibbons, 1972). Thus, given that the immune system was considered anti-bacterial, it stood to reason that the appendix, already identiﬁed as an immune ‘‘organ,’’ was also anti-bacterial in some way. In addition, Pasteur’s opinion about the mutualistic nature of the normal gut ﬂora was not shared by everyone in the early 1900’s, with some holding that the microbes within the mammalian gut were antagonists to our well being (Nencki, 1886; Metchnikoff, 1903; Gordon and Pesti, 1971). It was not until the mid 1900’s that Pasteur’s opinion about the beneﬁcial nature of the normal gut ﬂora became ﬁrmly established (Donaldson, 1964). A vast body of work during the late 1900’s provided a complex and evolving understanding of the function of the GALT, but why the GALT should be associated with the appendix remained a mystery. An important barrier to understanding the appendix was overcome in 2003 when it was realized that the immune system supports growth of beneﬁcial (mutualistic) bacteria in the mammalian 569 gut (Bollinger et al., 2003; Sonnenburg et al., 2004). This important shift in thinking made possible the deduction that the human appendix is a well-adapted ‘‘safe-house’’ for the maintenance of the mutualistic gut bacteria (Bollinger et al., 2007). Indeed, as long as the immune system is thought of as entirely anti-microbial, it is very difﬁcult to visualize a useful function of the appendix, or why the GALT would be associated with the long, narrow structure. THE EFFECT OF APPENDICITIS ON THINKING ABOUT THE FUNCTION OF THE APPENDIX Clinical experience with the appendix demonstrates that the human appendix is, in fact, a detriment to life in many cases. More than one in every 20 people in a typical industrialized country will be afﬂicted with an inﬂamed appendix during the course of their lifetime, with an expected mortality rate of about 50% in the absence of medical intervention. This observation might be viewed as evidence of a signiﬁcant Darwinian ﬁtness value of a ‘‘safe-house’’ to protect beneﬁcial bacteria. However, almost a century ago, Arthur Keith (Keith, 1912) proposed a much more likely explanation that would not be proven for another 60 years: ‘‘When we come to realize how slowly evolutionary processes have affected man’s body in past times, we can hardly expect our internal digestive system to adapt itself to the rapid pace demanded by the ever-accumulating resources of civilization.’’ It is now known that Keith was correct, and that in fact appendicitis is a disease associated with industrialized cultures but not developing cultures (Barker and Morris, 1988; Barker et al., 1988a,b; Bickler and DeMaio, 2008). For example, the typical incidence of childhood appendicitis in the United States appears to be about 35-fold greater than the incidence in segments of the African population that are unaffected by modern health care and sanitation practices (Bickler and DeMaio, 2008). Importantly, the incidence of appendicitis increases with the adoption of Western lifestyles in both African countries (Bickler and DeMaio, 2008) and in Europe (Barker and Morris, 1988; Barker et al., 1988a,b). In the 1980’s, two British epidemiologists, David Barker and David Strachan, found an explanation for the fact that the appendix can become inﬂamed in a lifethreatening manner in industrialized countries (Barker and Morris, 1988; Barker et al., 1988a,b; Strachan, 1989). They not only found a key to understanding the cause of appendicitis, but of a variety of other diseases related to over-reactivity of the immune system, including allergies and many autoimmune diseases. Providing the ﬁrst evidence for what became known as the ‘‘hygiene hypothesis,’’ Barker and Strachan independently found that immune system over-reactivity was a consequence of the hygienic environment associated with cultural changes following the industrial revolution (Barker and Morris, 1988; Barker et al., 1988a,b; Strachan, 1989). The conclusions of the hygiene hypothesis were opposed by the observation that certain components of 570 LAURIN ET AL. an ‘‘unhygienic environment’’ as deﬁned by post-industrial standards, such as dust-mite derived allergens and certain viral infections, can increase rather than lower the propensity for allergy or autoimmune disease (McGeady, 2004; Kivity et al., 2009). Indeed, ideas other than the hygiene hypothesis have been proposed which might, in part, account for over-reactive immune systems in developed countries (McGeady, 2004; Bickler and DeMaio, 2008). However, work during recent years has distinguished immunosuppressive environmental factors such as chronic colonization with helminths from immunostimulatory environmental factors such as acute viral infections and exposure to high levels of potential allergens (Bjorksten, 2009; Kivity et al., 2009). At the same time, strong support for the hygiene hypothesis came from a variety of directions (Yazdanbakhsh et al., 2001; Gale, 2002; Capron et al., 2004; Falcone et al., 2004; McGeady, 2004; Wilson and Maizels, 2004; Fumagalli et al., 2009), and the hypothesis has gained widespread support (Rook, 2009). The role of decreasing biome diversity in hygiene-associate hyperimmune responsiveness has become particularly well established, with most of the research focused on symbiotic worms, or helminths. Not only has helminth colonization in patients suffering from some immune-associated disorders been found to alleviate or halt the progression of those disorders (Summers et al., 2003; Correale et al., 2008; Reddy and Fried, 2009), but at least some of the underlying reasons why helminth colonization down-regulates the immune system have become evident. First, the helminth load in a typical mammal produces dozens if not hundreds of soluble molecules which down-regulate the immune system (Hewitson et al., 2009). Not only does helminth colonization actively down regulate the immune system as a means of evading destruction by the immune system (Hewitson et al., 2009), but increased activity of the immune system as a result of the colonization provides feedback inhibition that leads to a decreased sensitivity of the system (Lohr et al., 2009; Mizrahi and Ilan, 2009; Workman et al., 2009). Further, fossil evidence of helminth colonization in early Cretaceous dinosaurs (Poinar and Boucot, 2006) and even in Devonian jawless vertebrates (Lukševics et al., 2009) suggests that persistent colonization with helminths has resulted in selective pressures that must have inﬂuenced evolution of the immune system for at least 400 million years. Thus, based on a variety of observations made by medical science and conﬁrmed by other areas of investigation, it is not the immune system that is at fault for the incidence of appendicitis or of a variety of immune-associated diseases, but rather an incompatibility between human biology and post-industrial culture. It has been argued that the appendix may not have been lost during the course of natural selection because any decrease in the size of the appendix would make the structure even more dangerous than it is at present (Neese and Williams, 1996). This argument is refuted by several observations. First, the fact that appendicitis is caused by recent changes in human culture associated with industrialization (Barker and Morris, 1988; Barker et al., 1988a,b; Bickler and DeMaio, 2008) indicates that selection pressure due to appendicitis was probably not substantial prior to about 1860. Since the increased risk of appendicitis which occurred in the mid to late 1800’s was quickly followed by surgical procedures effectively preventing mortality from appendicitis (Drinkwater, 1924), it is doubtful that appendicitis will ever exert pressure for elimination of the appendix. Second, dozens of individuals have been identiﬁed patients who were born with the congenital absence of an appendix (Pester, 1965). Thus, loss of the appendix is a biological possibility in humans. Third, cladistic analyses suggest that the appendix has been lost several times during evolutionary history, accounting for the absence of an appendix in some rodents and possibly some primates (Smith et al., 2009). Such losses may result from a reduction in the selection pressure to maintain an appendix, perhaps due to changes in such factors as diet and social behavior that decrease the impact of intestinal pathogens on the health of the population. Although the exact nature of such changes remains unknown, the taxonomic distribution of the appendix (Fig. 2) clearly shows that the appendix has been lost more than once during the course of evolution. Finally, a number of taxa, including the wombat, the Cape dune mole-rat, the meadow vole, and the scaly-tailed ﬂying squirrel, have an appendix that is much smaller in terms of absolute size than that found in humans (Smith et al., 2009). Indeed, it is difﬁcult to imagine an appendix as large as the human appendix in a rodent that is <1% the size of a human. Thus, a very small appendix is not necessarily a dangerous appendix. THE EFFECT OF APPENDECTOMY ON THINKING ABOUT THE FUNCTION OF THE APPENDIX Perhaps, the most obvious and clearest evidence that the appendix has no signiﬁcant function in humans inhabiting developed countries is the fact that >5% of the population in these countries do not have their appendix due to appendectomy, and there are no apparent negative long-term side effects from that surgical procedure. In fact, until recently, the only clear clinical association with appendectomy was a slight decrease in the incidence of ulcerative colitis (Koutroubakis and Vlachonikolis, 2000). Of great interest is a recent, prospective study demonstrating that appendectomy signiﬁcantly improves the symptoms of 90% of patients with ulcerative proctitis, a type of ulcerative colitis, and leads to a complete remission of all symptoms in 40% of those patients (Bolin et al., 2009). Although it has long been known that ulcerative colitis, like appendicitis, is associated with modern hygiene, and that there is some connection between appendectomy and colitis (Koutroubakis and Vlachonikolis, 2000), the ﬁnding that the appendix apparently plays a direct role in ulcerative colitis is new. The apparent function of the appendix may, in fact, shed light on these observations if taken into account in light of the hygiene hypothesis: It is possible that the appendix associated with a hyperactive immune system helps maintain colitis by maintaining bacteria toward which the immune system is reacting in a hygiene-associated, pathogenic fashion. On the other hand, the considerable amounts of GALT housed in the appendix may be responsible for driving hygiene-associated, aberrant immune reactions that lead to ulcerative colitis even in regions of the gut distant from the appendix. Regardless of the mechanism(s) by which the appendix might contribute to ulcerative colitis, it is clear that the absence of Fig. 2. Phylogenetic tree of mammalian relationships with the most parsimonious evolutionary occurrence of the appendix and appendixlike structures mapped onto it. Taxa usually ranked as families were included as terminal operational taxonomic units (OTUs) as described by Smith and colleagues (Smith et al., 2009). For the source of the topology, see (Smith et al., 2009). The time calibration was taken mostly from a compilation of the paleontological literature that one of us (M. L.) used in previous and ongoing studies (Germain and Laurin, 2005; Pouydebat et al., 2008) in which references to the primary literature can be found. For some clades for which little paleontological data are available, ages were taken from a molecular study (Bininda-Emonds et al., 2007), but ages were decreased by about 20% to compensate for the fact that molecular ages are frequently much greater than paleontological ages of the same clades (Marjanovic and Laurin, 2007). A recent geological timescale (Gradstein et al., 2004) was introduced using the Stratigraphic Tools for Mesquite (Josse et al., 2006). Names of stages (Gradstein et al., 2004) were abbreviated and short stages were omitted because space is insufﬁcient to show the full names or even abbreviations at appropriate size. The optimization was performed in Mesquite 2.72 (Maddison and Maddison, 2009) using ordered states (taxa with variable expression of the appendix are considered as intermediate between taxa with and taxa without consistent expression of a cecal appendix); this differs slightly from the methods used by Smith et al. (2009). The phylogenetic signal in the distribution of the cecal appendix was assessed using a random taxon reshufﬂing analysis as described by Laurin (2004, 2005) using Mesquite, and was found to be highly signiﬁcant (P < 0.0001) for the overall tree. However, the phylogenetic signal was not signiﬁcant within the Euarchontoglires (P ¼ 0.34) or in Diprotodontia (P ¼ 0.20), perhaps reﬂecting the recurrent nature of that character within that clade. The rise of the appendix or propensity to evolve an appendix at the base of the Euarchontoglires and of Diprotodontia is indicated by red bars. 572 LAURIN ET AL. an appendix causes no obvious adverse effects in developed countries. Given the apparent function of the appendix, it is not surprising that appendectomy in the face of modern health care and hygiene has no ill effects. The apparent function of the appendix points toward a utility under conditions in which rapid recovery of the gut ﬂora following diarrheal illness is important. Such recovery is not expected to be particularly important in developed countries where advanced technology for handling sewage and producing drinking water are commonplace. In addition, the effective governmental regulation of food production in developed countries further reduces the impact of diarrheal illness on the population. These measures, combined, effectively eliminate the possibility of widespread epidemics of waterborne and foodborne gastrointestinal infection, and thus the possibility that the normal gut ﬂora will be contaminated or lost from a wide swath of the population. Finally, diarrheal illness is of only passing concern when the population is not struggling with issues such as starvation, malnutrition, or dehydration, and when medicines and nutritional support are readily available to prevent mortality due to infection of the gastrointestinal tract. Ironically, the only animal other than humans which has an appendix and which lives in an environment sufﬁciently hygienic to render the appendix useless is probably the laboratory rabbit. We suggest that the use of the laboratory rabbit in hygienic (i.e., biome-depleted) environments to study the function of the appendix may have contributed to the idea that the appendix had no important function. Although diarrheal disease does not signiﬁcantly affect some human populations due to the factors mentioned above, other human populations are less fortunate. For example, the World Health Organization reported that diarrheal disease was the fourth leading cause of disability-adjusted life years lost in the most populated African countries in 2000, lagging behind only HIV-AIDS, lower respiratory tract infections, and malaria (2001). Even more telling is the observation that diarrheal disease was the single greatest cause of disability-adjusted life years lost in 2000 in the most populated countries in South-East Asia (2001). Thus, acute diarrhea associated with contaminated drinking water or food was still a major factor in the survival of human populations in the very recent past, and was potentially one of the primary disease-related selection pressures on all human populations in the distant past. With this in mind, it seems very likely that any biological mechanism associated with the recovery from diarrheal illness would be highly advantageous to survival in a culture devoid of the medicine and technology associated with industrialization. The above argument provides an explanation for the observation that the loss of an appendix does no harm in human populations living in industrialized countries, and supports the conclusion that the appendix serves no apparent function in these populations. However, the same argument strongly suggests that the appendix may have been important for all human populations in the past, and indeed may still be important for many human populations today. THE EVOLUTION OF THE APPENDIX Charles Darwin noted the apparent lack of a function of the appendix in humans, and concluded that it must be an evolutionary remnant from a primate ancestor that ate leaves (Darwin, 1871). This idea was supported by the now-refuted (Fisher, 2000; Smith et al., 2009) view that in primates, the appendix is present only in hominids. The argument has been made that even though the rabbit also has an appendix, it is substantively different because it is large enough in size (about three times larger than the primate appendix, making it proportionately vastly larger in rabbits than in primates) to aid in digestion, and thus the primate appendix should still be considered unique. The erroneous ideas regarding the unique and vestigial nature of the appendix in hominids are still found in many textbooks, and may continue to remain widely held until the function and broad phylogenetic distribution (see below) of the appendix are widely appreciated. If indeed the appendix had no function, the most likely explanation might be that the structure is a vestige of evolution. Further, the high prevalence of appendicitis and the apparent absence of negative side-effects following appendectomy, described above, have been taken as direct evidence that the appendix is, in fact, a vestigial structure that is detrimental to life (Müller, 2002). It was not until recently that Smith et al. (2009) described in detail the various morphotypes and the phylogenetic distribution of the cecal appendix and appendix-like structures. The idea that the appendix is a harmful vestige of evolution was not supported by the comparative anatomical approach in a phylogenetic context (Smith et al., 2009). This approach revealed that species with an appendix belong to two clades (Fig. 2): A variety of Diprotodontia (diprodent marsupials) such as the wombat, the ringtailed possum, and the cuscus have an appendix. Second, a wide variety of Euarchontoglires, the clade including rodents, lagomorphs (rabbits and hares), and primates, also have appendices. Euarchontoglires with an appendix include a wide range of primates from both main clades (haplorhines and strepsirhines), rabbits, and a wide range of rodents from eight taxa traditionally considered families. Further, some species outside of Diprotodontia and Euarchontoglires, including all extant monotremes, have an appendix-like structure, although they do not have a true appendix. These observations clearly refute the idea that the primate appendix is unique, especially since the human appendix falls somewhere in the middle range when comparing the size and shape of human appendix with the appendices from other species. The appendix of the mole-rat, for example, is about 10 times smaller than the human appendix, whereas the appendix of the rabbit is about three times larger (Smith et al., 2009). Thus, although the term ‘‘vermiform’’ traditionally refers speciﬁcally to the primate appendix, it is unclear whether the primate appendix is particularly distinguished by anything but its name (Smith et al., 2009). More importantly, it is this evolutionary analysis, demonstrating the broad phylogenetic distribution of the appendix, which indicates most directly that the function of the cecal appendix at least compensates for the cost of building and maintaining the structure. An analysis of the phylogenetic distribution and morphological features of the appendix strongly suggest that the appendix can be described as a ‘‘recurrent phenotype’’ among Euarchontoglires and perhaps among Diprotodontia. Recurrent phenotypes are ‘‘exceedingly FUNCTION AND EVOLUTION OF THE CECAL APPENDIX common and phylogenetically widespread,’’ and have been described by a number of terms, including analogous variations, apomorphic tendencies, homiology, and underlying synapomorphy, among others (West-Eberhard, 2003, 2005). Such phenotypes are ‘‘similar or identical phenotypic traits with discontinuous phylogenetic distributions, which owe their similarity to common ancestry (homology)’’ (West-Eberhard, 2003). The cecal appendix has properties that are generally characteristic of recurrent phenotypes, including the following: (a) The appendix occurs sometimes as a ﬁxed character and sometimes as an alternative morphotype within a given species (Smith et al., 2009). Variability in the expression of the appendix will be described below. (b) The appendix not only shows variability in expression within some species, but when it is expressed, considerable variation in morphology within the same species, sex, and life stage can be observed, as will be described in detail below. (c) The distribution of the appendix within the clades in which it occurs (Euarchontoglires and Diprotodontia) does not necessarily reﬂect the phylogeny. This lack of a statistically signiﬁcant phylogenetic signal in these clades (Fig. 2) is not surprising given that 45% of the taxa traditionally ranked as families that contain species with an appendix also contain species without an appendix (Smith et al., 2009). It is thus possible that the appendices found in Euarchontoglires and perhaps also in Diprotodontia are homologous within their respective clades, due to common descent from an ancestor which had either developed a cecal appendix or perhaps a ‘‘constitutional responsiveness’’ (inherited ability to respond to environmental variation) (Darwin, 1859; West-Eberhard, 2003), which facilitates the relatively rapid evolution of a cecal appendix. At the same time, the appendix is apparently a highly labile character in Diprotodontia and Euarchontoglires, as with all recurrent traits (West-Eberhard, 2003), appearing to ‘‘blink on and off during evolution’’ within those clades with a propensity to possess the trait. The appendix or perhaps the constitutional responsiveness that facilitates the evolution of the appendix would potentially have evolved twice during the course of evolution, once near the base of Euarchontoglires, and once again at the base of Diprotodontia (Fig. 2). In this scenario, the appearance of the character that facilitated evolution of the appendix in marsupials probably occurred more than 50 million years ago, after the Diprotodont marsupial lineage split from other marsupial lineages (Nilsson et al., 2003; Bininda-Emonds et al., 2007). Of particular interest is the koala (Phascolarctos cinereus), a phascolarctid Diprotodont marsupial, which has an ‘‘appendix-like structure’’ that is long and narrow, with a closed end, like a true appendix. Like the human appendix, the koala’s appendix-like structure is full of microbial bioﬁlms (McKenzie, 1978). In the case of the koala, these bioﬁlms are formed by bacteria that are necessary for the koala to digest the eucalyptus leaf, its primary food source. However, the koala’s appendix-like structure is not a true appendix, since it is formed by a uniquely long and tapering cecum without a clear junction having been identiﬁed that would separate the cecum and the appendix. This unique structure may represent the evolution of a new morphotype derived from the much more common cecum with a true appendix that is present in some Diprotodont marsupials. 573 The appearance of the appendix or perhaps the constitutional responsiveness that facilitates the evolution of the appendix in Euarchontoglires was probably earlier than in marsupials. In one scenario, the acquisition potentially occurred between 85 and 120 million years ago, according to some molecular dating studies (Kumar and Hedges, 1998; Douzery et al., 2003), or between 60 and 65 Ma ago, according to the fossil record (Wible et al., 2007), before the Euarchonta separated from the Glires. Thus, the cecal appendix or the character causing its recurrence may have been preserved during natural selection for a large portion of mammalian evolutionary time, perhaps extending through the K–T extinction event and being maintained within at least three orders of extant mammals (primates, lagomorphs, and rodents). Although the morphology or morphologies that preceded the cecal appendices found in Euarchontoglires and in Diprotodontia remains unknown, observations made in monotremes indicate that a narrow, appendixlike structure might possibly precede the evolution of a larger cecum with an effective digestive function. Both extant monotremes, the platypus and the echidna, have small, tubular ceca that resemble an appendix in shape. The structure of the platypus cecum, for example, is 2.5 cm long and about 3 mm in diameter (Krause, 1975), about one-third the size but approximately equivalent in shape to the human appendix. The structure in the echidna cecum is even smaller, being slightly over 1 cm in length and 3–4 mm in diameter (Stevens and Hume, 1995). These structures are so small that it is unlikely that they contribute substantially to digestion as fermentation chambers. Thus, it seems possible that acquisition of a cecum with primarily a fermentation function might not be a prerequisite for the evolution of an appendix-like structure with primarily an immunologic function involving maintenance of the host–microbial relationship. Regardless of the morphological transitions that might occur during the evolution of the cecal appendix, the appendix appears to be ancient in origin (Fig. 2), apparently supporting a mutualistic relationship between bacteria and animals that is even more ancient. A centerpiece in this relationship, microbial bioﬁlms in the normal bowel, has been observed in the gut of earthworms (Jolly et al., 1993; Vinceslas-Akpa and Loquet, 1995; Mendez et al., 2003), in at least one amphibian, and in a variety of mammals ranging from koalas to mice and humans (Smith et al., 2009). SUBSTANTIAL VARIATION IN THE STRUCTURE OF THE APPENDIX One of the hallmarks of the cecal appendage is variability in its size. Such variability was suggested by Darwin as being an indicator that the appendix was a vestige. This argument is not especially strong, since high degrees of variability not related to vestigial structures are often observed. In humans, for example, substantial variation is seen in the size of the torso, several parameters associated with hair, pigmentation in the skin and eyes, and foot and hand size to name only a few. Nevertheless, considering the variability in size of the cecal appendix is potentially important and may provide insight into its function, and, as pointed out above, into the potentially recurrent nature of the structure in 574 LAURIN ET AL. evolution. The variability observed in the structure of the human appendix is less striking than variability observed in the appendix of some other species. In at least four primate species and in the laboratory mouse, the appendix is present in some individuals and absent in others (Smith et al., 2009). Most mammals with an appendix, however, express the appendix with more regularity, although this may, to an extent, reﬂect the much smaller sample size of studied individuals in those species. In humans, even though several dozen individuals have been identiﬁed who have been born without an appendix (Pester, 1965), the overwhelming majority of individuals do have an appendix. Although the length of the human appendix is thought to be highly variable, the diameter is considered to be relatively consistent (Odze and Goldblum, 2008). A relatively consistent diameter has been supported by several studies using computed tomography (CT) to evaluate the appendix size. For example, one study of the normal appendix in 167 patients revealed that the mean diameter of the normal appendix without apparent intraluminal content is 6.6 mm with a standard deviation of 1.0 mm. (Benjaminov et al., 2002). Another study of 305 patients showed that about 80% of all normal appendices have a diameter between 4 and 7 mm, and >90% have a diameter between 4 and 8 mm (Tamburrini et al., 2005). The degree of variation observed depended to some extent on the presence or absence of fecal material in the appendix, and thus some of the observed variation is not inherent in the tissue itself. Further, differences in body size or mass, which are generally not taken into account in reports of appendix size, may account to some extent for variation in appendix size. With this information in hand, it seems not unreasonable to conclude that the narrow lumen of the appendix, which is likely important in its apparent role as a safe house for beneﬁcial bacteria, is consistently maintained in the human population. In contrast to diameter, the length of the normal appendix is thought to be highly variable, with both very long (>20 cm) and very short (<2 cm) lengths being reported. A reasonably detailed discussion concerning the length of the appendix, as well as some quantitative assessment of its variability in length, was provided by Kelynack (1893) and again by Lake (1920). As described by Kelynack and again by Lake, several surgeons had reported the longest appendix they had encountered during the course of their work. Kelynack himself had found the maximum length of any appendix he had encountered to be about 15 cm, whereas three of his colleagues had identiﬁed maximum lengths of 15, 17, and 23 cm, respectively. Appendices reported by Lake as being ‘‘of such extreme length as to be worthy of particular notice’’ included structures with lengths of 21.5 cm, 22 cm, 23 cm, 29.4 cm, and 33 cm. On the basis of these observations, it seems likely that appendices longer than 17–20 cm are very rare. When examining the frequency of very short appendices, Kelynack reported that only two out of 177 (about 1%) of the appendices in his practice had a length of <2.54 cm. Thus, both very long (>15 cm) and very short (<2.54 cm) appendices are apparently uncommon. Although extremes in length are apparently rare, Kelynack did observe a substantial degree of variation in typical lengths, with about 7% of the appendices in his study being >12.7 cm in length, and about 11% being <5.1 cm in length. Although this variation in length might be seen as an indication that the function of the appendix is of little biological importance, the range of appendix lengths found throughout phylogeny (from about 1 cm in length in the lemming to >15 cm in the brushtail possum) is consistent with the idea that appendices with a wide range of lengths may all be quite functional. Thus, the quintessential structural feature of the appendix may well be the relatively narrow lumen rather than any particular length. With this in mind, there appears to be substantial selective pressure to keep the diameter of the appendix relatively constant, whereas its length appears to be under much less evolutionary pressure. IS THE APPENDIX A ‘‘VESTIGE?’’ Whether the cecal appendix should be considered a ‘‘vestige’’ of a larger cecum has been hotly debated, and the discovery of a function for which the appendix is well adapted brings renewed interest to this debate. By deﬁnition, a vestige has a reduced function, if any function at all, compared to the structure from which it evolved (Darwin, 1871; Fong et al., 1995). In addition, vestiges are typically reduced in size compared to their fully functional predecessors (Fong et al., 1995). However, simply because the size of a given structure is decreased and even the function has decreased does not necessarily indicate that a structure is a vestige. For example, when considering an ancestor with arms used for collecting food, grooming, and locomotion in an arboreal habitat, and a descendent with relatively smaller arms that are used for collecting food, grooming, but no longer for locomotion, the arms of the descendent would not be considered vestiges despite the fact that the relative size of the arm has decreased and at least one function has been lost. The same might be said of a cecum in an omnivore, which, although still of perfect utility and even necessity for the digestion of food, is smaller and of relatively less signiﬁcance for the omnivore than a larger cecum was for a putative folivore ancestor of that omnivore. By the same token, although the cecal appendix of humans has been considered a vestige, the cecum itself has not been considered a vestige in humans, despite its relatively small size. Thus, to some extent, the idea of a vestige is tied not so much to a decrease in size or even function, but to the idea that the phenotype is driven by evolutionary history at the expense of present utility. In other words, a vestige may well have some utility and may even be adapted to perform new functions, but the morphology is better explained when considering the function as seen in an ancestor rather than the function at present. In keeping with this idea, the reason that the identiﬁcation of vestigial structures played an important role in the founding of evolutionary theory was that those structures provided tangible traces of past generations (Müller, 2002). With that in mind, one indication that the appendix is not a vestige is that, based on what we know of the apparent function of the appendix, the narrow diameter is more effective than a large cecum in terms of efﬁciently providing a bacterial safe-house. It is the apparent improvement of this function as seen in the shape of the appendix, not so much the presence of some function, which is important in this discussion. Although vestiges can adapt to new functions, many structures FUNCTION AND EVOLUTION OF THE CECAL APPENDIX 575 (arms in land-dwelling vertebrates, for example) are in fact derived from structures that had different functions (lobe-ﬁns used for swimming, for example). Because such structures are apparently well adapted for their present function, they are not considered vestiges. On the other hand, because it is not known to what extent the function of the cecal appendix as a microbial safe house is improved over the same function which might be carried out by the cecum, it may be impossible to determine whether or not the appendix should be classiﬁed as a vestige of a larger cecum based strictly on considerations of function. However, evidence regarding function is not the only evidence which has a bearing on the issue. A second, more telling, indicator that the cecal appendix is not a vestige is the fact that a cecal appendix is in many cases associated with a large cecum used for digestion. In fact, the majority of species with an appendix, including most rodents with an appendix as well as some primates, possess this morphology (Smith et al., 2009). As pointed out by Darwin, if the appendix is a vestige of a larger cecum used for digestion, then the appendix and a large cecum should not co-occur in any species, since the vestige and the structure from which the vestige is derived should not exist simultaneously. Third, as described above, a modern analysis of the phylogenetic distribution, comparative anatomy, and evolution of the appendix refute the idea that a large cecum gives rise to an appendix plus a smaller cecum (Smith et al., 2009). In fact, those analyses indicate that a narrow, appendix-like structure may well have evolved in some cases prior to the evolution of a cecum used for fermentation. When considering the evolution of the appendix, the several phenotypes associated with the proximal large bowel can be considered. These phenotypes can be roughly classiﬁed into ﬁve categories, each expected to have unique functional properties: 1. A cecal appendix or appendix-like structure, with no evident cecum. This morphology appears sporadically in vertebrates, with all monotremes, some actinopterygians, some birds, and at least one marsupial having this morphology (Smith et al., 2009). Examples (Fig. 3A) include Ornithorhynchus anatinus (the platypus) and the Lasiorhinus latifrons (Southern hairy-nosed wombat). The morphology is consistent with an immunological function and a minimal digestive function. 2. A cecal appendix with a small cecum. This morphology is characteristic of hominids (Smith et al., 2009). Examples (Fig. 3B) include Homo sapiens (humans) and Pongo pygmaeus (orangutans). The morphology is consistent with an immunological function and a modest digestive function. 3. A cecal appendix with a long cecum. This morphology is found in those rodents that have an appendix, in all lagomorphs, in a few marsupials, and in primates other than hominids with an appendix (Smith et al., 2009). Examples (Fig. 3C) include Lemmus lemmus (lemmings) and Lepilemur leucopus (sportive lemurs). The morphology is consistent with an immunological function of the appendix coupled with an extensive digestive function of the cecum. Fig. 3. Five morphotypes at the junction between the large and small bowel. A: Cecal appendix or appendix-like structure, with no evident cecum of Ornithorhynchus anatinus, the platypus (a monotreme, left), and Lasiorhinus latifrons, the Southern hairynosed wombat (a marsupial, right). B: Cecal appendix with a small cecum, as seen in Homo sapiens (humans, left) and Pongo pygmaeus (orangutans, right). C: Cecal appendix with a long cecum, in Lepilemur leucopus (sportive lemurs, left) and Lemmus lemmus (lemmings, right). D: Cecum with no appendix of Rattus norvegicus (rats, left) and Sus scrofa (pigs, right). E: No cecum and no appendix, as in Desmodus rufus (vampire bats, left) and Physeter catodon (sperm whales, right). Drawings of the bowel of the platypus, the wombat, the human, the orangutan, and the sportive lemur are from Smith et al. (2009). The drawing of the bowel of the lemur is adapted from Behmann (Behmann, 1973). The drawings of the bowel from the rat, the pig, sperm whale, and the vampire bat are adapted from Stephens and Hume (1995). 4. A cecum with no appendix. This morphology is common in a wide range of mammals, and occurs in many birds. Examples (Fig. 3D) include Rattus norvegicus (rats) and Sus scrofa (pigs). This morphology is consistent with a limited immunological function and a digestive function dependent on cecum size. 576 LAURIN ET AL. 5. No cecum and no appendix. This morphology appears in a range of mammals, as well as in actinopterygians, reptiles and amphibians. Examples (Fig. 3E) include Desmodus rufus (vampire bats) and Physeter catodon (sperm whales). The morphology is consistent with a minimum cost to produce, although immunological and digestive functions must be maintained without a blind sac or pouch. Given these ﬁve morphological states and the potential for any one of these states to be most advantageous in a suitable environment, there is no reason to suppose that any one state must necessarily be preceded by another state; rather, the states may evolve as needed. Thus, a linear evolution leading from a large cecum to a smaller cecum plus an appendix may be only one possible scenario out of several by which an appendix may have evolved (Smith et al., 2009). This observation has a bearing on the vestigiality of the appendix: As pointed out by Müller, vestigial structures must be identiﬁed by the comparative method: ‘‘homology of the reduced character with a more fully developed, ancestral counterpart must be established, not only on the basis of structural and positional criteria, but also with regard to the continuous presence of the structure in the lineage leading from the ancestral to the derived form’’ (Müller, 2002). Our comparative data suggest such a continuous presence in human ancestors, going back at least to early apes in the Miocene (Fig. 2), but they do not suggest reduction in size, which is typical of vestigial structures (Darwin, 1859). The same data suggests multiple appearances of the appendix, and fewer losses, which in turn suggests a selective advantage of the appendix, an idea supported by epidemiological, immunological, and microbiological data. Thus, the cecal appendix of humans cannot be considered a vestige. CONFIDENCE REGARDING THE APPARENT FUNCTION OF THE CECAL APPENDIX, AND FUTURE WORK ON THAT FUNCTION The observation that microbial bioﬁlms are most concentrated in the proximal large bowel, in conjunction with information that has been widely known for some time, including the relative seclusion of the cecal appendix and the protective nature of bioﬁlms for resident bacteria, provides the means for a ‘‘proof by deduction’’ that the cecal appendix is a safe-house for bacteria. In light of current knowledge regarding the nature of microbial bioﬁlms, it would in fact be difﬁcult to imagine that regions of the bowel which harbor bioﬁlms provide no sanctuary for bacteria. Indeed, the deduced function of the cecal appendix as an immune-supported safe-house for beneﬁcial bacteria is supported by a host of observations in immunology and microbiology, and is consistent with observations made by medical science. Further, a wide range of clinical observations and experimental studies point toward the vital role of the normal ﬂora in protection against infection and disease, thus providing a biological motive for the maintenance of that ﬂora. Finally, studies in comparative anatomy as well as evolutionary biology demonstrate that some function must exist which at least compensates for the cost of producing the structure. Although some degree of conﬁdence can be ascribed to the idea that the cecal appendix is a safe-house for beneﬁcial bacteria, no experiment or study has directly demonstrated the function of the appendix. The study of patients in developing countries following appendectomy is one potential way to obtain direct evidence for a function, but hurdles to such studies abound. Finding appropriate patients may prove difﬁcult for several reasons. First, appendicitis in developing countries is relatively rare (see discussion above), and second, access to such patient populations for purposes of long-term studies is often difﬁcult. Third, the function of the appendix may be most important in children <5 years of age, when death due to diarrheal illness is most common, thus providing a considerable restriction on which patients may be important for such a study. Fifth, if the age-association of appendicitis in developing countries is similar to that seen in developed countries, then appendicitis is most common from 10 to 14 years of age. At that age, the function of the appendix may be less important because death due to diarrheal illness is less during those years than in younger years. To complicate the picture, the relatively rare cases of appendicitis that do occur in developing countries may have a much different root cause and pathogenesis than cases of appendicitis in developed countries, since appendicitis in developed countries is apparently due to hygiene-associated immune over-reactivity, which does not exist in developing countries. Further, even if it were possible to monitor the effects of appendectomy in developing countries, it would be difﬁcult to distinguish cause from effect: It is likely that the factors associated with appendicitis in developing countries may also have an effect on mortality. Although the design of studies in humans aimed at direct observation of the apparent function of the appendix seem problematic, studies in laboratory animals could be conducted. However, such studies might be expensive, and a suitable animal model with a human-like appendix may need to be found. Although the rabbit is commonly used in the laboratory and does have a cecal appendix, the morphology of the intestine as well as the nature of digestion in the herbivorous rabbit is substantially different than that found in humans, some other primates, and a variety of rodents. Further, removal of the rabbit’s appendix results in a relatively much more substantial reduction in immune tissue than does removal of a human appendix, and can have profound systemic effects (Dasso and Howell, 1997). Thus, the relative importance of the cecal appendix to the immune systems of rabbits and humans may be profoundly different, and removal of the rabbit appendix may have substantial effects on the immune system that are not evident in humans following appendectomy. With this in mind, it may prove difﬁcult to test directly the function of the appendix in humans given currently available laboratory animal models. Perhaps, one of the most interesting and promising approaches by which the function of the appendix may be further evaluated is through comparative anatomy and evolutionary biology. As described above, it was this approach which ﬁrst demonstrated conclusively that the appendix is indeed adapted for a speciﬁc function, and is in fact not a vestige of evolution. Several scenarios regarding the origins and function of the appendix may FUNCTION AND EVOLUTION OF THE CECAL APPENDIX lend themselves to direct testing by current approaches in evolutionary biology because they make different predictions about the order in which a caecum used for fermentation and an appendix appeared, and comparative data can be used to determine the order of appearance in various taxa, and reveal a regularity (if any) in such appearances. THE CECAL APPENDIX AS A COMPONENT OF THE IMMUNE SYSTEM UPENDED BY FACTORS ASSOCIATED WITH POST-INDUSTRIAL CULTURE Given a wide range of observations from medical science, evolutionary biology, immunology, and comparative anatomy described above, it seems likely that the appendix, like other components of the immune system designed to ﬁght threats that no longer apply to many humans, is not only made obsolete by factors associated with post-industrialized culture, but is overly reactive or sensitive due to an absence of stimulation. Indeed, given the ever-increasing evidence supporting the hygiene hypothesis and the fact that all of that evidence points towards certain biological components such as helminths and mutualistic bacteria as the critical factors mediating immunity (Matricardi et al., 2000; Kalliomaki et al., 2001; Schiffrin and Blum, 2002; Yazdanbakhsh et al., 2002; Capron et al., 2004; Imai and Fujita, 2004; Wilson and Maizels, 2004; Rook and Brunet, 2005; Hewitson et al., 2009; Rook, 2009), it could be argued that the traditional term ‘‘hygiene hypothesis’’ would be more descriptively labeled as the ‘‘biome depletion theory of immune disorders.’’ Examples of immune components other than the appendix which are apparently maladapted to the post-industrialized culture are the components designed to produce immunoglobulin E (IgE): production of ‘‘high’’ levels of IgE leads to detrimental side effects (allergy) in industrialized countries, but levels of IgE much greater than those found in allergic patients are generally present in pre-industrial populations as a response to parasitic infections (Scaglia et al., 1979; Gale, 2002; Devalapalli et al., 2006), and no allergy is produced. Thus, the appendix is apparently another immune component ill-suited to post-industrial culture. It is now on the shoulders of medical science to devise a way to stimulate the immune system in a way that averts diseases associated with biome depletion and avoids profound side effects. CONCLUSIONS The cecal appendix appears to be a ‘‘recurrent’’ trait, variably expressed in Euarchontoglires and in Diprotodontia. Changes in selection pressure which inﬂuence the presence or absence of the appendix could involve changes in such factors as diet and social behavior that decrease the impact of intestinal pathogens on the health of the population, but the exact nature of such factors remains unknown. Given the wide range of observations from medical science, evolutionary biology, immunology, and comparative anatomy described in this review, it seems likely that the appendix, like other components of the immune system designed to ﬁght threats that no longer apply to humans in a post-industrial soci- 577 ety has been made obsolete and even rendered harmful by factors associated with post-industrial culture. The appendix is ancient in origin, apparently supporting a mutualistic relationship between bacteria and animals that is even older. The support of the bacterial bioﬁlms within the appendix is mediated in large part by the innate and adaptive immune systems, which actively facilitate growth of beneﬁcial bacterial in the gut. Given the ancient nature of immune interactions with gut bacteria, it may be hypothesized that a small, appendix-like pouch which functioned primarily as an immune structure maintaining the gut ﬂora evolved prior to the evolution of a cecum that functioned effectively as a digestive structure. Although the morphology that preceded the cecal appendices found in Euarchontoglires and in Diprotodontia remains unknown, data on monotremes indicate that a narrow, appendix-like structure may have preceded the evolution of a larger cecum with an effective digestive function. In the gut of several mammals, including humans, mice, and rats, microbial bioﬁlms are associated with the proximal large bowel but not the small bowel or the distal large bowel. In those animals having a cecum but no cecal appendix, the apex of the cecum is associated with GALT, has the greatest concentration of microbial bioﬁlms within the gut, and is thought to perform the function performed by the cecal appendix (Berry, 1900; Smith et al., 2009). The distribution of microbial bioﬁlms in the gut of a frog, which lacks a cecum, reveals a pattern surprisingly similar to that seen in mammals with a cecum or an appendix (Smith et al., 2009). Thus, the proximal large bowel, even in the absence of a cecum or appendix, is apparently the region most used for support of mutualistic microbial bioﬁlms, and the observation of this pattern in an amphibian suggests the possibility of an ancient origin for the pattern, since the last common ancestor of lissamphibians and amniotes lived between 332 and 360 million years ago (Marjanovic and Laurin, 2007). 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