In a (somewhat) recent blog post entitled “Is there a crisis in Anolis taxonomy?”, Julian Velasco invited discussion on a perceived decline in the number of new anole taxonomists. While it was a fun look at the dynamics of anole taxonomy over time, I couldn’t help but feel like there is a more pressing taxonomic crisis going on right now, and it affects many of the researchers that frequent this blog.
I fear too many species of Anolis are being described based on questionable evidence. While this problem is not unique to anoles (a common term for it is “taxonomic inflation”; Isaac et al. 2004), a number of recently described anole species may be the result of overzealous taxonomic splitting. I will give some examples below and then briefly discuss two lines of evidence that I believe are often used to divide species inappropriately. Before I do so, it’s worth stating up front that I’ll focus on the work of Dr. Gunther Köhler and colleagues. This shouldn’t be surprising, as Dr. Köhler is the most prolific living describer of anole species. The following criticisms should not be seen as personal, as Köhler is not unique on any of the points I discuss below. But with many cryptic species described or resurrected over the past 10-15 years, his work has the largest impact on anole taxonomy and the science that depends on it.
I’ll start with the revision of the Anolis tropidonotus complex published in Mesoamerican Herpetology (Köhler et al. 2016). Below I provide a quick breakdown of the paper. I hope that others will contribute their own views on this work in the comments. The A. tropidonotus group is one that I am well-acquainted with, having spent months of field time collecting individuals across the distribution of the group. Köhler et al. (2016) raise a subspecies (A. tropidonotus spilorhipis) to species status while describing two new species, A. wilsoni and A. mccraniei. Unfortunately, the data presented–morphology and DNA–do not appear to strongly support the recognition of any new species level taxa. I argue that the inference of four species within A. tropidonotus sensu lato should require stronger evidence than that presented.
The authors sequenced 16S mitochondrial DNA for molecular analyses and present a consensus tree from Bayesian analyses of these data. This tree recovers four well-supported and geographically circumscribed mtDNA haplotype clades that correspond with the four new species. A table following the tree reveals the genetic distances between putatively new species topped out at 4.5%. This level of mitochondrial divergence is significantly less than intraspecific variation observed in other anoles (Malhotra & Thorpe 2000; Thorpe & Stenson 2003; Ng & Glor 2011). Moreover, Köhler et al.’s (2016) sampling map reflects sparse sampling of molecular data.
Based on Figure 3, morphology (other than perhaps hemipenes, which I discuss below) does not provide any support for delimitation of those populations characterized by distinct mtDNA haplotypes. The dewlap differences reported are slight and appear to fall within the type of variation observed within and among other populations of species in this group (see photos at the top of this post for an example of two spilorhipis males that came from the same locality; photos courtesy Luke Mahler). Bottom line–we see several populations with mitochondrial haplotypes that cluster together geographically with little to no morphological evidence for divergence.
The phylogenetic and morphological patterns displayed in Köhler et al. (2016) are consistent with patchy sampling of a widespread and continuously distributed species with potentially locally-adapted populations. The authors cite “the high degree of genetic distinctiveness… as evidence for a lack of gene flow, and conclude that these four lineages represent species-level units” (Köhler et al. 2016). This assumption is questionable, as researchers have long known of the pitfalls of using mtDNA to determine gene flow (Avise et al. 1983; Avise et al. 1984; Funk & Omland 2003) and supporting evidence from morphology is lacking. The different hemipenial types represent the strongest evidence for recognizing the lineages mtDNA haplotype groups. Below I will discuss the utility of those traits for species delimitation.
Finally, the authors did not compare their purported new tropidonotus-like species to Anolis wampuensis, a morphologically indistinguishable (McCranie & Kohler 2015) form that is potentially codistributed with the new species A. mccraniei. This should have been done to avoid the possibility that A. wampuensis is conspecific with one of the newly named forms.
Another example of taxonomic inflation in Anolis is from a 2014 monograph in Zootaxa (Köhler et al. 2014). I will focus on the treatment of the Anolis nebuloides group, which exemplifies the problems of this monograph and exhibits some of the issues I’ve seen repeated in species descriptions of late. In this paper, the authors use two mtDNA data sets: 16S and CO1. They present both trees for the nebuloides group; each tree is weakly supported and highly uncertain (below). The species are ultimately divided up on the basis of the molecular evidence.
As you can see on the map, there are five clusters of localities that appear to be separated by significant distances. These clusters correspond to each of the recognized species, and to OTUs in the phylogenetic trees. A reader unfamiliar with the region might interpret each species as a montane isolate, a common scenario involving species groups adapted to high elevations. However, this interpretation would be incorrect. Each of those clusters corresponds to a major road system that was sampled by the authors. In reality, the distribution of this group is likely to be continuous (or very nearly so) from the type locality of Anolis megapholidotus in Guerrero to at least the area in central Oaxaca where “zapotecorum” is reported. I have sampled within several of the putative gaps and have found populations of nebuloides-like anoles present at every single location I have checked within the outer boundaries of the distribution of the group, including very low elevations that would allow population connections between the sampled clusters. The authors should be aware of the elevational range of the group: they include on their map the type locality of the synonymized species Anolis simmonsi (=Anolis nebuloides), which is near 100 m elevation.
The authors cite the molecular evidence for splitting the group. The extent of the molecular evidence is that spatially proximal haplotypes cluster together in the authors’ phylogenetic analyses, which is exactly what one would expect from sparse sampling of a continuously distributed species. In other words, isolation by distance would explain their results perfectly, and sampling localities in-between could erase the artifactual pattern that the clusters are distinct evolutionary lineages. Once again, morphology does not corroborate the species delimited by the authors’ interpretation of the molecular data.
The tropidonotus and nebuloides anoles are only a couple examples. A careful reviewer might have raised some issues over the descriptions mentioned above, as well as many others. I suspect that reviewers may not be anole experts and therefore are unaware of the nuances of anole systematics. An example of this would be the relevance of a “lock-and-key” mechanism for reproductive isolation in the group. Generally, “lock and key” assumes differences in genital morphology evolved as a form of reproductive isolation between species (Shapiro & Porter 1989; Arnqvist 1998). While it is well-documented in some groups (especially many insects), we have yet to see published work that suggests an important role for “lock-and-key” reproductive isolation with respect to hemipenial morphology in anoles, and the literature is littered with examples that do not follow the expected pattern of isolation (Shapiro & Porter 1989).
What appears to be happening is that some authors are essentially defining species as the smallest group they can diagnose according to hemipenes or mitochondrial divergence/clustering alone. This is a dangerous way to approach taxonomy, as many species have diagnosable populations that freely share genetic material (Thorpe & Stenson 2003; Ng & Glor 2011; Ng et al. 2016). There is also a precedent for mtDNA markers misinforming researchers on the extent of gene flow between anole populations (Thorpe et al. 2008; Ng & Glor 2011). When dealing with putative cryptic species, an accurate assessment of gene flow is critical. And without demonstrating some level of reproductive isolation, the populations should not be recognized as species.
On that note: care should be taken when a single locus is used as the main line of evidence for splitting a species, especially if that locus is mtDNA. It’s become clear that mtDNA (or any single locus) can be misleading when trying to determine whether populations are reproductively isolated (Funk & Omland 2003; Petit & Excoffier 2009). In the case of mtDNA, this may be related to the male-biased nature of anole dispersal and mitochondria being a matrilineally inherited trait (Thorpe et al. 2008). Regardless, there are several examples of mitochondrially divergent populations of anoles having significant gene flow between them (Thorpe et al. 2008; Ng & Glor 2011), which should preclude species designations based on morphological differences the populations might have. Mitochondrial divergence of over 10% has been observed in multiple population pairings, yet extensive ongoing nuclear gene flow has ruled out the possibility that these mitochondrial lineages represent valid species (Thorpe et al. 2008; Ng & Glor 2011). Had those authors relied primarily on mtDNA divergence to delimit species, flawed conclusions would have resulted despite morphological distinctiveness of some populations. If adding nuclear DNA evidence is not an option, mtDNA evidence should be strengthened by other traits that corroborate splits (such as dewlap color divergence, which has a well-documented history of usefulness in species delimitation in anoles; Losos 2009; Glor & Laport 2012) and should never stand alone.
Hemipenial variation, in particular, has been cited as the single diagnostic trait for many recently described anole species. However, to date, we have no evidence that divergence in hemipenial traits between populations leads to reproductive isolation, which would be necessary for hemipenial traits to be used in this way. In fact, the evidence is building against such a claim. As many of the readers of this blog are aware, hemipenes were recently found to evolve faster within anoles than any other trait (Klaczko et al. 2015). This result is consistent with other studies in animals that show rapid divergent evolution of male genitalia (Arnqvist 1998; Eberhard 2009). When dealing with such fast evolving traits, we should expect to see hemipenial polymorphism within species just as we see polymorphism in scale traits, etc. In fact, I and others have found exactly that–in a recently accepted manuscript, we report on hemipenial polymorphism within Anolis sericeus (Lara-Tufiño et al. 2016). Work from Dr. Köhler’s lab has shown one species diagnosed solely by hemipenial morphology to be invalid (Anolis osa; Köhler et al. 2010; Köhler et al. 2012). My own work on the A. sericeus group (in prep.) stands in strong contrast to the results of Köhler and Vesely (2010), which used hemipenes to divide up the group. Phillips et al.’s (2015) work on the A. humilis group showed that a species diagnosed solely by hemipenial structure (A. quaggulus) was found not to warrant species status.
Other similarly diagnosed species may await a similar fate. Anolis cryptolimifrons, for example, was diagnosed from A. limifrons only in hemipenial structure and is geographically surrounded by A. limifrons (Köhler & Sunyer 2008). Pending evidence of minor hemipenial differences causing reproductive isolation, it seems prudent to regard species such as A. cryptolimifrons with skepticism. A more parsimonious interpretation simply acknowledges the presence of hemipenial polymorphism in the species.
A final note on hemipenes and sampling: many of the species descriptions that utilize hemipenial morphology to justify taxonomic splits include relatively few examined samples (population-wise and overall number of individuals). For instance, large gaps exist where hemipene morphotypes are assumed in Köhler and Vesely (2010; see map below)–the authors did not include samples from a large portion of the group’s distribution (hemipenial sampling shown by filled in shapes in the map). This practice is less important in descriptions where hemipenes are unused or ancillary evidence, but critical in papers like Köhler and Vesely (2010) where hemipenial structure is the primary diagnostic trait used to identify purported species. In some papers–including the tropidonotus work discussed above (Köhler et al. 2016)–detailed information on the number of hemipenes sampled per locality is lacking, so we are unable to judge the distribution of this trait at all. These data are important when a fast-evolving trait such as hemipenes is cited as primary evidence for species status.
In the future, we may find cases where divergent hemipenes restrict gene flow between closely-related populations. But given current evidence, differences in hemipenial morphology should not be accepted as the primary evidence to delimit species.
I have described just a few cases of taxonomic inflation in anoles. There are many others that have occurred over the past 15 years. This influx of potentially illegitimate names poses real challenges for anole researchers. If your research depends on accurate diversity estimates in clades or accurate distributions for species units, results can be compromised. Conservation decisions become muddled, and even getting permits to do ecological work can become more problematic due to the fact that many of the new species mentioned in this post are believed to have small distributions and considered endangered by the authors (Köhler et al. 2014; McCranie and Köhler 2015; Köhler and Hedges 2016). When describing cryptic species in continuously distributed groups like nebuloides and tropidonotus, it is vital to demonstrate a lack of gene flow before changing the taxonomic status of populations. I hope that more rigorous testing for species boundaries will be provided in future taxonomic overhauls, as these changes have real consequences for other anole researchers. An easy fix to this problem would be to encourage the recognition of subspecies, as Dr. Hillis has suggested before on this blog. Recognizing taxonomic diversity below species level can be plenty useful for a plethora of research topics and is less harmful to other scientific ventures. Another option would be to treat new discovered mitochondrial haplotype clades as “Unconfirmed Candidate Species” (Padial et al. 2010) pending evidence of reproductive isolation.
Let’s hear your thoughts and comments!
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