The Multispecies Coalescent Over-Splits Species in The Case of Geographically Widespread Taxa

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The Multispecies Coalescent Over-splits Species in the Case of Geographically Widespread

Taxa

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E. Anne Chambers1* and David M. Hillis1
1Department of Integrative Biology and Biodiversity Center, The University of Texas at Austin,

Austin TX 78712 USA

*Corresponding author: E. Anne Chambers: [email protected]

Short Title: Multispecies Coalescent and Species Delimitation

© The Author(s) 2019. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved.
For Permissions, please email: [email protected]
CHAMBERS AND HILLIS

Abstract.---Many recent species delimitation studies rely exclusively on limited analyses of

genetic data analyzed under the multispecies coalescent (MSC) model, and results from these

studies often are regarded as conclusive support for taxonomic changes. However, most MSC-

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based species delimitation methods have well-known and often unmet assumptions. Uncritical

application of these genetic-based approaches (without due consideration of sampling design, the

effects of a priori group designations, isolation by distance, cytoplasmic–nuclear mismatch, and

population structure) can lead to over-splitting of species. Here, we argue that in many common

biological scenarios, researchers must be particularly cautious regarding these limitations,

especially in cases of well-studied, geographically variable, and parapatrically-distributed

species complexes. We consider these points with respect to a historically controversial species

group, the American milksnakes (Lampropeltis triangulum complex), using genetic data from a

recent analysis (Ruane et al. 2014; Syst. Biol. 63:231-250). We show that over-reliance on the

program BPP, without adequate consideration of its assumptions and of sampling limitations,

resulted in over-splitting of species in this study. Several of the hypothesized species of

milksnakes instead appear to represent arbitrary slices of continuous geographic clines. We

conclude that the best available evidence supports three, rather than seven, species within this

complex. More generally, we recommend that coalescent-based species delimitation studies

incorporate thorough analyses of geographic variation and carefully examine putative contact

zones among delimited species before making taxonomic changes.

Keywords: Speciation, species concepts, species delimitation, taxonomy, classification

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MULTISPECIES COALESCENT AND SPECIES DELIMITATION

Systematists attempt to understand and organize the diversity of life using two

fundamental concepts: species and trees of relationships among species. Under this framework,

species are viewed as individual, independently evolving metapopulation lineages, within which

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organisms typically mate and exchange genes (Wiley 1978; Mayden 1997; de Queiroz 1998,

2007). Species lineages split and give rise to new independent lineages, forming phylogenetic

trees of species in the process. Within those trees, monophyletic groups of species, or clades,

represent historical groups that share a common evolutionary origin.

The boundary between species and clades is not arbitrary, as life is clearly not organized

in a continuum. Instead, there are clear reproductive and genetic breaks that allow different

lineages to evolve on independent evolutionary pathways. Within sexual species, gene flow

typically maintains cohesion such that lineages evolve as units through time (Ghiselin 1974;

Templeton 1989). Ecological circumstances (selection for particular ecological roles) may also

play a role in maintaining species, even in the case of asexual organisms (Fontaneto et al. 2007;

Hillis 2007; Fontaneto and Barraclough 2015).

Although the theoretical distinction between species and clades is clear, the origins of

new species are necessarily fuzzy, as are the beginnings of all ontological individuals (Ghiselin

1974; Frost and Hillis 1990; de Queiroz 1998). Species rarely split instantaneously into

descendant lineages, and different biologists may use different operational criteria to detect a

splitting event (de Queiroz 1998, 2007). Widespread, geographically variable, but continuously

distributed species and species complexes present a particularly difficult problem for

systematists, as their members may exhibit considerable biological divergence at continental

scales. In some cases, this variation can be clinal and essentially continuous, with gene flow

across the entire species range (e.g., Slatkin and Maddison 1990; Slatkin 1991). In other cases, a

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CHAMBERS AND HILLIS

species complex might consist of multiple geographically, genetically cohesive, parapatric taxa

with little or no gene flow between species where they come into contact (e.g., Hillis 1988).

Intermediate conditions are also possible, such that gene flow is restricted but not entirely

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lacking between particular regional lineages, and such groups present a particular challenge for

species delimitation (Ensatina salamanders provide a textbook example of such complexity and

controversy; Wake and Schneider 1998).

Here, we explore the limitations of a commonly used approach for species delimitation

that relies on the multispecies coalescent model (hereafter, MSC-based methods). Despite the

known assumptions and limitations of these methods (Leaché and Fujita 2010; Olave et al. 2014;

Eberle et al. 2016; Luo et al. 2018; Barley et al. 2018), they are often used in isolation for species

delimitation and taxonomic change. We illustrate, using a case study, problems that may arise

from inadequate consideration of a priori group designations, limited sampling, and lack of

attention to contact zones in the context of one MSC-based species delimitation method.

LIMITATIONS OF THE MULTISPECIES COALESCENT MODEL FOR SPECIES DELIMITATION

Misapplication of the Multispecies Coalescent Model

The multispecies coalescent has become an important conceptual framework for inferring

relationships among species (species trees) from relationships among different genes (gene

trees), while taking into account incongruence among gene trees that results from incomplete

lineage sorting (Maddison 1997). Because genes trees are not always monophyletic within

species lineages, the multispecies coalescent was introduced as a way to detect recently divergent

lineages from collections of gene trees (Knowles and Carstens 2007). However, several

biological processes other than incomplete lineage sorting (including hybridization and

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MULTISPECIES COALESCENT AND SPECIES DELIMITATION

geographic structuring of populations) can also contribute to discordance among gene trees, and

the extent to which the multispecies coalescent is able to estimate a species tree depends in part

on how much discordance is limited to the process of incomplete lineage sorting within species

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(Sukumaran and Knowles 2017; Barley et al. 2018; Leaché et al. 2018).

The multispecies coalescent has been implemented in several methods for species

delimitation (e.g., Yang and Rannala 2010; Ence and Carstens 2011; Camargo et al. 2012; Fujita

et al. 2012; Leaché et al. 2014), and some authors have argued that these methods present a more

objective approach for testing species hypotheses compared to traditional methods of species

delimitation (Leaché and Fujita 2010; Fujita et al. 2012). One commonly used method is

Bayesian Phylogenetics and Phylogeography (BPP; Yang and Rannala 2010), which we examine

here. Recently, as the limitations of BPP have been explored (Sukumaran and Knowles 2017;

Barley et al. 2018; Leaché et al. 2018), it has become evident that this method does not

necessarily delimit species boundaries, but may also identify other kinds of genetic structure

within species.

Reliance on A Priori Grouping and Problems with Limited Sampling

Many MSC-based methods (including BPP) use clustering algorithms for initial

population-level assignment of individuals to groups which are subsequently validated using the

MSC-based method (see Carstens et al. 2013 for a full review). The number of individuals and

loci sampled play a significant role in ensuring programs such as Structure or Structurama

(Pritchard et al. 2000; Huelsenbeck et al. 2011) infer appropriate groups for testing (Rittmeyer

and Austin 2012; Olave et al. 2014; Hime et al. 2016). Limited geographic sampling can produce

the appearance of distinct genetic clusters, even when samples are drawn from continuous clines

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CHAMBERS AND HILLIS

or geographically structured populations (Hedin et al. 2015; Barley et al. 2018). Consider, for

example, two extreme alternatives. In one case, distinct species lineages have a narrow contact

zone with little to no gene flow or hybridization. In another case, a single species exhibits a

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geographic cline with gradual genetic change across geographic space. Distinguishing these two

scenarios requires thorough sampling across the cline or contact zone. If sampling is limited and

genetic information is obtained only from geographically distant populations, clustering methods

may be incapable of distinguishing between these two scenarios (Irwin 2002; Schwartz and

McKelvey 2008; Rittmeyer and Austin 2012; Puechmaille 2016; Bradburd et al. 2018).

There has been extensive discussion of the limitations of MSC-based methods. Overall,

depending on taxonomic, geographic, and genetic sampling, BPP can yield variable results in

delimitation (Setiadi et al. 2011; Olave et al. 2014; Reid et al. 2014; Zhang et al. 2014; Hime et

al. 2016; Barley et al. 2018). Here, we extend this literature by providing a re-analysis of an

existing dataset from a published study (Ruane et al. 2014) to illustrate the impact of using

limited data on BPP’s ability to delimit species. Particularly, we focus on the ramifications of

using nuclear genetic datasets with limited sampling and little phylogenetic signal, combined

with a strong conflicting signal from interspecific introgression of mitochondrial DNA, on

species delimitation. We emphasize that our analysis is not necessarily a criticism of the MSC-

based method BPP itself, but rather its application to inappropriate datasets in species

delimitation studies.

CASE STUDY: THE LAMPROPELTIS TRIANGULUM COMPLEX

Ruane et al. (2014) sought to clarify species boundaries and relationships in the

Lampropeltis triangulum complex (American milksnakes) using an MSC-based approach and

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concluded that genetic evidence supported the recognition of seven species in what had

traditionally been considered a single species (Williams 1988). Based primarily on results from

BPP, Ruane et al. (2014) elevated seven groups in the L. triangulum complex to full species

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status.

Using the Ruane et al. (2014) dataset, we show that sparse geographic sampling,

combined with a conflicting signal from interspecific introgression of mitochondrial DNA, led to

over-splitting of the American milksnake complex. We first detail inconsistencies observed in

the a priori clustering analyses and consider the information that can be inferred from such

analyses, and then examine the insights that can be gained from an examination of gene trees.

We then propose reasons that species splits were recognized despite the lack of supporting

evidence from the clustering analyses or evidence of any genetic or reproductive gaps between

species. Finally, we perform additional tests on two of the newly recognized species that

demonstrate the tendency of BPP to over-split species in the case of limited sampling across

broad geographic ranges.

A Priori Grouping

As discussed above, individuals are often assigned to groups (or putative species) before

input into MSC-based methods like BPP. This is usually accomplished using clustering methods

that report the relative support for each individual’s assignment into different clusters. Ruane et

al. (2014) assigned individuals to clusters in two different ways. First, they used the program

Structurama (Huelsenbeck et al. 2011), which searches for deviations from Hardy–Weinberg

equilibrium expectations across sampled gene loci, and then assigns individuals to genetic

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CHAMBERS AND HILLIS

groups that minimize these deviations. Ruane et al. (2014) also constructed a mitochondrial

DNA gene tree, from which they identified groups for subsequent population assignment.

Ruane et al. (2014) found that Structurama did not distinguish between their a priori

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geographic groups gentilis (western milksnakes) and triangulum (eastern milksnakes). When we

repeated the Ruane et al. (2014) Structurama analysis (see online Appendix 1, available on

Dryad at http://dx.doi.org/10.5061/dryad.[NNNN]), the highest support was given to different

cluster numbers depending on the run, indicating the data were not informative enough to

provide consistent and robust results across different runs (see online Appendix 1). However,

there were a few consistencies. We observed that Structurama almost always grouped Ruane et

al.’s (2014) nominal taxa polyzona, abnorma, and micropholis into a single cluster; that gentilis

and triangulum were always assigned to the same cluster, and that elapsoides and annulata were

generally shown as composites of multiple genetic clusters (Fig. S1). The fact that Structurama

consistently showed no division between gentilis and triangulum is especially noteworthy, as this

indicates that the samples of these putative taxa, collected thousands of kilometers apart from

New York to Montana to Arizona, do not deviate significantly from Hardy–Weinberg

equilibrium expectations (for the data examined) across the breadth of North America. Because

of this observation, we will be focusing on these two purported taxa for our subsequent analysis.

Gene Tree Phylogenies and Introgression of Mitochondrial Genomes

Ruane et al. (2014) collected data on 11 nuclear genes and one mitochondrial gene. Using

the same dataset reported by Ruane et al. (2014), we constructed gene trees for all 11 nuclear

genes (one representative nuclear gene is shown in Fig. 1, and the rest are shown in Fig. S2; see

online Appendix 2), as well as the single mitochondrial gene (Fig. S3). The overall amount of

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divergence between the 11 nuclear genes was low (0.017–0.076 substitutions per site between

the most divergent samples of the L. triangulum complex), especially compared to the higher

divergence of the single mitochondrial gene (0.225 substitutions per site for samples within the

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L. triangulum complex). Among recently divergent species, we would not expect congruence

among all gene trees, and some gene trees would not be expected to be monophyletic within

species lineages. However, there is no evidence of any consistent nuclear genetic divergence, nor

evidence for any reproductive isolation, at the contact zones between some of the purported

species recognized by Ruane et al. (2014). For example, geographically closest individuals of

gentilis and triangulum are genetically indistinguishable at every nuclear locus (Fig. S2). The

lack of any genetic break at the contact zone of these purported species suggests that their

division is an arbitrary split in a population continuum, rather than a break between distinct

species.

Closely related species are expected to retain some shared interspecific polymorphisms.

Indeed, humans and chimpanzees are known to share genetic polymorphisms that are thought to

have arisen in their common ancestor (e.g., Fan et al. 1989). Nonetheless, humans and

chimpanzees are also estimated to be diagnostically distinct across 4% of their genomes (Varki

and Altheide 2005). Interspecific differences between humans and chimpanzees (which total

approximately 125 million nucleotides) far exceed all intraspecific polymorphisms, and only a

small percentage of the latter are shared across these species (Varki and Altheide 2005). Georges

et al. (2018) emphasized the importance of such diagnostic differences as evidence for species

boundaries and lineage independence. In contrast, there are no diagnostic nucleotide differences

among the nuclear genes sampled by Ruane et al. (2014) between gentilis and triangulum. Given

that there is no evidence of deviations from Hardy–Weinberg equilibrium expectations (as shown

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in the Structurama analyses), and no evidence of even a single nuclear gene that consistently

differs between these two purported species, then what is the basis for hypothesizing the

existence of these species lineages? Is there any reason to expect any biological differences

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between two “species” that exhibit no known genetic differences?

In contrast to the low levels of nuclear divergence discussed above, upon construction of

the mitochondrial gene tree, Ruane et al. (2014) found clear evidence for multiple captures of L.

alterna mitochondrial DNA within western North American populations of the L. triangulum

complex (i.e., the populations referred to as the forms gentilis and annulata; Fig. S3). These

western populations of L. triangulum have mitochondrial haplotypes that are deeply embedded

within those of L. alterna, which in turn has a mitochondrial genome that is more closely related

to species of the L. getula complex and L. extenuata than to the eastern North American

populations of L. triangulum (Fig. S3). These introgression events appear to have happened

several times and are still ongoing (note the nearly identical mitochondrial DNA haplotypes of L.

alterna and L. triangulum where the two co-exist in Val Verde County, Texas; Fig. S3). Indeed,

the only consistent genetic difference between gentilis and triangulum is that individuals

assigned to gentilis have introgressed mitochondrial DNA from L. alterna, whereas individuals

assigned to triangulum do not. No single nucleotide from any of the sampled nuclear genes

follows this same pattern.

BPP Analysis

Ruane et al. (2014) first ran BPP using Structurama assignments as terminal lineages on

guide trees, resulting in high support for six lineages within the L. triangulum complex (recall

that gentilis and triangulum were initially treated as a single lineage by Ruane et al. [2014] based

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MULTISPECIES COALESCENT AND SPECIES DELIMITATION

on their Structurama assignments). We found the same result when we performed the same

analysis using unguided BPP (Yang and Rannala 2014; PP=99.2%; Table S1; online Appendix

3).

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Given the divergent mitochondrial DNA haplotypes in western populations of the

combined triangulum–gentilis lineage (the introgressed haplotypes from L. alterna; Fig. S3),

Ruane et al. (2014) next tested whether BPP would support a division between triangulum and

gentilis, despite their Structurama results. To conduct this test, they ran BPP with a guide tree

generated from their mitochondrial gene tree, assigning these two lineages to different groups.

BPP strongly supported this split as well, while still differentiating L. alterna, thus leading

Ruane et al. (2014) to propose L. gentilis and L. triangulum as distinct species.

Given that no nuclear genes (Figs. 1 and S2) show evidence of genetic differentiation

between gentilis and triangulum, and even Structurama fails to separate individuals of these taxa,

the only basis for distinguishing these taxa appears to be the introgressed mitochondrial DNA.

Similar cases of mitochondrial DNA capture have confounded species delimitation in other taxa

(e.g., polar bears versus brown bears: Miller et al. 2012; freshwater mussels: Chong et al. 2016),

and deep intraspecific polymorphisms of mitochondrial DNA have similarly affected species

delimitation in other studies (e.g., Folt et al. 2019). Without the introgressed mitochondrial

DNA, there would have been no basis for testing a split between gentilis and triangulum.

Therefore, we tested if the support from BPP for the division of gentilis and triangulum was

limited to the distribution of the introgressed L. alterna DNA (as examined by Ruane et al. 2014;

see Fig. 2a), or if it was simply a reflection of the geographic proximity of samples taken from

across a broad geographic distribution. In other words, does BPP support any east–west split of

the gentilis–triangulum populations at any point in the combined continental distribution of these

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forms, or is the split tested by Ruane et al. (2014) at the break in introgressed mitochondrial

DNA distinctive?

Using the nuclear dataset from Ruane et al. (2014), we tested five east–west splits of the

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gentilis–triangulum populations using BPP, including the split tested by Ruane et al. (2014; split

3 in Fig. 2b), as well as two splits farther west (splits 1 and 2 in Fig. 2b), and two splits farther

east (splits 4 and 5 in Fig. 2b; online Appendix 3). If the support from BPP reported by Ruane et

al. (2014) for split 3 reflects a real split between species, and is not simply a reflection of genetic

similarity of geographically proximate populations on either side of an arbitrary line, then we

would expect much stronger support from BPP for split 3 than for splits 1, 2, 4, or 5. In contrast,

if BPP is simply supporting any split that results in clustering of two groups of geographically

proximate samples from a broad distribution of a single species, we would expect to see support

for all five splits in Fig. 2b. We found the latter result: regardless of the geographic split between

populations, BPP indicated very high support (PP = 100% for splits 1–4, and PP > 96% for split

5; Table S1) for all five of the east–west splits of the gentilis–triangulum cline.

Our empirical results support the simulations of Barley et al. (2018), who demonstrated

that if samples are taken from separated geographic localities from a single species that exhibits

isolation by distance, BPP consistently supports the separated geographic clusters as distinct

species. That result is in contrast to the simulations of Zhang et al. (2011), who simulated a

stepping-stone model and found that only in cases of relatively high migration rates did BPP

falsely recover low support for a single species. As noted by Barley et al. (2018), the results from

theoretical studies depend largely on parameters used in the respective simulations. Our results

suggest that the simulations conducted by Barley et al. (2018) better match the empirical system

studied by Ruane et al. (2014) than do the simulations of Zhang et al. (2011). Note that even if

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the split between gentilis and triangulum reported by Ruane et al. (2014) represented an actual

species split, BPP also supports all the other east–west geographic splits shown in Figure 2b.

We do not suggest that any of the alternative species splits in Fig. 2b represent “better”

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species delimitation in the L. triangulum complex compared to those examined by Ruane et al.

(2014). Rather, our analysis merely demonstrates that BPP supports virtually any geographic

partition of samples in this potential continental cline as “species.” But clearly, splits 1–5 in Fig.

2b cannot all be true species splits, as they each are mutually inconsistent with one another. BPP

does not provide stronger support for the gentilis–triangulum split than it does for other east–

west splits of the samples.

THE IMPORTANCE OF CONTACT ZONES

When splits are hypothesized within an otherwise continuous distribution, contact zone

analyses have traditionally been used to assess the degree of genetic isolation and gene flow

between the putative taxa (Barton and Hewitt 1985; Durand et al. 2009). Systematists need to

distinguish between widespread, clinal geographic variation within a species on one hand, versus

distinct genetic and reproductive breaks between species on the other. This is especially

important when species are thought to be distributed parapatrically, such that the species contact

one another along narrow zones of potential gene flow. In such cases, the study of contact zones

can reveal if (a) hybridization between the putative species is absent or rare; (b) the contact zones

act as “genetic sinks” (thus restricting gene flow between the putative species); or (c) there is

broad gene flow and integration between the putative species at the contact zone. Case (a) is

uncontroversial, as it is consistent with virtually any concept of species (i.e., there is clear

evidence that the taxa are reproductively isolated, evolutionary distinct, and independent

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lineages). In recent decades, many biologists have argued that case (b), or evidence of a narrow

hybrid zone that acts as a “genetic sink” that strongly restricts gene flow between species, is also

consistent with the hypothesis of distinct species (e.g., Sage and Selander 1979; Hafner et al.

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1983; Yanchukov et al. 2006). In contrast, case (c) refutes the hypothesis that the lineages are

evolving independently from one another (as there are no reproductive or genetic breaks between

the lineages to support their independent evolution).

Examining the population genetic structure at contact zones can also determine selective

forces that may be playing roles in driving, or maintaining, divergence (Sobel and Streisfeld

2015; Bertrand et al. 2016). Many approaches, genetic and otherwise, have been developed for

examining contact zone interactions (e.g., Gompert and Buerkle 2010; Derryberry et al. 2014),

although many species delimitation studies may require additional sampling for such an analysis.

SPECIES DELIMITATION AND TAXONOMIC RECOMMENDATIONS

Ruane et al. (2014) stated that they followed the “general lineage species concept” of de

Queiroz (1998, 2007). In these two papers, de Queiroz argued that virtually all species concepts

treat species as “separately evolving metapopulation lineages” that simply use different lines of

evidence to assess the independence and isolation of lineages. In other words, virtually all

“species concepts” are conceptualizing the same entities—namely, the individual, independent,

evolving lineages of life, within which organisms typically mate and exchange genes (as we

described in the opening of this paper).

Species delimitation is typically a two-step process (see Hillis 2019). Taxonomists first

group organisms into putative taxa using one of several criteria. These include (1) correlated

diagnostic characters (including morphological, genetic, or behavioral attributes), which are

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often assessed in a hierarchical phylogenetic analysis; (2) deviations from Hardy–Weinberg

equilibrium (as conducted, for example, by the programs Structure and Structurama); and (3)

multivariate analyses that assess overall divergence, such as principal components analysis.

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These tests are all ways of identifying groups of individuals that appear to be different from one

another. However, differences arise within species as well as between them, so a second step is

needed to assess if the observed differences are evidence of independently evolving lineages, or

if the observed variation simply represents geographic or population variation within species. If

the groups in question come into geographic contact, then taxonomists typically assess lineage

independence by looking for direct or indirect evidence of reproductive barriers between the

groups at contact zones. Indirect evidence may include sharp geographic breaks in suites of

morphological, genetic, and/or behavioral characters at the contact zones; direct evidence may

include behavioral assessments of reproductive interactions between the putative species.

Multispecies coalescent-based approaches have also been used to assess the evolutionary

independence of lineages (as in Ruane et al. 2014), but as shown in Barley et al. (2018), BPP

does not appear to discriminate adequately between geographic clinal structure versus species

boundaries. Although our results appear to be an empirical example of this scenario (Fig. 2b),

without adequate sampling at purported contact zones there is no way to distinguish these two

possibilities using BPP alone.

Despite the inability of BPP to distinguish between clinal variation versus speciation, we

can use the data collected by Ruane et al. (2014) to ask if there is any evidence for sharp genetic

or reproductive breaks among the various groups that they examined. Ruane et al. (2014) also

presented an analysis to summarize their data, in the form of a SplitsTree analysis (Fig. 3; Huson

and Bryant 2006). This tree does not represent any single gene tree, but is instead a summary of

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support and counter-support for various clusters of individuals examined by Ruane et al. (2014)

across all examined loci. Individuals that are nearly identical across all loci are located adjacent

to one another (separated by small branch lengths) on this tree; in contrast, individuals that differ

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across many loci are well separated in this tree. Therefore, we can look at the contact zones

between each purported taxon, and ask if geographically adjacent individuals in different

purported taxa exhibit any evidence for the genetic or reproductive breaks that are expected from

separately evolving lineages. If there are none, then there is no reason to hypothesize a break

between distinct species rather than a continuous geographic cline.

Figure 3 shows three hypotheses for species delimitation in American milksnakes

projected on the Ruane et al. (2014) SplitsTree analysis, with a depiction of the distribution of

the purported taxa. The one-species hypothesis of Williams (1988; shown in Fig. 3a) is refuted

by two lines of evidence: first, there are far larger genetic gaps among subgroups of his L.

triangulum than there are between those subgroups and other well differentiated, sympatric

species (i.e., L. alterna). Second, where these subgroups of Williams’ L. triangulum come into

contact, they are sympatric, and yet maintain large genetic gaps between individuals. Thus, we

agree with Ruane et al. (2014) in rejecting the one-species hypothesis of Williams (1988).

Figure 3b presents an alternative taxonomic hypothesis that addresses the problems noted

above, and divides L. triangulum of Williams (1988) into three distinct species: L. triangulum, L.

elapsoides, and L. polyzona. This hypothesis is almost identical to the arrangement proposed by

Blanchard (1921), although he noted that collections at the time were not sufficient in lower

Central America to firmly establish the relationship between the nominal forms L. polyzona and

L. micropholis, and he tentatively treated those two species as distinct as well, pending further

collection of intermediate populations. There are substantial, consistent genetic breaks across

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MULTISPECIES COALESCENT AND SPECIES DELIMITATION

multiple loci among all three of the species recognized in this hypothesis. In addition, where any

two of these three species come into geographic contact, there are areas of known sympatry,

accompanied by genetic divergence across multiple loci. Lampropeltis triangulum and L.

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elapsoides are known to occur sympatrically, with little or no hybridization, across a broad area

of parts of Kentucky, Tennessee, Alabama, Georgia, North Carolina, and Virginia in the United

States (indeed, this region of sympatry was discussed by Williams 1988). The known area of

sympatry between L. triangulum and L. polyzona in northern Veracruz, Mexico is much smaller,

with the two species reported together from just a single locality (also reported by Williams

1988). Thus, all the genetic and geographic data appear to support the recognition of these three

species.

In contrast, the remaining taxa recognized by Ruane et al. (2014; Fig. 3c) exhibit no

known areas of sympatry or any evidence of sharp genetic breaks at or near purported contact

zones. Instead, individuals on either side of purported contact zones (other than the ones noted in

Fig. 3b) are genetically much more similar to one another than they are to other geographically

distant individuals in their own taxon. This is not consistent with the expectation for

independently evolving lineages. As with the human/chimpanzee example discussed earlier, we

might expect similarities in occasional genes through independent lineage sorting, but we would

still expect large genetic gaps across most loci in comparisons of individuals drawn from

different species. No such genetic gaps exist between geographically adjacent samples of

micropholis–abnorma–polyzona, or between geographically adjacent samples of annulata–

gentilis–triangulum (Figs. 2b, 3c, and S2). These findings are also largely consistent with the

Structurama results (Fig. S1), except that annulata does appear to show significant Hardy–

Weinberg equilibrium deviations from the gentilis–triangulum grouping. However, such

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CHAMBERS AND HILLIS

deviations are not surprising given the large geographic distance between the samples examined

by Ruane et al. (2014) of annulata versus gentilis–triangulum (for example, the distance between

the closest samples examined for nuclear genes of annulata and gentilis–triangulum is

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approximately 485 km). Better sampling is needed to determine if the relatively small genetic

differences and Hardy–Weinberg deviations between these groups are indicative of geographic

clines or species breaks.

Despite frequent discussion of integrative approaches for species delimitation (Dayrat

2005; Leaché et al. 2009; Padial et al. 2010; Schlick-Steiner et al. 2010; Fujita et al. 2012;

Derkarabetian and Hedin 2014; Huang and Knowles 2016; Renner 2016), researchers sometimes

use limited data and rely on results generated by a single analysis to delimit species.

Consideration of morphological, behavioral, and ecological data, particularly including analyses

at contact zones, is a critical part of testing species hypotheses (Zhang et al. 2011; Edwards and

Knowles 2014; Pante et al. 2015; Solís-Lemus et al. 2015). We recommend against taxonomic

changes on the basis of analyses of limited samples, and demonstrate that BPP analyses of

limited geographic samples can support many groupings that are inconsistent with species.

Although we present an alternative hypothesis to that presented by Ruane et al. (2014) in

Figure 3b, we emphasize that the data presented by Ruane et al. (2014) are inadequate to fully

examine the species boundaries in this group. The existing data do appear to support the species

delimited in Figure 3b, but it is certainly possible that additional genetic and geographic

sampling will demonstrate the existence of additional species boundaries in this group. However,

we see no convincing evidence from the data presented by Ruane et al. (2014) to support the

additional species recognized in Figure 3c.

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MULTISPECIES COALESCENT AND SPECIES DELIMITATION

Species delimitation is not a simple process. In well studied, widely distributed taxa,

species designations should incorporate multiple sources of evidence regarding geographic

variation (of genes, morphology, and behavior), reproductive isolation, and gene flow. New

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species designations, especially of well-studied groups, are best made after careful consideration

of all sources of relevant evidence. Nomenclatural changes to well-studied groups should be

made only after due consideration of all available data (e.g., Setiadi et al. 2011; Barley et al.

2013; Hedin et al. 2015; Pante et al. 2015; Pyron et al. 2016; Folt et al. 2019). Although a

conservative approach to taxonomic change can risk underestimating diversity (Padial et al.

2010), this is preferable to making poorly supported taxonomic changes with each new dataset

and analysis (Hillis 2019).

SUPPLEMENTARY MATERIAL

Supplementary material, including data and online appendices, are available from the

Dryad Data Repository:http://dx.doi.org/10.5061/dryad.[NNNN].

ACKNOWLEDGMENTS

We thank Sara Ruane and Frank Burbrink for guidance on the milksnake re-analysis. We

also thank Carole Baldwin, Anthony Barley, Peter Beerli, David Cannatella, Kevin de Queiroz,

Harry Greene, Tracy Heath, Aleta Quinn, Jordan Satler, Robert Thomson, and April Wright for

comments and conversation related to the manuscript. Finally, we would like to thank Richard

Glor, Adam Leaché, and 13 anonymous reviewers for helpful suggestions and critique that

improved this manuscript.

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FIGURE CAPTIONS

Figure 1. Majority-rule consensus gene tree constructed with nuclear gene 2CL8, used as a

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representative tree to illustrate consistencies observed across all 11 nuclear gene trees. From this

tree, it is clear that Central and South American milksnake lineages (polyzona, abnorma, and

micropholis) form a monophyletic cluster with little resolution. L. elapsoides is consistently

recovered as a monophyletic lineage, while remaining U.S. lineages (triangulum, gentilis, and

annulata) are rarely resolved and exhibit no diagnostic differences. Remaining gene trees are

given in Figure S2.

Figure 2. Results and group assignment from five runs of unguided BPP. a) Points represent

samples with nuclear gene data from Ruane et al. (2014), with the same data used here, and

ranges shaded according to the Ruane et al. (2014) final population assignment. b) Population

assignment between each of five runs of BPP (see online Appendix 1 for details).

Figure 3. Three hypotheses for species delimitation in milksnakes (Lampropeltis triangulum

complex) with SplitsTree networks (Huson and Bryant 2006) and ranges colored based on the

proposed species given in each hypothesis (adapted from Ruane et al. 2014). The Lampropeltis

alterna lineage shown in grey (not indicated on range map) is included because of its relevance

to mitochondrial introgression events. a) Hypothesis 1: American milksnakes represent a single,

polytypic species across their entire range (Williams 1988); b) Hypothesis 2: three species, L.

triangulum, L. elapsoides, and L. polyzona (similar to Blanchard 1921); c) Hypothesis 3: the

seven species proposed by Ruane et al. (2014).

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