How to fail at species delimitation

Species delimitation is the act of identifying species‐level biological diversity. In recent years, the field has witnessed a dramatic increase in the number of methods available for delimiting species. However, most recent investigations only utilize a handful (i.e. 2–3) of the available methods, often for unstated reasons. Because the parameter space that is potentially relevant to species delimitation far exceeds the parameterization of any existing method, a given method necessarily makes a number of simplifying assumptions, any one of which could be violated in a particular system. We suggest that researchers should apply a wide range of species delimitation analyses to their data and place their trust in delimitations that are congruent across methods. Incongruence across the results from different methods is evidence of either a difference in the power to detect cryptic lineages across one or more of the approaches used to delimit species and could indicate that assumptions of one or more of the methods have been violated. In either case, the inferences drawn from species delimitation studies should be conservative, for in most contexts it is better to fail to delimit species than it is to falsely delimit entities that do not represent actual evolutionary lineages.     

Species concepts and species delimitation

The issue of species delimitation has long been confused with that of species conceptualization, leading to a half century of controversy concerning both the definition of the species category and methods for inferring the boundaries and numbers of species. Alternative species concepts agree in treating existence as a separately evolving metapopulation lineage as the primary defining property of the species category, but they disagree in adopting different properties acquired by lineages during the course of divergence (e.g., intrinsic reproductive isolation, diagnosability, monophyly) as secondary defining properties (secondary species criteria). A unified species concept can be achieved by treating existence as a separately evolving metapopulation lineage as the only necessary property of species and the former secondary species criteria as different lines of evidence (operational criteria) relevant to assessing lineage separation. This unified concept of species has several consequences for species delimitation, including the following: First, the issues of species conceptualization and species delimitation are clearly separated; the former secondary species criteria are no longer considered relevant to species conceptualization but only to species delimitation. Second, all of the properties formerly treated as secondary species criteria are relevant to species delimitation to the extent that they provide evidence of lineage separation. Third, the presence of any one of the properties (if appropriately interpreted) is evidence for the existence of a species, though more properties and thus more lines of evidence are associated with a higher degree of corroboration. Fourth, and perhaps most significantly, a unified species concept shifts emphasis away from the traditional species criteria, encouraging biologists to develop new methods of species delimitation that are not tied to those properties.     

New Metrics for Comparison of Taxonomies Reveal Striking Discrepancies among Species Delimitation Methods in Madascincus Lizards

Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C_tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R_tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.     

Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander,Plethodon kentucki: Incongruence among Data Sets and Methods

Species are a fundamental unit of biodiversity, yet can be challenging to delimit objectively. This is particularly true of species complexes characterized by high levels of population genetic structure, hybridization between genetic groups, isolation by distance, and limited phenotypic variation. Previous work on the Cumberland Plateau Salamander, Plethodon kentucki, suggested that it might constitute a species complex despite occupying a relatively small geographic range. To examine this hypothesis, we sampled 135 individuals from 43 populations, and used four mitochondrial loci and five nuclear loci (5693 base pairs) to quantify phylogeographic structure and probe for cryptic species diversity. Rates of evolution for each locus were inferred using the multidistribute package, and time calibrated gene trees and species trees were inferred using BEAST 2 and *BEAST 2, respectively. Because the parameter space relevant for species delimitation is large and complex, and all methods make simplifying assumptions that may lead them to fail, we conducted an array of analyses. Our assumption was that strongly supported species would be congruent across methods. Putative species were first delimited using a Bayesian implementation of the GMYC model (bGMYC), Geneland, and Brownie. We then validated these species using the genealogical sorting index and BPP. We found substantial phylogeographic diversity using mtDNA, including four divergent clades and an inferred common ancestor at 14.9 myr (95% HPD: 10.8–19.7 myr). By contrast, this diversity was not corroborated by nuclear sequence data, which exhibited low levels of variation and weak phylogeographic structure. Species trees estimated a far younger root than did the mtDNA data, closer to 1.0 myr old. Mutually exclusive putative species were identified by the different approaches. Possible causes of data set discordance, and the problem of species delimitation in complexes with high levels of population structure and introgressive hybridization, are discussed.     

Nuclear simple sequence repeat markers are superior to DNA barcodes for identification of closely related Rhododendron species on the same mountain

Accurate species delimitation of sampled biological material is critical for a range of studies. Although the DNA barcodes developed in recent years are useful for identifying numerous well differentiated species that have not experienced frequent gene flow, they fail to delimit recently diverged species, especially those with extensive introgressions. Here we use five Rhododendron species growing together on the same mountain as a model system to compare the species delimitation effectiveness of the DNA barcodes (internal transcribed spacer, matK, psbA‐trnH, and rbcL) previously proposed versus 15 pairs of microsatellite markers. Using these markers, we genotyped 129 individuals, which were members of five species according to morphological identification. We identified five simple sequence repeat genetic clusters (independently evolving lineages) corresponding to the morphological identification. However, we found that numerous individuals contained cryptic hybrid introgressions from the other species. The four DNA barcodes could not delimit three out of four closely related species that showed clear morphological differentiation and cryptic introgressions. Even after excluding all cryptic hybrids, two closely related species could not be successfully identified. The low discrimination ability of the DNA barcodes for closely related Rhododendron species could result from two, not mutually exclusive factors: introgressive hybridization and incomplete lineage sorting. Our results highlight the importance of simple sequence repeat markers in delimiting closely related species and identifying cryptic introgressions in the absence of morphological changes.     

An empirical comparison of character‐based and coalescent‐based approaches to species delimitation in a young avian complex

The process of discovering species is a fundamental responsibility of systematics. Recently, there has been a growing interest in coalescent‐based methods of species delimitation aimed at objectively identifying species early in the divergence process. However, few empirical studies have compared these new methods with character‐based approaches for discovering species. In this study, we applied both a character‐based and a coalescent‐based approaches to delimit species in a closely related avian complex, the light‐vented/Taiwan bulbul (Pycnonotus sinensis/Pycnonotus taivanus). Population aggregation analyses of plumage, mitochondrial and 13 nuclear intron character data sets produced conflicting species hypotheses with plumage data suggesting three species, mitochondrial data suggesting two species, and nuclear intron data suggesting one species. Such conflict is expected among recently diverged species, and by integrating all sources of data, we delimited three species verified with independently congruent character evidence as well as a more weakly supported fourth species identified by a single character. Attempts to validate species hypothesis using Bayesian Phylogenetics and Phylogeography (BPP), a coalescent‐based method of species delimitation, revealed several issues that can seemingly affect statistical support for species recognition. We found that θ priors had a dramatic impact on speciation probabilities, with lower values consistently favouring splitting and higher values consistently favouring lumping. More resolved guide trees also resulted in overall higher speciation probabilities. Finally, we found suggestive evidence that BPP is sensitive to the divergent effects of nonrandom mating caused by intraspecific processes such as isolation‐with‐distance, and therefore, BPP may not be a conservative method for delimiting independently evolving population lineages. Based on these concerns, we questioned the reliability of BPP results and based our conclusions about species limits exclusively on character data.     

Methods for species delimitation in bumblebees (Hymenoptera,Apidae, Bombus): towards an integrative approach

Delimitation of closely related species is often hindered by the lack of discrete diagnostic morphological characters. This is exemplified in bumblebees (genus Bombus). There have been many attempts to clarify bumblebee taxonomy by using alternative features to discrete morphological characters such as wing shape, DNA, or eco‐chemical traits. Nevertheless each approach has its own limitations. Recent studies have used a multisource approach to gather different lines of speciation evidence in order to draw a strongly supported taxonomic hypothesis in bumblebees. Yet, the resulting taxonomic status is not independent of selected evidence and of consensus methodology (i.e. unanimous procedure, majority, different weighting of evidence). In this article, we compare taxonomic conclusions for a group of taxonomically doubtful species (the Bombus lapidarius‐group) obtained from the four commonly used lines of evidence for species delimitation in bumblebees (geometric morphometric of wing shape, genetic differentiation assessment, sequence‐based species delimitation methods and differentiation of cephalic labial gland secretions). We ultimately aim to assess the usefulness of these lines of evidence as components of an integrative decision framework to delimit bumblebee species. Our results show that analyses based on wing shape do not delineate any obvious cluster. In contrast, nuclear/mitochondrial, sequence‐based species delimitation methods, and analyses based on cephalic labial gland secretions are congruent with each other. This allows setting up an integrative decision framework to establish strongly supported species and subspecies status within bumblebees.     

Species delimitation and geography

Despite the importance of the geographical arrangement of populations for the inference of species boundaries, only a few approaches that integrate spatial information into species delimitation have thus far been developed. Persistent differentiation of sympatric groups of individuals is the best criterion for species status. Species delimitation becomes more prone to error if allopatric metapopulations are considered because it is often difficult to assess whether observed differences between allopatric metapopulations would be sufficient to prevent the fusion of these metapopulations upon contact. We propose a novel approach for testing the hypothesis that the multilocus genetic distances between individuals or populations belonging to two different candidate species are not larger than expected based on their geographical distances and the relationship of genetic and geographical distances within the candidate species. A rejection of this null hypothesis is an argument for classifying the two studied candidate species as distinct species. Case studies show that the proposed tests are suitable to distinguish between intra‐ and interspecific differentiation. The regression approach proposed here is more appropriate for testing species hypotheses with regard to isolation by distance than (partial) Mantel tests. Our tests assume a linear relationship between genetic and (transformed) geographical distances. This assumption can be compromised by a high genetic variability within populations as found in a case study with microsatellite markers.     

The Biosemiotic Concept of the Species

Any biological species of biparental organisms necessarily includes, and is fundamentally dependent on, sign processes between individuals. In this case, the natural category of the species is based on family resemblances (in the Wittgensteinian sense), which is why a species is not a natural kind. We describe the mechanism that generates the family resemblance. An individual recognition window and biparental reproduction almost suffice as conditions to produce species naturally. This is due to assortativity of mating which is not based on certain individual traits, but on the difference between individuals. The biosemiotic model described here explains what holds a species together. It also implies that boundaries of a species are fundamentally fuzzy, and that character displacement occurs in case of sympatry. Speciation is a special case of discretisation that is an inevitable result of any communication system in work. The biosemiotic mechanism provides the conditions and communicative restrictions for the origin and persistence of diversity in the realm of living (communicative and semiotic) systems.     

Species boundaries in the messy middle—A genome‐scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.     

Comparing Multiple Criteria for Species Identification in Two Recently Diverged Seabirds

Correct species identification is a crucial issue in systematics with key implications for prioritising conservation effort. However, it can be particularly challenging in recently diverged species due to their strong similarity and relatedness. In such cases, species identification requires multiple and integrative approaches. In this study we used multiple criteria, namely plumage colouration, biometric measurements, geometric morphometrics, stable isotopes analysis (SIA) and genetics (mtDNA), to identify the species of 107 bycatch birds from two closely related seabird species, the Balearic (Puffinus mauretanicus) and Yelkouan (P. yelkouan) shearwaters. Biometric measurements, stable isotopes and genetic data produced two stable clusters of bycatch birds matching the two study species, as indicated by reference birds of known origin. Geometric morphometrics was excluded as a species identification criterion since the two clusters were not stable. The combination of plumage colouration, linear biometrics, stable isotope and genetic criteria was crucial to infer the species of 103 of the bycatch specimens. In the present study, particularly SIA emerged as a powerful criterion for species identification, but temporal stability of the isotopic values is critical for this purpose. Indeed, we found some variability in stable isotope values over the years within each species, but species differences explained most of the variance in the isotopic data. Yet this result pinpoints the importance of examining sources of variability in the isotopic data in a case-by-case basis prior to the cross-application of the SIA approach to other species. Our findings illustrate how the integration of several methodological approaches can help to correctly identify individuals from recently diverged species, as each criterion measures different biological phenomena and species divergence is not expressed simultaneously in all biological traits.     

Considering gene flow when using coalescent methods to delimit lineages of North American pitvipers of the genus Agkistrodon

Examining species diversity and mechanisms of speciation using coalescent models provides a framework for how regional diversity is accrued, even in well‐studied areas such as the Nearctic. It is likely, that gene flow among closely‐related species with adjacent distributions may be common. However, the absence of gene flow is a primary assumption of many phylogeographical methods that produce species trees and delimit species using Bayesian or likelihood functions in a coalescent framework. In the present study, we examine delimitation when gene flow between species is present using empirical datasets from two species of North American pitvipers of the genus Agkistrodon. We also use niche modelling to determine whether these young lineages occur in distinct environmental niches. To manage the problem of gene flow between species, we first identify admixed individuals, demonstrate that gene flow has occurred, and then identify the impact of alternative population assignments of admixed individuals on delimitation posterior probabilities. In addition, we examine the influence of mitochondrial genes relative to other loci combined in coalescent analyses that delimit species. Here, we find that the copperheads (Agkistrodon contortrix) and the cottonmouths (Agkistrodon piscivorus) are each composed of two distinct species, with each occupying different niches. Importantly, we find that species can be delimited when the amount of gene flow between lineages is low, although the methods are acutely sensitive to population assignment of individuals. © 2014 The Linnean Society of London     

Species delimitation through DNA barcoding of freshwater leeches of the Glossiphonia genus (Hirudinea: Glossiphoniidae) from Eastern Siberia,Russia

The study of biodiversity is a priority task of biological science. The structural unit of biodiversity is a species that has a clear identification in a taxonomic system. Morphological features are traditionally the main criteria for species discrimination in zoological studies. However, the presence of inter- and intraspecific polymorphism and phenotypic plasticity makes it difficult to identify species in many groups of invertebrates. To solve this problem, in this research, we analyzed morphological and genetic data in combination to delimit species among the Eastern Siberia Glossiphonia leeches using different approaches. Morphology analysis revealed phenetically distinct groups, suggesting the existence of at least two species in the region, G. verrucata, a rare Palaearctic species, and a potentially new species Glossiphonia sp. Moreover, sequence-based species delimitation methods congruently supported eight distinct species groups (including two Siberian species) within the available molecular dataset of the Glossiphonia world fauna, using phylogenetic (ML and BI), coalescent (ABGD and GMYC) methods, and pairwise analysis of sequences. The detected p-distances (modal value of 0.11) between these 8 groups and the level of genetic polymorphism (max. 0.0041) within groups indicate that the groups are 8 independent species according to the DNA barcoding. Our results once again proved the usefulness of molecular systematics. At the same time, we detected several inaccuracies in the leech species identification, as well as many ambiguous sites in sequences uploaded on GenBank, which affects the analysis and impedes progress of DNA barcoding technology.     

Species limits in butterflies (Lepidoptera: Nymphalidae): reconciling classical taxonomy with the multispecies coalescent

Species delimitation is at the core of biological sciences. During the last decade, molecular‐based approaches have advanced the field by providing additional sources of evidence to classical, morphology‐based taxonomy. However, taxonomy has not yet fully embraced molecular species delimitation beyond threshold‐based, single‐gene approaches, and taxonomic knowledge is not commonly integrated into multilocus species delimitation models. Here we aim to bridge empirical data (taxonomic and genetic) with recently developed coalescent‐based species delimitation approaches. We use the multispecies coalescent model as implemented in two Bayesian methods (dissect/stacey and bp&p ) to infer species hypotheses. In both cases, we account for phylogenetic uncertainty (by not using any guide tree) and taxonomic uncertainty (by measuring the impact of using a priori taxonomic assignments to specimens). We focus on an entire Neotropical tribe of butterflies, the Haeterini (Nymphalidae: Satyrinae). We contrast divergent taxonomic opinion – splitting, lumping and misclassifying species – in the light of different phenotypic classifications proposed to date. Our results provide a solid background for the recognition of 22 species. The synergistic approach presented here overcomes limitations in both traditional taxonomy (e.g. by recognizing cryptic species) and molecular‐based methods (e.g. by recognizing structured populations, and not raising them to species). Our framework provides a step forward towards standardization and increasing reproducibility of species delimitations.     

Species delimitation under the general lineage concept: an empirical example using wild North American hops (Cannabaceae: Humulus lupulus)

There is an emerging consensus that the intent of most species concepts is to identify evolutionarily distinct lineages. However, the criteria used to identify lineages differ among concepts depending on the perceived importance of various attributes of evolving populations. We have examined five different species criteria to ask whether the three taxonomic varieties of Humulus lupulus (hops) native to North America are distinct lineages. Three criteria (monophyly, absence of genetic intermediates, and diagnosability) focus on evolutionary patterns and two (intrinsic reproductive isolation and niche specialization) consider evolutionary processes. Phylogenetic analysis of amplified fragment length polymorphism (AFLP) data under a relaxed molecular clock, a stochastic Dollo substitution model, and parsimony identified all varieties as monophyletic, thus they satisfy the monophyly criterion for species delimitation. Principal coordinate analysis and a Bayesian assignment procedure revealed deep genetic subdivisions and little admixture between varieties, indicating an absence of genetic intermediates and compliance with the genotypic cluster species criterion. Diagnostic morphological and AFLP characters were found for all varieties, thus they meet the diagnosability criterion. Natural history information suggests that reproductive isolating barriers may have evolved in var. pubescens, potentially qualifying it as a species under a criterion of intrinsic reproductive isolation. Environmental niche modeling showed that the preferred habitat of var. neomexicanus is climatically unique, suggesting niche specialization and thus compliance with an ecological species criterion. Isolation by distance coupled with imperfect sampling can lead to erroneous lineage identification using some species criteria. Compliance with complementary pattern- and process-oriented criteria provides powerful corroboration for a species hypothesis and mitigates the necessity for comprehensive sampling of the entire species range, a practical impossibility in many systems. We hypothesize that var. pubescens maintains its genetic identity, despite substantial niche overlap with var. lupuloides, via the evolution of partial reproductive isolating mechanisms. Variety neomexicanus, conversely, will likely persist as a distinct lineage, regardless of limited gene flow with vars. lupuloides and pubescens because of ecological isolation--adaptation to the unique conditions of the Rocky Mountain cordillera. Thus, we support recognition of vars. neomexicanus and pubescens as species, but delay making a recommendation for var. lupuloides until sampling of genetic variation is complete or a stable biological process can be identified to explain its observed genetic divergence.     

Integration of conflict into integrative taxonomy: fitting hybridization in species delimitation of Mesocarabus (Coleoptera: Carabidae)

In species differentiation, characters may not diverge synchronously, and there are also processes that shuffle character states in lineages descendant from a common ancestor. Species are thus expected to show some degree of incongruence among characters; therefore, taxonomic delimitation can benefit from integrative approaches and objective strategies that account for character conflict. We illustrate the potential of exploiting conflict for species delimitation in a study case of ground beetles of the subgenus Carabus (Mesocarabus), where traditional taxonomy does not accurately delimit species. The molecular phylogenies of four mitochondrial and three nuclear genes, cladistic analysis of the aedeagus, ecological niche divergence and morphometry of pronotal shape in more than 500 specimens of Mesocarabus show that these characters are not fully congruent. For these data, a three‐step operational strategy is proposed for species delimitation by (i) delineating candidate species based on the integration of incongruence among conclusive lines of evidence, (ii) corroborating candidate species with inconclusive lines of evidence and (iii) refining a final species proposal based on an integrated characterization of candidate species based on the evolutionary analysis of incongruence. This procedure provided a general understanding of the reticulate process of hybridization and introgression acting on Mesocarabus and generated the hypothesis of seven Mesocarabus species, including two putative hybrid lineages. Our work emphasizes the importance of incorporating critical analyses of character and phylogenetic conflict to infer both the evolutionary history and species boundaries through an integrative taxonomic approach.     

Species concepts and species reality: salvaging a Linnaean rank

The validity of the species category (rank) as a distinct level of biological organization has been questioned. Phenetic, cohesion and monophyletic species concepts do not delimit species-level taxa that are qualitatively distinct from lower or higher taxa: all organisms throughout the tree of life exhibit varying degrees of similarity, cohesion, and monophyly. In contrast, interbreeding concepts delimit species-level taxa characterized by a phenomenon (regular gene flow) not found in higher taxa, making the species category a distinct level of biological organization. Only interbreeding concepts delimit species-level taxa that are all comparable according to a biologically meaningful criterion and qualitatively distinct from entities assigned to other taxonomic categories. Consistent application of interbreeding concepts can result in counterintuitive taxonomies--e.g. many wide polytypic species in plants and narrow cryptic species in animals. However, far from being problematic, such differences are biologically illuminating--reflecting differing barriers to gene flow in different clades. Empirical problems with interbreeding concepts exist, but many of these also apply to other species concepts, whereas others are not as severe as some have argued. A monistic view of species using interbreeding concepts will encounter strong historical inertia, but can save the species category from redundancy with other categories, and thus justify continued recognition of the species category.     

A protocol for the delimitation of areas of endemism and the historical regionalization of the Brazilian Atlantic Rain Forest using harvestmen distribution data

The concept of areas of endemism (AoEs) has rarely been discussed in the literature, even though the use of methods to ascertain them has recently increased. We introduce a grid‐based protocol for delimiting AoEs using alternative criteria for the recognition of AoEs that are empirically tested with harvestmen species distributions in the Atlantic Rain Forest. Our data, comprising 778 records of 123 species, were analysed using parsimony analysis of endemicity and endemicity analysis on four different grids (two cell sizes and two cell placements). Additionally, we employed six qualitative combined criteria for the delimitation of AoEs and applied them to the results of the numerical analyses in a new protocol to objectively delimit AoEs. Twelve AoEs (the most detailed delimitation of the Atlantic Rain Forest so far) were delimited, partially corroborating the main divisions previously established in the literature. The results obtained with the grid‐based methods were contradictory and were plagued by artefacts, probably due to the existence of different endemism patterns in one cell or to a biogeographical barrier set obliquely to latitudinal and longitudinal axes, for example. Consequently, the congruence patterns found by them should not be considered alone; qualitative characteristics of species and clade distributions and abiotic factors should be evaluated together. Mountain slopes are the main regions of endemism, and large river valleys are the main divisions. Refuges, marine transgressions and tectonic activity seem to have played an important role in the evolution of the Atlantic Rain Forest.     

Unpacking the species conundrum: philosophy,practice and a way forward

The history of ecology and evolutionary biology is rife with attempts to define and delimit species. However, there has been confusion between concepts and criteria, which has led to discussion, debate, and conflict, eventually leading to lack of consistency in delimitation. Here, we provide a broad review of species concepts, a clarification of category versus concept, an account of the general lineage concept (GLC), and finally a way forward for species discovery and delimitation. Historically, species were considered as varieties bound together by reproduction. After over 200 years of uncertainty, Mayr attempted to bring coherence to the definition of species through the biological species concept (BSC). This has, however, received much criticism, and the last half century has spawned at least 20 other concepts. A central philosophical problem is that concepts treat species as ‘individuals’ while the criteria for categorization treats them as ‘classes’. While not getting away from this problem entirely, the GLC attempts to provide a framework where lineage divergence is influenced by a number of different factors (and correlated to different traits) which relate to the different species concepts. We also introduce an ‘inclusive’ probabilistic approach for understanding and delimiting species. Finally, we provide a Wallacean (geography related) approach to the Linnaean problem of identifying and delimiting species, particularly for cases of allopatric divergence, and map this to the GLC. Going one step further, we take a morphometric terrain approach to visualizing and understanding differences between lineages. In summary, we argue that while generalized frameworks may work well for concepts of what species are, plurality and ‘inclusive’ probabilistic approaches may work best for delimitation.