THE CHALLENGE OF SPECIES DELIMITATION AT THE EXTREMES: DIVERSIFICATION WITHOUT MORPHOLOGICAL CHANGE IN PHILIPPINE SUN SKINKS

An accurate understanding of species diversity is essential to studies across a wide range of biological subdisciplines. However, delimiting species remains challenging in evolutionary radiations where morphological diversification is rapid and accompanied by little genetic differentiation or when genetic lineage divergence is not accompanied by morphological change. We investigate the utility of a variety of recently developed approaches to examine genetic and morphological diversity, and delimit species in a morphologically conserved group of Southeast Asian lizards. We find that species diversity is vastly underestimated in this unique evolutionary radiation, and find an extreme case where extensive genetic divergence among lineages has been accompanied by little to no differentiation in external morphology. Although we note that different conclusions can be drawn when species are delimited using molecular phylogenetics, coalescent‐based methods, or morphological data, it is clear that the use of a pluralistic approach leads to a more comprehensive appraisal of biodiversity, and greater appreciation for processes of diversification in this biologically important geographic region. Similarly, our approach demonstrates how recently developed methodologies can be used to obtain robust estimates of species limits in “nonadaptive” or “cryptic” evolutionary radiations.     

Impacts of climate warming on hybrid zone movement: Geographically diffuse and biologically porous “species borders”

Abstract The ecology and evolutionary biology of insect–plant associations has realized extensive attention, especially during the past 60 years. The classifications (categorical designations) of continuous variation in biodiversity, ranging from global patterns (e.g., latitudinal gradients in species richness/diversity and degree of herbivore feeding specialization) to localized insect–plant associations that span the biospectrum from polyphenisms, polymorphisms, biotypes, demes, host races, to cryptic species, remain academically contentious. Semantic and biosystematic (taxonomical) disagreements sometimes detract from more important ecological and evolutionary processes that drive diversification, the dynamics of gene flow and local extinctions. This review addresses several aspects of insect specialization, host‐associated divergence and ecological (including “hybrid”) speciation, with special reference to the climate warming impacts on species borders of hybridizing swallowtail butterflies (Papilionidae). Interspecific hybrid introgression may result in collapse of multi‐species communities or increase species numbers via homoploid hybrid speciation. We may see diverging, merging, or emerging genotypes across hybrid zones, all part of the ongoing processes of evolution. Molecular analyses of genetic mosaics and genomic dynamics with “divergence hitchhiking”, combined with ecological, ethological and physiological studies of “species porosity”, have already begun to unveil some answers for some important ecological/evolutionary questions. (i) How rapidly can host‐associated divergence lead to new species (and why doesn't it always do so, e.g., resulting in “incomplete” speciation)? (ii) How might “speciation genes” function, and how/where would we find them? (iii) Can oscillations from specialists to generalists and back to specialists help explain global diversity in herbivorous insects? (iv) How could recombinant interspecific hybridization lead to divergence and speciation? From ancient phytochemically defined angiosperm affiliations to recent and very local geographical mosaics, the Papilionidae (swallowtail butterflies) have provided a model for enhanced understanding of ecological patterns and evolutionary processes, including host‐associated genetic divergence, genomic mosaics, genetic hitchhiking and sex‐linked speciation genes. Apparent homoploid hybrid speciation in Papilio appears to have been catalyzed by climate warming‐induced interspecific introgression of some, but not all, species diagnostic traits, reflecting strong divergent selection (discordant), especially on the Z (= X) chromosome. Reproductive isolation of these novel recombinant hybrid genotypes appears to be accomplished via a delayed post‐diapause emergence or temporal isolation, and is perhaps aided by the thermal landscape. Changing thermal landscapes appear to have created (and may destroy) novel recombinant hybrid genotypes and hybrid species.     

Ephemeral ecological speciation and the latitudinal biodiversity gradient

The richness of biodiversity in the tropics compared to high‐latitude parts of the world forms one of the most globally conspicuous patterns in biology, and yet few hypotheses aim to explain this phenomenon in terms of explicit microevolutionary mechanisms of speciation and extinction. We link population genetic processes of selection and adaptation to speciation and extinction by way of their interaction with environmental factors to drive global scale macroecological patterns. High‐latitude regions are both cradle and grave with respect to species diversification. In particular, we point to a conceptual equivalence of “environmental harshness” and “hard selection” as eco‐evolutionary drivers of local adaptation and ecological speciation. By describing how ecological speciation likely occurs more readily at high latitudes, with such nascent species especially prone to extinction by fusion, we derive the ephemeral ecological speciation hypothesis as an integrative mechanistic explanation for latitudinal gradients in species turnover and the net accumulation of biodiversity.     

The speciation view: Disentangling multiple causes of adaptive and non-adaptive radiation in terms of speciation

Biological diversification often includes burst of lineage splitting. Such “radiation” has been known to act as evolutionary arenas with the potential to generate unique phylogenetic clusters and further novel groups. Although these radiations when accompanied by ecological diversification, so-called “adaptive radiation” have persisted as a central premise in evolutionary biology, the ecological and genetic mechanism of such rapid diversification has remained unclear. There are several critical definitions for the pattern of adaptive radiation, and those provide delimitation of adaptive and non-adaptive radiation. That being said, only a few studies have provided any clear demarcations in our understanding of the adaptive and non-adaptive causes of radiation from the mechanism of speciation. Here, we review the current consensus for the causes of adaptive radiation, especially along with the recent theoretical synthesis of “ecological speciation.” Further, we suggest the signature of adaptive and non-adaptive radiation in the earliest stages of diversification from the viewpoint of speciation. These criteria from the speciation view are useful to find the cases with the signatures of adaptive/non-adaptive radiation.     

Investigating the timing of origin and evolutionary processes shaping regional species diversity: Insights from simulated data and neotropical butterfly diversification rates

Different diversification scenarios have been proposed to explain the origin of extant biodiversity. However, most existing meta‐analyses of time‐calibrated phylogenies rely on approaches that do not quantitatively test alternative diversification processes. Here, I highlight the shortcomings of using species divergence ranks, which is a method widely used in meta‐analyses. Divergence ranks consist of categorizing cladogenetic events to certain periods of time, typically to either Pleistocene or to pre‐Pleistocene ages. This approach has been claimed to shed light on the origin of most extant species and the timing and dynamics of diversification in any biogeographical region. However, interpretations drawn from such method often confound two fundamental questions in macroevolutionary studies, tempo (timing of evolutionary rate shifts) and mode (“how” and “why” of speciation). By using simulated phylogenies under four diversification scenarios, constant‐rate, diversity‐dependence, high extinction, and high speciation rates in the Pleistocene, I showed that interpretations based on species divergence ranks might have been seriously misleading. Future meta‐analyses of dated phylogenies need to be aware of the impacts of incomplete taxonomic sampling, tree topology, and divergence time uncertainties, as well as they might be benefited by including quantitative tests of alternative diversification models that acknowledge extinction and diversity dependence.     

Genomic data reveal two distinct species from the widespread alpine ginger Roscoea tibetica Batalin (Zingiberaceae)

Species delimitation is a key foundation for exploring biodiversity. However, the existence of continuous phenotypic variation in widespread species challenges accurate species delimitation based on classical taxonomy. In this study, we investigated the cryptic diversity of a widespread herb (Roscoea tibetica Batalin) in a biodiversity hotspot (the Hengduan Mountains, China) using genotyping by sequencing, examining morphological traits, developing species distribution models, and simulating demographic history. Phylogenomic reconstruction, principal component analysis, and genetic structure inferences indicated that previously reported R. tibetica comprised two monophyletic lineages with a deep divergence. Several morphological diagnostic characteristics were discovered from field and common garden that corresponded to these independent evolutionary lineages. Species distribution models illustrated significant ecological divergence between both lineages. All evidence strongly supported that R. tibetica, as described in previous taxonomy, actually comprises two distinct species. Model test of gene flow and effective population size changes in fastsimcoal2, and a negative Tajima's D-value suggested that recent contact likely occurred between the two lineages. Our results proposed that cryptic diversity in previously reported R. tibetica was possibly associated with phenotypic plasticity in heterogeneous environments and morphological convergence in similar habitats. This study suggests that caution should be exercised when attempting to gain biological insight into species with large-scale morphological variation, and species delimitation should be done in advance.     

AFLP markers provide insights into the evolutionary relationships and diversification of New Caledonian Araucaria species (Araucariaceae)

? Premise of the study: Despite its small size, New Caledonia is characterized by a very diverse flora and striking environmental gradients, which make it an ideal setting to study species diversification. Thirteen of the 19 Araucaria species are endemic to the territory and form a monophyletic group, but patterns and processes that lead to such a high species richness are largely unexplored. ? Methods: We used 142 polymorphic AFLP markers and performed analyses based on Bayesian clustering algorithms, genetic distances, and cladistics on 71 samples representing all New Caledonian Araucaria species. We examined correlations between the inferred evolutionary relationships and shared morphological, ecological, or geographic parameters among species, to investigate evolutionary processes that may have driven speciation. ? Key results: We showed that genetic divergence among the present New Caledonian Araucaria species is low, suggesting recent diversification rather than pre-existence on Gondwana. We identified three genetic groups that included small-leaved, large-leaved, and coastal species, but detected no association with soil preference, ecological habitat, or rainfall. The observed patterns suggested that speciation events resulted from both differential adaptation and vicariance. Last, we hypothesize that speciation is ongoing and/or there are cryptic species in some genetically (sometimes also morphologically) divergent populations. ? Conclusions: Further data are required to provide better resolution and understanding of the diversification of New Caledonian Araucaria species. Nevertheless, our study allowed insights into their evolutionary relationships and provides a framework for future investigations on the evolution of this emblematic group of plants in one of the world's biodiversity hotspots.     

Diversification rates in a temperate legume clade: Are there “so many species” of Astragalus (Fabaceae)?

Astragalus, the largest genus of flowering plants, contains upwards of 2500 species. Explanations for this exceptional species diversity have pointed to unusual population structure or modes of speciation. Surprisingly, however, three different statistical analyses indicate that diversification rates in Astragalus are not exceptionally high compared to its closest relatives. Instead, rates are high throughout the “Astragalean clade,” a much broader radiation distributed throughout the temperate zone. The increase in diversification rate is associated with the origin and divergence of this clade from common ancestors of it and several much less diverse and more narrowly distributed Asian genera. This suggests that causal factors in the shift toward higher rates of diversification must be due not to factors unique to Astragalus, but to characteristics common to the entire Astragalean clade. However, this larger clade has never been circumscribed in classifications based on morphological data. This raises the possibility that the causes of increased diversification may not be due to morphological innovation, but may instead be related to ecological factors or cryptic physiological or biochemical features.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

Cryptic species in the Neotropical fish genus Curimatopsis (Teleostei,Characiformes)

Detritivores of the fish family Curimatidae are assigned to eight genera, one of which, the Curimatopsis, with only five species, is the least speciose genus and sister to other seven genera in the family. Ongoing morphological investigations reveal, however, the likely existence of additional species. In this study, fifty‐one specimens of Curimatopsis from multiple rivers of the Amazon, Paraguay and Suriname drainages were identified morphologically according to the present species concepts and then barcoded using the universal cytochrome c oxidase subunit I (COI) mitochondrial marker. Species delimitation analyses were conducted using Bayesian methods through the general mixed Yule‐coalescent analysis combined with conventional likelihood, genetic distance and haplotypic diversity approaches. We found eleven well‐supported clusters that represent four of the named species and seven cryptic, undescribed species of Curimatopsis. Our results show a clear delimitation of species boundaries constrained by distinct Amazonian river ecotones that may have promoted intrageneric lineage diversification. This is the first of a series of genetic studies applicable to future taxonomic, phylogenetic and evolutionary studies across the Curimatidae.     

Coalescent-based species delimitation in an integrative taxonomy

The statistical rigor of species delimitation has increased dramatically over the past decade. Coalescent theory provides powerful models for population genetic inference, and is now increasingly important in phylogenetics and speciation research. By applying probabilistic models, coalescent-based species delimitation provides clear and objective testing of alternative hypotheses of evolutionary independence. As acquisition of multilocus data becomes increasingly automated, coalescent-based species delimitation will improve the discovery, resolution, consistency, and stability of the taxonomy of species. Along with other tools and data types, coalescent-based species delimitation will play an important role in an integrative taxonomy that emphasizes the identification of species limits and the processes that have promoted lineage diversification.     

Cryptic speciation in the Acari: a function of species lifestyles or our ability to separate species?

There are approximately 55,000 described Acari species, accounting for almost half of all known Arachnida species, but total estimated Acari diversity is reckoned to be far greater. One important source of currently hidden Acari diversity is cryptic speciation, which poses challenges to taxonomists documenting biodiversity assessment as well as to researchers in medicine and agriculture. In this review, we revisit the subject of biodiversity in the Acari and investigate what is currently known about cryptic species within this group. Based on a thorough literature search, we show that the probability of occurrence of cryptic species is mainly related to the number of attempts made to detect them. The use of, both, DNA tools and bioassays significantly increased the probability of cryptic species detection. We did not confirm the generally-accepted idea that species lifestyle (i.e. free-living vs. symbiotic) affects the number of cryptic species. To increase detection of cryptic lineages and to understand the processes leading to cryptic speciation in Acari, integrative approaches including multivariate morphometrics, molecular tools, crossing, ecological assays, intensive sampling, and experimental evolution are recommended. We conclude that there is a demonstrable need for future investigations focusing on potentially hidden mite and tick species and addressing evolutionary mechanisms behind cryptic speciation within Acari.     

Phylogenetic approaches for studying diversification

Estimating rates of speciation and extinction, and understanding how and why they vary over evolutionary time, geographical space and species groups, is a key to understanding how ecological and evolutionary processes generate biological diversity. Such inferences will increasingly benefit from phylogenetic approaches given the ever‐accelerating rates of genetic sequencing. In the last few years, models designed to understand diversification from phylogenetic data have advanced significantly. Here, I review these approaches and what they have revealed about diversification in the natural world. I focus on key distinctions between different models, and I clarify the conclusions that can be drawn from each model. I identify promising areas for future research. A major challenge ahead is to develop models that more explicitly take into account ecology, in particular the interaction of species with each other and with their environment. This will not only improve our understanding of diversification; it will also present a new perspective to the use of phylogenies in community ecology, the science of interaction networks and conservation biology, and might shift the current focus in ecology on equilibrium biodiversity theories to non‐equilibrium theories recognising the crucial role of history.     

Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

A Combination of Divergence and Conservatism in the Niche Evolution of the Moorish Gecko,Tarentola mauritanica (Gekkota: Phyllodactylidae)

The quantification of realized niche overlap and the integration of species distribution models (SDMs) with calibrated phylogenies to study niche evolution are becoming not only powerful tools to understand speciation events, but can also be used as proxies regarding the delimitation of cryptic species. We applied these techniques in order to unravel how the fundamental niche evolved during cladogenesis within the Tarentola mauritanica species-complex. Our results suggest that diversification within this complex, during the Miocene and Pleistocene, is associated with both niche divergence and niche conservatism, with a pattern that varies depending on whether the variables involved are related to the mean or seasonality of temperature and humidity. Moreover, climatic variables related to humidity and temperature seasonality were involved in the niche shift and genetic diversification of the European/North African clade during the Pleistocene and in its maintenance in a fundamental niche distinct from that of the remaining members of the group. This study further highlights the need for a taxonomic revision of the T. mauritanica species-complex.     

Both temperature fluctuations and East Asian monsoons have driven plant diversification in the karst ecosystems from southern China

Karst ecosystems in southern China are species‐rich and have high levels of endemism, yet little is known regarding the evolutionary processes responsible for the origin and diversification of karst biodiversity. The genus Primulina (Gesneriaceae) comprises ca. 170 species endemic to southern China with high levels of ecological (edaphic) specialization, providing an exceptional model to study the plant diversification in karsts. We used molecular data from nine chloroplast and 11 nuclear regions and macroevolutionary analyses to assess the origin and cause of species diversification due to palaeoenvironmental changes and edaphic specialization in Primulina. We found that speciation was positively associated with changes in past temperatures and East Asian monsoons through the evolutionary history of Primulina. Climatic change around the mid‐Miocene triggered an early burst followed by a slowdown of diversification rate towards the present with the climate cooling. We detected different speciation rates among edaphic types, and transitions among soil types were infrequently and did not impact the overall speciation rate. Our findings suggest that both global temperature changes and East Asian monsoons have played crucial roles in floristic diversification within the karst ecosystems in southern China, such that speciation was higher when climate was warmer and wetter. This is the first study to directly demonstrate that past monsoon activity is positively correlated with speciation rate in East Asia. This case study could motivate further investigations to assess the impacts of past environmental changes on the origin and diversification of biodiversity in global karst ecosystems, most of which are under threat.     

Genomic data reveal deep genetic structure but no support for current taxonomic designation in a grasshopper species complex

Taxonomy has traditionally relied on morphological and ecological traits to interpret and classify biological diversity. Over the last decade, technological advances and conceptual developments in the field of molecular ecology and systematics have eased the generation of genomic data and changed the paradigm of biodiversity analysis. Here we illustrate how traditional taxonomy has led to species designations that are supported neither by high throughput sequencing data nor by the quantitative integration of genomic information with other sources of evidence. Specifically, we focus on Omocestus antigai and Omocestus navasi, two montane grasshoppers from the Pyrenean region that were originally described based on quantitative phenotypic differences and distinct habitat associations (alpine vs. Mediterranean‐montane habitats). To validate current taxonomic designations, test species boundaries, and understand the factors that have contributed to genetic divergence, we obtained phenotypic (geometric morphometrics) and genome‐wide SNP data (ddRADSeq) from populations covering the entire known distribution of the two taxa. Coalescent‐based phylogenetic reconstructions, integrative Bayesian model‐based species delimitation, and landscape genetic analyses revealed that populations assigned to the two taxa show a spatial distribution of genetic variation that do not match with current taxonomic designations and is incompatible with ecological/environmental speciation. Our results support little phenotypic variation among populations and a marked genetic structure that is mostly explained by geographic distances and limited population connectivity across the abrupt landscapes characterizing the study region. Overall, this study highlights the importance of integrative approaches to identify taxonomic units and elucidate the evolutionary history of species.     

Fire as a key driver of Earth's biodiversity

Many terrestrial ecosystems are fire prone, such that their composition and structure are largely due to their fire regime. Regions subject to regular fire have exceptionally high levels of species richness and endemism, and fire has been proposed as a major driver of their diversity, within the context of climate, resource availability and environmental heterogeneity. However, current fire‐management practices rarely take into account the ecological and evolutionary roles of fire in maintaining biodiversity. Here, we focus on the mechanisms that enable fire to act as a major ecological and evolutionary force that promotes and maintains biodiversity over numerous spatiotemporal scales. From an ecological perspective, the vegetation, topography and local weather conditions during a fire generate a landscape with spatial and temporal variation in fire‐related patches (pyrodiversity), and these produce the biotic and environmental heterogeneity that drives biodiversity across local and regional scales. There have been few empirical tests of the proposition that ‘pyrodiversity begets biodiversity’ but we show that biodiversity should peak at moderately high levels of pyrodiversity. Overall species richness is greatest immediately after fire and declines monotonically over time, with postfire successional pathways dictated by animal habitat preferences and varying lifespans among resident plants. Theory and data support the ‘intermediate disturbance hypothesis’ when mean patch species diversity is correlated with mean fire intervals. Postfire persistence, recruitment and immigration allow species with different life histories to coexist. From an evolutionary perspective, fire drives population turnover and diversification by promoting a wide range of adaptive responses to particular fire regimes. Among 39 comparisons, the number of species in 26 fire‐prone lineages is much higher than that in their non‐fire‐prone sister lineages. Fire and its byproducts may have direct mutagenic effects, producing novel genotypes that can lead to trait innovation and even speciation. A paradigm shift aimed at restoring biodiversity‐maintaining fire regimes across broad landscapes is required among the fire research and management communities. This will require ecologists and other professionals to spread the burgeoning fire‐science knowledge beyond scientific publications to the broader public, politicians and media.     

Intricate evolutionary histories in montane species: a phylogenetic window into craniodental discrimination in the Peromyscus mexicanus species group (Mammalia: Rodentia: Cricetidae)

Mountain‐associated species, which exhibit allopatric distributions associated with elevation, endemisms and complex evolutionary histories, pose challenging evolutionary scenarios in which to discern the diversification of species. The Peromyscus mexicanus mice group, distributed along mountains in southern Mexico and Central America, is morphometrically variable, a key rationale for the ongoing controversy regarding its species delimitation. Based on the recognized 15 mitochondrial lineages for the group, we analysed external and craniodental morphometric variables to test whether lineages can be differentiated morphometrically and allow for the delimitation of species. We also aimed to test the prediction that the phylogenetic structure of the morphometric data is concordant with that of the molecular information. Based on 19 craniodental measurements from 521 specimens, multivariate and discriminant analyses showed that lineages are morphometrically discernible, representing distinct phenotypes, and that overall size and mandible measurements are significant features that discriminate lineages, supporting hypotheses about differences in feeding habits between species. Also, a pattern of increasing size with elevation was observed, further supported by specific morphological differences exhibited between highland and lowland lineages inhabiting the same mountain. Our results demonstrate that P. mexicanus is both genetically and morphometrically variable, where most highland montane species are differentiated from lowland species; also, a significant correlation between mitochondrial and morphometric information is indicative of phenetic concordance, altogether in agreement with a recent taxonomic proposal for the group. We suggest that the group's intricate diversification responds to ecological diversification and adaptation to a variety of mountain habitats and Pleistocene biogeographic climatic dynamics.     

Delimitation despite discordance: Evaluating the species limits of a confounding species complex in the face of mitonuclear discordance

The delimitation of species is an essential pursuit of biology, and proper taxonomies are crucial for the assessment and conservation management of organismal diversity. However, delimiting species can be hindered by a number of factors including highly conserved morphologies (e.g., cryptic species), differences in criteria of species concepts, lineages being in the early stages of the speciation or divergence process, and discordance between gene topologies (e.g., mitonuclear discordance). Here we use a taxonomically confounded species complex of toads in Central America that exhibits extensive mitonuclear discordance to test delimitation hypotheses. Our investigation integrates mitochondrial sequences, nuclear SNPs, morphology, and macroecological data to determine which taxonomy best explains the divergence and evolutionary relationships among these toads. We found that a three species taxonomy following the distributions of the nuclear SNP haplotypes offers the best explanation of the species in this complex based off of the integrated data types. Due to the taxonomic instability of this group, we also discuss conservation concerns in the face of improper taxonomic delimitation. Our study provides an empirical and integrative hypothesis testing framework to assess species delimitation hypotheses in the face of cryptic morphology and mitonuclear discordance and highlights the importance that a stable taxonomy has over conservation‐related actions.