Determining the exact taxonomic status of creatures like the Mexican gray wolf and the red wolf hinges on a clear understanding of “species” and, crucially, “What Is A Subspecies”. This understanding must be combined with access to genomic, ecological, and morphological data. This article will explore species and subspecies concepts, with a particular emphasis on how they apply to North American canids, especially in light of modern discoveries about gene flow between species.
Defining Species and Subspecies: Evolving Concepts
The classification of species has long been rooted in the idea that species are distinct biological entities. The way we define species has evolved significantly, driven by the availability of more data and new analytical methods. Early classification systems, such as Aristotle’s, relied on morphological similarities and differences. Species were considered static and distinct. Linnaeus, who developed the hierarchical naming system still in use today, was one of the first to recognize hybridization as a potential mechanism for the formation of new species. In the mid-1800s, Darwin suggested that species arise through gradual change, which is why evolutionary relationships among organisms can be depicted using a phylogenetic tree.
Darwin’s concept of evolutionary relationships remains the basis for modern taxonomy. Modern species concepts have shifted away from the “type-specimen” approach of early taxonomists, focusing instead on population-level characteristics (Coyne and Orr, 2004; Howard and Berlocher, 1998; Mayr, 1963), particularly the range of individual phenotypes. A key focus is on defining independently evolving lineages (Hey, 2001; Mace, 2004).
Ecological, morphological, and behavioral data all play a role in identifying these lineages. Discrepancies can occur when different types of data point to different conclusions. For instance, organisms sharing ecological roles may encounter and mate with each other, while still maintaining distinct genetic groups. Closely related organisms tend to share similar morphology and behavior, but these similarities don’t necessarily indicate a close genetic relationship due to environmental influences.
Modern genetic data and analytical tools are vital for resolving these challenges. DNA sequence data complements ecological, morphological, and behavioral data, providing information on genetic makeup and enabling inferences about genetic relatedness and history. Analyzing genomic data from ancient samples, gathered through paleontology and archaeology, alongside modern population data, can reveal key insights into population history. Collecting this data is still difficult, especially for North American canids, because while modern samples are available, there is a scarcity of data from older samples.
The challenge for contemporary taxonomy is to use diverse data to assess the taxonomic status of organism groups under modern species concepts. Despite differing emphases, these concepts aim to identify groups with reproductive compatibility that sustains genetic continuity. This can be assessed using ecological, phenotypic, and genomic data, as detailed later. The next sections will focus on three species concepts specifically relevant to canid systematics.
Biological Species Concept
Ernst Mayr (1942) defined the biological species concept as groups of potentially or actually interbreeding individuals reproductively isolated from other such groups. Most biologists agree with this approach, while also understanding the complexities given increasing evidence that hybridization is a key part of the history of recognized species.
Reproductive isolation can be either pre-mating or post-mating. Pre-mating isolation prevents mating between populations due to geographic barriers, or ecological or behavioral differences, setting them on separate evolutionary paths. Post-mating isolation is the inability of different species to produce fertile offspring due to factors like fertilization failure, non-viable fertilized eggs, or sterile offspring. In this case, contemporary biologists would agree that the populations potentially belong to different species. Directly testing for these post-mating isolating mechanisms is not always feasible, as assessing hybrid viability and fertility requires observation in the wild or controlled settings.
Even with some levels of pre- and post-mating isolation, gene flow can still occur between populations. This indicates that complete reproductive isolation might be too strict a requirement for defining species. When classifying North American canids, it’s crucial to remember that hybridization is common in the history of accepted species due to the fact that they are evolutionarily young and geographically overlapping (in part due to human habitat alteration).
Phylogenetic Species Concept
The phylogenetic species concept aligns with the biological species concept, with added emphasis on shared ancestry. It suggests individuals belong to the same species if they descend from a common ancestor and share lineage-specific mutations. Because of mutation and genetic drift, reproductively isolated species diverge at the DNA level over time, regardless of changes in morphology or behavior. Reduced gene flow causes divergence, which is accelerated by natural selection favoring different mutations. Natural selection can also preserve similar phenotypes in divergent lineages, even with underlying DNA sequence changes. This results in cryptic species that are morphologically similar but genetically divergent, rendering them reproductively incompatible and on different evolutionary trajectories.
Chronospecies Concept
Changes can accumulate across generations of a lineage, making modern members distinct from their predecessors despite genetic continuity (Stanley, 1978). The concept of chronospecies refers to a group of organisms at one point in time evolving into a later group distinct enough to be considered a separate species. Chronospecies determination is generally based on time-series documentation using morphological data. However, defining a chronospecies is challenging because historical samples are often limited, and assessing the influence of environmental factors on organismal phenotypes is difficult. DNA extracted from paleontological specimens can offer unprecedented opportunities to assess genetic cohesion between past and present generations. While DNA from historical North American canid individuals is not always available, some key specimens and data have been obtained from skeletal remains.
In summary, new species constantly arise and existing ones constantly change (Hey, 2001; Mace, 2004). While no species concept is perfect, the principles underlying the biological, phylogenetic, and chronospecies concepts—reproductive isolation mediated by genetic and ecological factors, and phylogenetic continuity mediated by mutation, drift, selection, and inheritance—provide a robust platform for identifying species. Making genomic, ecological, and behavioral observations transcends the limitations of individual species concepts and provides a strong framework for evaluating the taxonomic status of North American canids.
What is a Subspecies? The Designation Explained
Classification schemes often recognize taxonomic groups below the species level, prompting the question: what is a subspecies? Early definitions of subspecies distinguished populations sharing pattern, color, or morphological attributes not found in other, geographically separated populations of the same species. Modern concepts (Haig et al., 2006), including Darwin’s suggestion, emphasize a partial restriction of gene flow. Virtually all modern definitions follow this spirit, viewing subspecies as potentially interbreeding populations phylogenetically distinguishable from, but reproductively compatible with, other such groups (Mayr, 1953, 1963; Mayr and Ashlock, 1991).
“Species” Definition Under the Endangered Species Act
The U.S. Endangered Species Act (ESA) defines “species” broadly, including not only a species but also “any subspecies of fish or wildlife or plants, and any distinct population segment of any species or vertebrate fish or wildlife which interbreeds when mature.” This means a subspecies occupying a distinct geographic area can be treated as a “species” for restoration and recovery purposes if its survival is threatened. The ESA allows legal protection for subspecies and “distinct population segments” (DPSs) of vertebrates as if they were recognized taxonomic species. DPSs are defined based on discreteness and significance to the species (U.S. FWS and NMFS, 1996). The ESA’s broader use of “species” provides stability for conservation policy, even amidst changing taxonomic designations and species concepts.
Understanding “Species” in Light of Hybridization
Contemporary species concepts are based on the idea of distinct species as independently evolving lineages. This works when gene flow is minimal or nonexistent between species. However, genomic data increasingly shows that cross-species exchange of genetic material through hybridization is a key feature of the evolutionary history of many accepted species (Arnold, 2006; Grant and Grant, 1992; Mallet, 2005), which raises challenges for strict application of existing species concepts.
Hybridization, Introgression, and Admixture
Several terms describe gene flow across taxonomic groups. “Hybridization,” “introgression,” and “admixture” are sometimes used interchangeably. In this report, hybridization refers to the mating of two individuals from different species to yield offspring with ancestry from both parents (Abbott et al., 2013) (see Figure 2-1).
Interspecific mating doesn’t always result in sustained hybridization. Sometimes, individuals from different species cannot produce offspring, or their offspring are infertile. Reduced hybrid viability or fertility creates a barrier to gene flow between hybridizing species. It can also drive the evolution of pre-mating isolation due to the higher reproductive success of individuals that mate exclusively with members of their own species.
If hybrid individuals produce fertile offspring, an admixed population can develop. These individuals can have varying genetic contributions from each parental species. Several long-term outcomes are possible. Survival and mating of hybrid individuals with a parental species can result in introgression, moving specific regions of one parental species’ genome into the chromosomes of some individuals of the other species. If these chromosome regions contain differences between species (different alleles), the presence of the new regions may affect the fitness of the individuals that possess them. These differences will remain in the population if they provide advantage or are not detrimental to the individuals who carry them. Chromosomal regions conferring alleles that only moderately reduce fitness can linger for a long time or even become fixed, especially in small populations, where natural selection is weak as compared to genetic drift.
Molecular Patterns of Hybridization
Hybridization produces specific molecular patterns. Mammalian cells contain two copies of each chromosome, one from each parent. A hybrid individual would have one chromosome in each pair from parental species 1 and the other from parental species 2. During the production of eggs and sperm, the two chromosomes within a pair trade segments through genetic recombination, producing new chromosome copies composed of new combinations of the DNA from the original chromosomes. If hybrids are fertile, their offspring inherit individual chromosomes containing segments from both parental species. With each successive generation, each segment is broken into smaller pieces, producing increasingly complex mosaics of DNA from the two parental species.
Admixture Examples
Admixture, the formation of novel genetic combinations through hybridization, is now recognized as a core component of the history of many taxonomically recognized species. For example, genomic analyses indicate that modern Europeans and Asians inherited a fraction of their genome from Neanderthals and Denisovans. On average, 2–3 percent of the genomes of humans originating from areas outside sub-Saharan Africa consists of DNA that introgressed from Neanderthals (Green et al., 2010), while Melanesians and aboriginal Australians trace 3–4 percent of their DNA to introgression with Denisovans (Reich et al., 2010).
Indeed, hybridization is known to have occurred in all major lineages of non-human primates (Tung and Barreiro, 2017; Zinner et al., 2011), including mangabeys (Rungucebus) (Zinner et al., 2009b), baboons (Papio) (Zinner et al., 2009a), guenons (Guschanski et al., 2013), macaques (Fan et al., 2014; Guschanski et al., 2013; Tosi et al., 2003), langurs (Ting et al., 2008), howler monkeys (Cortés-Ortiz et al., 2007; Mourthe et al., 2018), marmosets (Malukiewicz et al., 2015), and lemurs (Wyner et al., 2002).
Introgression Across Diverged Lineages
Introgression is possible even across highly diverged lineages. Pigs (Sus scrofa) show evidence of gene flow from an extinct species outside the Sus genus that had diverged from pigs an estimated 8.5 million years ago (Ai et al., 2015). The divergent fragment of introgressed DNA harbors genetic variation associated with climate adaptation and may be an example of adaptive introgression.
In summary, genome sequencing has revealed evidence that introgression between phenotypically and genotypically divergent species has occurred in many mammalian lineages. As such, complete genetic separation and a complete absence of admixture appears no longer to be a strict criterion for defining species.
Hybridization Context
Hybridization is possible when populations that have been geographically isolated for some time regain contact due to natural processes or anthropogenic effects. For example, the pet trade in Brazil has led to the introduction of some marmoset species (genus Callithrix) into urban areas historically inhabited only by other marmoset species (Malukiewicz et al., 2015). These introductions have resulted in hybridization among several species of marmosets that were adapted to different ecological and social environments.
Implications of Hybridization
Hybridization can have diverse implications for the conservation and evolutionary trajectories of the species involved. These outcomes are potentially relevant to the evolutionary history of canids in North America.
Strict Genetic Separation
Species may remain genetically distinct following hybridization if they harbor genetic incompatibilities reducing the fitness or fertility of hybrid individuals. In extreme cases, this can result in complete inviability or sterility of hybrids. Even without complete sterility, hybrids can be unfit in ways that limit introgression. Features may evolve that reduce or prevent interspecific matings, such as frog calls that allow individuals to identify potential mates (Butlin, 1987; Butlin and Smadja, 2018). These features can limit introgression and potentially eliminate introgressed genes, leaving negligible evidence of hybridization.
Partial Genetic Separation with Introgression
Species may remain distinct following hybridization but still retain the capacity for occasional exchange of genetic material. Genomes are likely mosaics, with individuals of one species having segments of DNA derived from another (Ellegren et al., 2012). The genetic elements exchanged may be adaptive, allowing the recipient species to adapt to new environmental conditions through shared genetic variation.
There is evidence for adaptive introgression in several species (Whitney et al., 2006), including modern humans. Genomic regions that introgressed from Neanderthals and Denisovans into modern human populations include genes associated with immune defense functions (Quach et al., 2016) and with human survival in high-altitude environments (Huerta-Sánchez et al., 2014). Similar examples exist in other lineages. Wu et al. (2018) inferred introgression among yak (Bos grunniens), domesticated cattle (Bos taurus), the wisent (Bison bonasus), the gayal (Bos frontalis), and the banteng (Bos javanicus). The introgressed regions contain genes that regulate red blood cell production and high-altitude adaptation in both the yak and Tibetan cattle (Wu et al., 2018).
Stable Hybrid Zones
Stable hybrid zones can form when species adapted to different environments inhabit neighboring or overlapping ranges, or when formerly geographically separated species meet due to range expansion. Ecological selection may counteract hybridization and maintain distinct species. Varying degrees of introgression can occur where the species meet. If selection is strong, separate species can be maintained despite gene flow. At the genomic level, the species will eventually become genetically similar except for the regions harboring genes associated with local adaptations (Barton and Hewitt, 1981). When species experience secondary contact, stable hybrid zones can also be maintained by a balance between selection against hybrids and the dispersal of parental species into the area of contact. In such cases, neutral introgression is expected, and adaptive introgression is possible.
Formation of Hybrid Species
If hybridization is common and there are no strong ecological selection or genetic incompatibilities, an entirely new hybrid species can emerge (Mallet, 2007). This speciation by hybridization may result in a third species replacing the two parental species or coexisting with them. Many plant species (e.g., wild sunflowers, cf. Ungerer et al., 1998) and some animal species (e.g., Heliconius butterflies, see Mavárez et al., 2006; virgin chub, DeMarais et al., 1992; and Caribbean bats, DeMarais et al., 1992; Larsen et al., 2010) have originated from hybridization.
Establishing Guidelines for Determining Taxonomic Status
Increasing evidence of gene flow among taxonomic groups conflicts with earlier views that strict reproductive isolation is a defining feature of taxonomically valid species. All modern species concepts focus on whether a given group of organisms constitutes a distinct, independently evolving lineage that merits recognition as a taxonomically valid species. Combining several approaches is often essential. Morphological and paleontological analyses can be informative in assessing the continuity of organismal phenotypes over time and geographical areas, but they are often limited by the availability of fossils, which may be few in number, in poor condition, or from a limited geographic range. Genetic and genomic data can provide an enormous number of quantifiable characters, but they are not always available for specimens of diverse ages. Behavioral traits and ecological roles have long been recognized as important in the designation of taxa, especially below the species level (Crandall et al., 2000; Haig et al., 2006), as they explicitly address differences in adaptive characters, but they are not readily useful for assessing continuity between historical and contemporary populations.
A Framework for Establishing Taxonomic Designations
Although modern species concepts do have some differences, they are united by the goal of identifying groups of organisms whose reproductive compatibility sustains genetic continuity. The levels of genetic differentiation do not need to be the sole—or even the primary—evidence considered. It is tempting to prioritize molecular data, as they are readily quantifiable and can be used to define phylogenetic and past population relationships. However, exclusively focusing on molecular characters risks over-splitting taxa or, conversely, failing to recognize as distinct those that have diverged recently and now occupy different ecological niches but have not yet had time to accrue substantial genetic differentiation (Coates et al., 2018; Haig et al., 2006).
Combining genomic data with morphological and ecological data can provide a more complete picture of the taxonomy and evolutionary history of species and subspecies.
Strong support for the validity of a taxonomic designation of a species may include the following:
- Morphological, paleontological, or fossil evidence that the taxon under consideration is evolving independently or morphological evidence that specimens possess a phenotype that is distinct from other defined species.
- Evidence of genetic or genomic distinctiveness, based on data from several independently segregating genetic loci, with evidence that extant subpopulations are connected by gene flow. If hybridization with other defined species is also detected, it must be clear that introgression does not substantially affect the discreteness of the taxon under consideration.
- Ecological or behavioral data indicating adaptive differences and reproductive incompatibilities separating the taxon under consideration from other closely related species. Relevant differences may be ecological, behavioral, or physiological. Genomic data, if available, may reveal genetic variation underlying these differences but are not required to confirm the importance of ecological or behavioral differences.
Strong support for the validity of a taxonomic designation of what is a subspecies may include the following:
- Morphological, paleontological, or fossil evidence of a geographically and historically isolated lineage within the species to which it belongs. Morphological evidence may be especially useful for identifying distinct, locally adapted phenotypes that evolved during isolation from other lineages of the same species.
- Genetic or genomic evidence of distinctness based on data from multiple independently inherited genetic loci, with no evidence of reproductive isolation from other populations of the same species in regions of range overlap. Phylogeographic analyses may be especially useful in assessing the distinctness of the lineage under consideration from other populations belonging to the same lineage.
- Ecological, behavioral, or physiological characters that provide evidence of adaptive differences between the lineage and other groups belonging to the same species. Available genetic or genomic data may reveal the presence of alleles, or of differences in allele frequencies, at loci that underlie these differences.
