A Retrospection on the Phylogeny of Bats

Introduction

The bat (specifically, the family Chiroptera) is among the most peculiar of mammals, as well as one of the most diverse. 1,200 species of bat are currently recognized, forming nearly 25% of named mammal species (3). Though sometimes colloquially called “flying rats” and sharing perhaps a passing resemblance to Murids, bats are distinct from rodents both genetically and morphologically— the family Chiroptera can be immediately distinguished from all other organisms (extant and extinct, as far as we currently know) by their “hand-wings”, formed from skin sketched between multiple elongated phalanges. These are structurally analogous to the wings of birds or extinct Pterosaurs, with the only similarity being that in all groups, wings were derived from the front limbs. However, the universal similarities between Chiroptera species essentially ends there. Some traits that we may culturally associate with the “essence” of what makes a bat— complex vocalizations, “blood-sucking” or, at the least, carnivory, nocturnal behavior, and communal roosting— are far from the norm when it comes to individual genera. In fact, there is almost no “norm” that can be applied to Chiroptera, and the lack of fossil records that can be definitively stated to be ancestral bats (there is a particular lack of anything resembling an intermediate wing; rather, wings appear fully-formed in the scant record) merely adds to the confusion (5). Over the decades, there have been several attempts to unravel the mysteries of the bat phylogeny, employing robust morphological research in eyes and dentition, genetic relatedness, fossil traces, and cytochrome b. Arguments have been made that Chiropetra is monophyletic, paraphyletic, and polyphyletic, and oftentimes the newest leading theory is intended to directly refute or support the claims made by an author years or decades prior. This paper will seek to review the most monumental cornerstones in our understanding of bat phylogeny, explore the ways in which they built upon one another to form our most recent, popularly-accepted theory, and reflect on what mysteries remain to be uncovered in the future. In particular, literature will be reviewed based on which evidence was being studied to create the authors’ phylogeny, and consider ways in which their scope could be adjusted or expanded to account for other studies or unaddressed mysteries.

The “Flying Primate”

Perhaps the foundation of all debate that followed was a controversial 1991 paper by John D. Pettigrew (14).. Pettrigew’s bold claim was to state that “megabats” and “microbats” were actually a polyphyletic group of organisms, and that Chiroptera was not a true taxonomic classification. The differences between the carnivorous, echolocating microbats and frugivorous, diurnal, and non-echolocating megabats (4) was widely acknowledged in the scientific community, with the assumption both groups derived from the same winged ancestor. (Whether this ancestor was echolocating or not was, and would remain for many years, a contentious subject.) However, Pettigrew took this distinction another step further— megabats, he argued, actually shared a more recent common ancestor with modern primates, and that the hand-wings for which bats were so distinct were actually analogously evolved structures. His evidence for this convergent evolution was an intense examination of the flight adaptations in both groups; megabats have differently arranged ankle bones than microbats, and dissimilar formations of nerves in the wing membrane. This, he argued, was evidence towards the theory that hand-wings and upside-down roosting had evolved twice in different lineages, rather than a derived trait of Chiroptera. As for close ancestry with primates, Pettigrew paid particular attention to the morphology of the megabat’s eye. These eyes contain extraordinarily specialized neural and ocular adaptations seen nowhere else in Mammalia excepting primates, and certainly not in microbats. These adaptations are unique, highly complex, and remarkably ineffectual in practice; Pettigrew argued that such morphology had a very slim chance of convergent evolution, because the adaptations themselves gave no obvious fitness advantage, and were more likely inherited from a common ancestor. Thus, megabats were the cousins of primates, and Chiroptera was no more.

Or so Pettigrew claimed. But his assertions were based almost exclusively on morphological data, which, though convincing, is ultimately not sufficient for constructing an accurate phylogeny. Pettigrew seemed unwilling to explore other explanations for the eyes of the megabat (perhaps such unique organization of optic nerves is one of the “spandrels” (8) of certain frugivorous behaviors.) And while his observations of ankle bone orientation were astute, the convergent arising of hand-wings in two independently-evolving lineages was not supported by any genomic or fossil evidence. Neither was the ecology of these animals considered— could perhaps the “odd” ankles of the megabats be to accommodate their greater mass while feeding upside-down, something that insectivorous microbats do not do? This is an original, unsubstantiated conjecture, but provides an example of the type of questions that were not posed by Pettigrew when he assessed the morphology of these bats. Yet, despite the flaws inherent in his limited scope, Pettigrew’s attempt to reclassify the messy, poorly understood Chiroptera provides a solid morphological foundation for the more microscopic innovations that followed.

Divisions by Genomic Data

As research shifted away from purely morphological data, many of the next major advancements in bat research were published in direct response to Pettigrew’s “flying primate” hypothesis. One particularly important paper was by Emma Teeling et. all, in 2002 (18).. Teeling and her co-authors posited two hypotheses: that “megabats” should be removed from the superordinal group Archonta, and that the “microbat” label was actually a paraphyletic classification. These researchers had access to genomic and mitochondrial data that Pettigrew did not, opening the gateway for an analysis that extended beyond the morphological. Bats have high frequencies of mitochondrial heteroplasmy in female germ lines (13), which allowed for robust mitochondrial research across multiple genera. This data revealed that megabats have minimal genomic evidence to support their placement in Archonta— the now-abandoned classification for primates, treeshrews, and colugos— but are closely related to microbats; bootstrapping models found a 0% reliability for the Archonta placement of extant bats. All bat species were subsequently reclassified into the superorder Laurasiatheria (11), but Teeling’s arguments did not end there. Molecular data had begun to shed doubt on the monophyly of microbats; through the study of four nuclear and three mitochondrial genes, a convincing case for the reclassification of the megabat superfamily Rhinolophidae into the “microbat” label, due now to their evolutionary relatedness rather than morphological similarities, was laid out (20). Teeling favored the “echolocation-first” theory of bat evolution over the “wing-first theory”, and suggested that highly specialized larynxes were present in the last common ancestor of extant bats and subsequently lost in megabats. These results were supported by a follow-up study on the morphology of Eocene taxa that concluded laryngeal echolocation evolved in a common ancestor 52-55 million years ago (16). That same year, however, microbat paraphyly was contested by a follow-up study on the molecular phylogenetics of a single species of Rhinolophidae (one which had been previously unstudied by Teeling and her team) which provided no evidence for a closer relationship with microbats than megabats (12). The reconciliation of these ideas was to claim that neither the superfamily Rhinolophidae or the microbat classification was monophyletic, and that Rhinolophidae actually contained a polyphyly of species that had descended from both lineages (18). This was an adequate solution to the immediate confusion of molecular data, but skirted any addressment of the larger issue: the current taxa for bats was confused, inaccurate, and archaic, based on an outdated morphological division between “micro” and “mega” that was unraveling on the genetic level. As with Pettigrew, ecological and life history components that inform the adaptation of individual species were not considered. Then, in complete antithesis of Pettigrew, morphological relatedness was completely ignored in favor of mitochondrial DNA. This was certainly illuminating for the few species of Rhinolophidae that were studied, but cast doubt on the entire phylogeny of the reunited order Chiroptera.

Phylogeny from Ecology

It is clear that neither morphological nor genetic data alone are able to create a satisfying phylogeny, and so now ecology, fossil evidence, and life history needed to be additionally considered. Scholar J. Findley said that bats were “among the most difficult vertebrates to study” (6) and claimed that no mammal group had undergone greater adaptive radiation than the bat, which would lead to many attempts to understand the group as a whole. One of the most comprehensive efforts to reconcile existing data with confused phylogeny was done by Patricia Freeman, who at last brought an element of ecology to a combined morphological and genetic perspective (7). Freeman tracked a highly detailed pattern of adaptation through both extinct and extant bat species via the examination of cranial and dental morphology and ribosomal DNA. Her research proposed a third option in the wing-first/echolocation-first debate: insectivory first. Freeman concluded that the earliest common ancestor of all bats (wholeheartedly reinforcing the bat monophyly) was an insectivore that looked similar to today’s “microbat” classification. Rather than branching into two “megabat” and “microbat” lineages, this insectivorous ancestor underwent rapid adaptive radiation into multiple ecological niches, including carnivory (from which blood-lapping vampire bats derived (17)), durophagy, nectarivory, and frugivory. Throughout these branching lineages, insectivory remained either the exclusive diet of certain genera, or was merely supplemented by these food sources. From this, Freeman suggested that a small ancestor species, capable of both insect and fruit eating, diverged into both the huge, fruit-eating megabats and smaller insectivores, explaining the close relationship between certain “microbat” groups and the odd, diurnal megabats. Echolocation, too, arose, atrophied, and radiated throughout the phylogeny (9) based on ecological pressures. In extant bats, two modes of echolocation— nasal-calls and oral-calls— exist in almost all species except the so-called megabats, and those genera most likely lost the laryngeal adaptations formerly present in a common ancestor. Freeman admits to some flaws in her study, especially the total lack of fossil evidence for the transition from insectivorous teeth to those of a frugivore (which is not, in itself, a rebuttal of her hypothesis, due to the rarity of bat fossils in general). The unique dentition of vampire bats is also not reflected in any fossils, nor are there similarities between them and another living groups, making Freeman’s placement of the vampire subfamily into Phyllostomidae— a family of nasally-echolocating New World bats— based more on the morphology of their “leaf-noses” and the geographical range of extant species than fossil or genomic evidence. Additionally, while this was the most robust examination of the bat phylogeny since Pettigrew’s “flying primate” hypothesis was debunked, it still offered no concrete solutions to the state of Chiroptera’s jumbled phylogenies. However, the addition of ecology and biogeography to molecular analysis was beginning to shed light on mysteries that no discipline alone could unravel.

Yangochiroptera and Yinpterochiroptera

Continued research led by Teeling, Springer, and other authors formerly cited presented a molecular phylogeny that, combined with morphological data, supported the hypothesis that megabat genera were actually nested into four microbat lineages which began their rapid radiation in the Eocene, coinciding with a global increase in temperature and diversification of insect and plant species (20). “Megabat” as a classification was officially about as cladistically accurate as “fishes”— it could refer to certain morphological features such as increased size, non-echolocating larynxes, and frugivory, but it had no true, phylogenetic meaning. Additionally, new groupings were finally suggested and rapidly codified by the scientific community: Yangochiroptera, which encloses twelve formerly-microbat species, and Yinpterochiroptera, which consists of the Old World fruit bats (“megabats”) and six of the microbat families from Rhinolophidae (15). These groupings were supported by the sequencing of previously unstudied genes and in a much broader range of species than was ever attempted before, addressing some of the uncertainty prior studies had introduced by only analyzing some members of a likely polyphyletic group. Molecular phylogenies constructed from this data also supported the convergent emergence of laryngeal echolocation in both Yinpterochiroptera and Yangochiroptera (10), though the methods and dates by which these adaptations arose were still shrouded in mystery. The authors suggested that whole-genome sequencing of bat species, three-dimensional imaging to allow a clearer understanding of the morphology of their tiny and delicate laryngeal organs, and comparison to similar vocalizing genes in other mammals may provide more insight in the future (10). I believe a more geographic and ecological approach to the Yangochiroptera and Yinpterochiroptera division could also illuminate the evolutionary motivation of these two lineages.

A More Robust Phylogeny

Fortunately, further research was conducted (and is ongoing) to solidify these classifications, as well as situate the lineages in the evolutionary framework. In 2011, the cytochrome b genealogy of half of all bat species (648 terminal taxa) was studied with the goal of constructing a new phylogenetic tree. Interestingly, initial results weakly supported the archaic megabat and microbat distinctions, rather than the newer and now widely-recognized Yangochiroptera and Yinpterochiroptera. However, a time-calibrated, pruned dataset (using only taxa that had the full 1140bp cytochrome b length) strongly supported a monophyletic Yinpterochiroptera (1). Broader family classifications were also supported, but relatedness of families and accuracy of smaller taxonomic groupings were either only weakly supported or considered to be paraphyletic in their current organizations, mostly due to the lack of available information on several nodes. Their study also became an extraordinarily valuable resource for the study of the actual divergence times of different lineages in history, which may yet be the key to understanding why extant bats are so distinct, diverse, and seemingly prone to convergent evolution (diets, body plans, and echolocation, for example.) The utility of cytochrome b genealogies additionally allowed the solidification of several smaller taxonomic groups; it isn’t perfect, but a 2018 project that analyzed nine nuclear and mitochondrial DNA sequence markers of 804 bat terminals found strong evidence not only for existence of Yinpterochiroptera and Yangochiroptera as accurate classifications, but for the monophyly of all polytypic families currently recognized under Chiroptera (2). Many smaller groups— such as subfamilies, genera, and species groups— were found to be relatively accurate with only minor necessary adjustments (with the exception of Molossidae, which the authors state needs a complete revision.) Chiroptera is in a more accurate state than it has ever been, and advancements in whole-genome sequencing, imaging technologies, and fossil discoveries will only continue to hone the smaller taxa into more accurate forms. From there, our understanding of the evolution and adaptations of these amazing animals will deepen as well; the relatedness of some bat families raises interesting biogeographical questions about mass migration and the age of lineage splits, as do the unique adaptations that are either convergently evolved or seemed to arise from “nowhere” (such as Pettigrew’s primate-eye observation, for which we still have no reasonable explanation.) All of these new theories, questions, and discoveries are informed by the phylogeny of these marvelous animals, which has proven to be quite the decade-spanning challenge to unravel.

Reflections

The story of the bat is one of reworked and revisited theory, technological advancement, and interdisciplinary collaboration. Research that focuses on only one method of phylogeny-building has proven to yield unsatisfactory or contradictory results, forcing a more holistic approach that can and should be applied to families beyond Chiroptera, especially those that have undergone adaptive radiation. Because while it may be tempting to pin blame on Pettigrew as the sole perpetrator for decades of confusion due to his focusing so heavily on morphology alone, and it is true that the current literature suggests that morphology is a misleading metric by which to base phylogenies without the support of fossil and genomic evidence, we must also acknowledge that genomic data alone was equally insufficient at constructing an accurate phylogeny when uniformed by research in other forms of species relatedness. With time comes not only better technology for studying these animals, but also an increased understanding of the extraordinarily complex facets of evolution. The bat has taught us that the phylogenies of the future will be more complex undertakings to construct, but also more accurate reflections of the oft-surprising twists and branchings of the tree of life.

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