Speciation Rates Research Articles

The genomic signatures of evolutionary stasis.

Evolutionary stasis characterizes lineages that seldom speciate and show little phenotypic change over long stretches of geological time. Although lineages that appear to exhibit evolutionary stasis are often called living fossils, no single mechanism is thought to be responsible for their slow rates of morphological evolution and low species diversity. Some analyses of molecular evolutionary rates in a handful of living fossil lineages have indicated that these clades exhibit slow rates of genomic change. Here, we investigate mechanisms of evolutionary stasis using a dataset of 1,105 exons for 481 vertebrate species. We demonstrate that two ancient clades of ray-finned fishes classically called living fossils, gars and sturgeons, exhibit the lowest rates of molecular substitution in protein-coding genes among all jawed vertebrates. Comparably low rates of evolution are observed at fourfold degenerate sites in gars and sturgeons, implying a mechanism of stasis decoupled from selection that we speculate is linked to a highly effective DNA repair apparatus. We show that two gar species last sharing common ancestry over 100 million years ago produce morphologically intermediate and fertile hybrids in the wild. This makes gars the oldest naturally hybridizing divergence among eukaryotes and supports a theoretical prediction that slow rates of nucleotide substitution across the genome slow the accumulation of genetic incompatibilities, enabling hybridization across deeply divergent lineages and slowing the rate of speciation over geological timescales. Our results help establish molecular stasis as a barrier to speciation and phenotypic innovation and provide a mechanism to explain the low species diversity in living fossil lineages.

Blocking then stinging as a case of two-step evolution of defensive cage architectures in herbivore-driven ecosystems.

Dense branching and spines are common features of plant species in ecosystems with high mammalian herbivory pressure. While dense branching and spines can inhibit herbivory independently, when combined, they form a powerful defensive cage architecture. However, how cage architecture evolved under mammalian pressure has remained unexplored. Here we show how dense branching and spines emerged during the age of mammalian radiation in the Combretaceae family and diversified in herbivore-driven ecosystems in the tropics. Phylogenetic comparative methods revealed that modern plant architectural strategies defending against large mammals evolved via a stepwise process. First, dense branching emerged under intermediate herbivory pressure, followed by the acquisition of spines that supported higher speciation rates under high herbivory pressure. Our study highlights the adaptive value of dense branching as part of a herbivore defence strategy and identifies large mammal herbivory as a major selective force shaping the whole plant architecture of woody plants.

The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Escarpment evolution drives the diversification of the Madagascar flora.

Madagascar exhibits high endemic biodiversity that has evolved with sustained and stable rates of speciation over the past several tens of millions of years. The topography of Madagascar is dominated by a mountainous continental rift escarpment, with the highest plant diversity and rarity found along the steep, eastern side of this geographic feature. Using a process-explicit model, we show that precipitation-driven erosion and landward retreat of this high-relief topography creates transient habitat organization through multiple mechanisms, including catchment expansion, isolation of highland remnants, and formation of topographic barriers. Habitat isolation and reconnection on a million-year timescale serves as an allopatric speciation pump creating the observed biodiversity.

Repeated upslope biome shifts in Saxifraga during late-Cenozoic climate cooling.

Mountains are among the most biodiverse places on Earth, and plant lineages that inhabit them have some of the highest speciation rates ever recorded. Plant diversity within the alpine zone - the elevation above which trees cannot grow-contributes significantly to overall diversity within mountain systems, but the origins of alpine plant diversity are poorly understood. Here, we quantify the processes that generate alpine plant diversity and their changing dynamics through time in Saxifraga (Saxifragaceae), an angiosperm genus that occurs predominantly in mountain systems. We present a time-calibrated molecular phylogenetic tree for the genus that is inferred from 329 low-copy nuclear loci and incorporates 73% (407) of known species. We show that upslope biome shifts into the alpine zone are considerably more prevalent than dispersal of alpine specialists between regions, and that the rate of upslope biome shifts increased markedly in the last 5 Myr, a timeframe concordant with a cooling and fluctuating climate that is likely to have increased the extent of the alpine zone. Furthermore, alpine zone specialists have lower speciation rates than generalists that occur inside and outside the alpine zone, and major speciation rate increases within Saxifraga significantly pre-date increased rates of upslope biome shifts. Specialisation to the alpine zone is not therefore associated with speciation rate increases. Taken together, this study presents a quantified and broad scale perspective of processes underpinning alpine plant diversity.

Diversification and dispersal in the Americas revealed by new phylogenies of the wrens and allies (Passeriformes: Certhioidea)

Abstract The passerine superfamily Certhioidea lacks a complete phylogeny despite decades of recognition as a clade and extensive systematic work within all its constituent families. Here, we inferred a near-complete species-level phylogeny of Certhioidea from a molecular supermatrix, including the first comprehensive sampling of the wrens (Troglodytidae), and used this phylogeny to infer its biogeographic and diversification histories. We also inferred an expanded phylogeny including nearly 100 putative phylospecies previously documented in the literature, and we found that including this diversity had notable impacts on the inferred evolutionary history of Certhioidea. This phylospecies-level tree documented a few instances of species paraphyly, some previously described in the literature and some novel. We found that Certhioidea originated largely in Eurasia and dispersed into North America five times in the last 20 million years, including at the origin of the “New World certhioids,” wrens and gnatcatchers, a clade herein named Orthourae. After this initial dispersal event, both wrens and gnatcatchers diversified extensively across the hemisphere, with both lineages repeatedly crossing between continents. However, we detected no notable impact of the formation of the Isthmus of Panama on the frequency of dispersal events between North and South America. The inclusion of phylospecies altered this biogeographic inference in some portions of the tree but overall was largely consistent. With species-level sampling, we found that diversification rates within Certhioidea were largely constant through time with a detectable deceleration toward the present. By contrast, phylospecies-level sampling recovered a different diversification history with a significant rate increase at the crown node of Orthourae after dispersing into the Americas and increased speciation rates particularly within the genera Polioptila and Henicorhina. This largely resolved phylogeny for Certhioidea has yielded important insights into the evolutionary history of this group and provides a framework for future comparative work on this fascinating clade.

Climatic‐niche breadth, niche position, and speciation in lizards and snakes

AbstractAimThe climatic niche is associated with diversification in many groups of animals and plants. However, the relationships between climatic‐niche breadth evolution, climatic‐niche position evolution, and speciation remain underexplored. It is particularly unclear whether changes in climatic‐niche breadth are related to diversification. We tested two hypotheses relating niche breadth, niche position, and speciation using climatic data in Squamata (lizards and snakes), one of the largest radiations of tetrapods. These hypotheses were: (1) the oscillation hypothesis (niche breadth changes along with niche position and speciation) and (2) the musical‐chairs hypothesis (niche breadth remains relatively constant when niche position changes during speciation).LocationGlobal.TaxonSquamata (lizards and snakes).MethodsWe estimated rates of speciation and evolutionary rates for both climatic‐niche position and climatic‐niche breadth for 5320 squamate species. We tested relationships among these rates using Bayesian phylogenetic generalised linear‐mixed models.ResultsHigher speciation rates were associated with higher rates of evolution in niche position and in niche breadth. Faster rates of change in niche breadth were related to narrower niches and faster rates of change in niche position.Main ConclusionsOur results support the oscillation hypothesis to explain the relationships between speciation and changes in climatic‐niche position and climatic‐niche breadth. We found that species that changed climatic‐niche breadths more rapidly: (1) speciated faster; (2) evolved towards narrower niche breadths; and (3) changed climatic‐niche positions more rapidly. These results suggest that oscillation between wider and narrower niches is coupled with climatic‐niche divergence and speciation. These conclusions may apply to many other groups of plants and animals in which speciation is often related to climatic‐niche divergence.

An Elevational Phylogeographic Diversity Gradient in Neotropical Birds Is Decoupled from Speciation Rates.

AbstractA key question about macroevolutionary speciation rates is whether they are controlled by microevolutionary processes operating at the population level. For example, does spatial variation in population genetic differentiation underlie geographical gradients in speciation rates? Previous work suggests that speciation rates increase with elevation in Neotropical birds, but underlying population-level gradients remain unexplored. Here, we characterize elevational phylogeographic diversity between montane and lowland birds in the megadiverse Andes-Amazonian system and assess its relationship to speciation rates to evaluate the link between population-level differentiation and species-level diversification. We aggregated and georeferenced nearly 7,000 mitochondrial DNA sequences across 103 species or species complexes in the Andes and Amazonia and used these sequences to describe phylogeographic differentiation across both regions. Our results show increased levels of both discrete and continuous metrics of population structure in the Andean mountains compared with the Amazonian lowlands. However, higher levels of population differentiation do not predict higher rates of speciation in our dataset. Multiple potential factors may lead to our observed decoupling of initial population divergence and speciation rates, including the ephemerality of incipient species and the multifaceted nature of the speciation process, as well as methodological challenges associated with estimating rates of population differentiation and speciation.

Diversifying in the mountains: spatiotemporal diversification of frogs in the Western Ghats biodiversity hotspot.

Mountain ranges are hotspots of biodiversity. However, the mechanisms that generate biodiversity patterns in different mountainous regions and taxa are not apparent. The Western Ghats (WG) escarpment in India is a globally recognized biodiversity hotspot with high species richness and endemism. Most studies have either invoked paleoclimatic conditions or climatic stability in the southern WG refugium to explain this high diversity and endemism. However, the factors driving macroevolutionary change remain unexplored for most taxa. Here, we generated the most comprehensive dated phylogeny to date for ranoid frogs in the WG and tested the role of paleoclimatic events or climatic stability in influencing frog diversification. We found that the diversity of different ranoid frog clades in the WG either accumulated at a constant rate through time or underwent a decrease in speciation rates around 3-2.5 Ma during the Pleistocene glaciation cycles. We also find no significant difference in diversification rate estimates across elevational gradients and the three broad biogeographic zones in the WG (northern, central, and southern WG). However, time-for-speciation explained regional species richness within clades, wherein older lineages have more extant species diversity. Overall, we find that global paleoclimatic events have had little impact on WG frog diversification throughout most of its early history until the Quaternary and that the WG may have been climatically stable allowing lineages to accumulate and persist over evolutionary time.

Punctuated evolution of bill morphology in the largest family of songbirds (Thraupidae)

Abstract We assessed correlations between speciation rate and bill morphology in an ecologically diverse and continental-scale songbird radiation, the tanagers (Thraupidae). Our analyses showed that bill size, shape, and their evolutionary rates are not correlated with speciation rate. However, we did find evidence that each axis of variation in bill morphology diversifies at speciation events interspersed with periods of gradual evolution, consistent with a punctuated equilibrium model of character change. To determine correlations, we incorporated a time-calibrated molecular phylogeny and high-resolution three-dimensional surface scans of bill structure from museum study skins. Overall, our findings suggest that bill size and shape diversify in association with the speciation process, but not through any effect on the rate of speciation. Previous studies have shown other traits, such as song, ecological niche, and plumage do influence speciation and, ultimately, species richness in tanagers.

ReMASTER: improved phylodynamic simulation for BEAST 2.7.

Phylodynamic models link phylogenetic trees to biologically-relevant parameters such as speciation and extinction rates (macroevolution), effective population sizes and migration rates (ecology and phylogeography), and transmission and removal/recovery rates (epidemiology) to name a few. Being able to simulate phylogenetic trees and population dynamics under these models is the basis for (i) developing and testing of phylodynamic inference algorithms, (ii) performing simulation studies which quantify the biases stemming from model-misspecification, and (iii) performing so-called model adequacy assessments by simulating samples from the posterior predictive distribution. Here I introduce ReMASTER, a package for the phylogenetic inference platform BEAST 2 that provides a simple and efficient approach to specifying and simulating the phylogenetic trees and population dynamics arising from phylodynamic models. Being a component of BEAST 2 allows ReMASTER to also form the basis of joint simulation and inference analyses. ReMASTER is a complete rewrite of an earlier package, MASTER, and boasts improved efficiency, ease of use, flexibility of model specification, and deeper integration with BEAST 2. ReMASTER can be installed directly from the BEAST 2 package manager, and its documentation is available online at https://tgvaughan.github.io/remaster. ReMASTER is free software, and is distributed under version 3 of the GNU General Public License. The Java source code for ReMASTER is available from https://github.com/tgvaughan/remaster.

Explaining extreme differences in species richness among co-occurring palm clades in Madagascar

Abstract Imbalance in species richness among related clades is a pervasive, yet incompletely understood feature of biodiversity. Comparison of species-poor and species-rich clades that have evolved within the same region can shed light on the mechanisms underlying this phenomenon. The long-isolated island of Madagascar is an ideal place for doing this. Madagascar harbours at least ten clades of palms (Arecaceae) that have colonized the island independently and diversified to widely differing degrees, ranging from one to 180 known species. We estimated colonization times and diversification rates for these clades based on an extensive phylogenomic dataset and tested the degree to which clades that arrived in Madagascar earlier have more species (time-for-speciation effect), finding a moderate effect. For context, we tested for time-for-speciation effects in other plant and animal lineages, finding variable but qualitatively similar results. Our findings suggest that variation in diversification rate (i.e. speciation and/or extinction rate) is a major driver of species richness imbalance among Malagasy clades, both in palms and elsewhere. We demonstrate that in palms, differences in diversification rates originated long before colonization of the island, suggesting a minor role of classical ‘island radiation’ and a stronger role of heritable traits driving diversification rate. Ability to colonize new climates also appears to play a role. Future work should address the interplay between the dynamic environment of Madagascar and the inherited traits of colonizing lineages to fully explain the island’s intriguing mix of species-poor and species-rich clades.

Lokiceratops rangiformis gen. et sp. nov. (Ceratopsidae: Centrosaurinae) from the Campanian Judith River Formation of Montana reveals rapid regional radiations and extreme endemism within centrosaurine dinosaurs.

The Late Cretaceous of western North America supported diverse dinosaur assemblages, though understanding patterns of dinosaur diversity, evolution, and extinction has been historically limited by unequal geographic and temporal sampling. In particular, the existence and extent of faunal endemism along the eastern coastal plain of Laramidia continues to generate debate, and finer scale regional patterns remain elusive. Here, we report a new centrosaurine ceratopsid, Lokiceratops rangiformis, from the lower portion of the McClelland Ferry Member of the Judith River Formation in the Kennedy Coulee region along the Canada-USA border. Dinosaurs from the same small geographic region, and from nearby, stratigraphically equivalent horizons of the lower Oldman Formation in Canada, reveal unprecedented ceratopsid richness, with four sympatric centrosaurine taxa and one chasmosaurine taxon. Phylogenetic results show that Lokiceratops, together with Albertaceratops and Medusaceratops, was part of a clade restricted to a small portion of northern Laramidia approximately 78 million years ago. This group, Albertaceratopsini, was one of multiple centrosaurine clades to undergo geographically restricted radiations, with Nasutuceratopsini restricted to the south and Centrosaurini and Pachyrostra restricted to the north. High regional endemism in centrosaurs is associated with, and may have been driven by, high speciation rates and diversity, with competition between dinosaurs limiting their geographic range. High speciation rates may in turn have been driven in part by sexual selection or latitudinally uneven climatic and floral gradients. The high endemism seen in centrosaurines and other dinosaurs implies that dinosaur diversity is underestimated and contrasts with the large geographic ranges seen in most extant mammalian megafauna.

Completing a molecular timetree of apes and monkeys.

The primate infraorder Simiiformes, comprising Old and New World monkeys and apes, includes the most well-studied species on earth. Their most comprehensive molecular timetree, assembled from thousands of published studies, is found in the TimeTree database and contains 268 simiiform species. It is, however, missing 38 out of 306 named species in the NCBI taxonomy for which at least one molecular sequence exists in the NCBI GenBank. We developed a three-pronged approach to expanding the timetree of Simiiformes to contain 306 species. First, molecular divergence times were searched and found for 21 missing species in timetrees published across 15 studies. Second, untimed molecular phylogenies were searched and scaled to time using relaxed clocks to add four more species. Third, we reconstructed ten new timetrees from genetic data in GenBank, allowing us to incorporate 13 more species. Finally, we assembled the most comprehensive molecular timetree of Simiiformes containing all 306 species for which any molecular data exists. We compared the species divergence times with those previously imputed using statistical approaches in the absence of molecular data. The latter data-less imputed times were not significantly correlated with those derived from the molecular data. Also, using phylogenies containing imputed times produced different trends of evolutionary distinctiveness and speciation rates over time than those produced using the molecular timetree. These results demonstrate that more complete clade-specific timetrees can be produced by analyzing existing information, which we hope will encourage future efforts to fill in the missing taxa in the global timetree of life.

Evolution of Seed Dispersal Modes in the Orchidaceae: Has the Vanilla Mystery Been Solved?

Orchid seeds are predominantly wind-dispersed, often developed within dry, dehiscent fruits that typically release millions of dust-like seeds into the air. Animal-mediated seed dispersal is a lesser-known phenomenon in the family and predominantly occurs in groups belonging to early-diverging lineages bearing indehiscent, fleshy fruits with hard, rounded, dark seeds. In this review, we explore the evolutionary trends of seed dispersal mechanisms in Orchidaceae, focusing on the pantropical genus Vanilla. Notably, certain Neotropical species of Vanilla produce vanillin-aromatic compounds synthesized naturally in their fruits, which plays a pivotal role in seed dispersal. Ectozoochory occurs in dry, dehiscent fruits, whose seeds are dispersed by (i) male euglossine bees collecting the fruit’s vanillin aromatic compounds and (ii) female stingless bees collecting the fruit’s mesocarp. Endozoochory occurs in (iii) highly nutritious, indehiscent fruits consumed by terrestrial mammals or (iv) fleshy, dehiscent fruits whose mesocarp is consumed by arboreal mammals. Wind dispersal appears to be a derived state in Orchidaceae and, given its predominance, a trait likely associated with enhanced speciation rates. Zoochory primarily occurs in groups derived from early-diverging lineages; occasional reversions suggest a link between dispersal mode and fruit and seed traits. Interestingly, fruit dehiscence and fleshiness in Vanilla lack phylogenetic signal despite their role in determining dispersal modes, suggesting potential environmental adaptability.

Phylogeny and biogeography of the Cryptocaryeae (Lauraceae)

AbstractThe biogeographical history of many lineages within the Lauraceae remains poorly known because of the difficulty of assigning macrofossils to living genera, poor pollen preservation, and the absence of sufficiently resolved or well‐supported phylogenies. Here, we utilize plastid genome sequencing to reinvestigate the phylogenetic and biogeographic history of trees in the tribe Cryptocaryeae, an important component of broad‐leaved forests worldwide, with around 800 species that rely on vertebrate frugivores to disperse their seeds. A new time‐calibrated phylogeny with high support for 176 species was used to infer the biogeographic history and speciation rates based on inferences of BAMM analyses. A monophyletic Cryptocaryeae with Aspidostemon, Beilschmiedia, Cryptocarya, Dahlgrenodendron, and Eusideroxylon clades was confirmed. The five clades of Cryptocaryeae were estimated to share a common ancestor in tropical Africa or Asia in the Early Cretaceous around 123 Ma. The Beilschmiedia and Cryptocarya clades were estimated to have originated in South America around 66 Ma. Extant subclades of the Beilschmiedia clade could colonize Australia by 35 Ma and extant subclades of the Cryptocarya clade could colonize Australia by 42 Ma. Diversification rates of the Beilschmiedia clade accelerated 14 and 12 Ma after its origin and diversification rates of the Cryptocarya clade accelerated 18 Ma after its origin. Over 45% extant Cryptocaryeae species originated in the three periods of accelerated differentiation in the Miocene of Asia and Australia. Long‐distance dispersal has had a major influence on biogeography, with dispersal to Asia likely occurring seven times, Zealandia six times, America three times, Australia three times, Africa twice, and Oceania at least once. Over 70% extant Cryptocaryeae species which diverged before the Quaternary grow in the Southern Hemisphere, while 90% extant Cryptocaryeae species which diverged in the Quaternary grow in the Northern Hemisphere. The Cryptocaryeae originated in and diversified with the first angiosperm‐dominated broad‐leaved evergreen forests, from the Cretaceous to the Paleogene. Long‐distance seed dispersal, probably by birds, although possibly also by flotation, has allowed the tribe to track these forests in space and time, despite a failure to adapt to cold, dry, or highly seasonal environments.

Dispersal modes affect Rhamnaceae diversification rates in a differentiated manner.

The evolution of dispersal modes has been proposed to promote the diversification of angiosperms. However, little is known about the relative impact of different dispersal modes on plant diversification. We test the association between dispersal modes and diversification rates using Rhamnaceae, the cosmopolitan buckthorn family, as a model. We found that species with diplochory have the highest diversification rates followed by those with myrmecochory and ballistic dispersal, while lineages dispersed by vertebrates and wind have relatively low diversification rates. The difference in diversification rates may be closely linked to the difference in dispersal distance and ecological interactions implied by each dispersal mode. Species which disperse over larger geographical distances may have much higher speciation rates due to the increased chance of establishing isolated populations due to geological barriers or habitat fragmentation. However, long-distance dispersal may also increase the chance of extinction. By contrast, species with short-distance dispersal modes may have low speciation rates. Complex interactions with the surrounding environment may, however, impact diversification rates positively by increasing plant survival and reproductive success.

Colour polymorphism and conspicuousness do not increase speciation rates in Lacertids

Colour polymorphism and conspicuousness do not increase speciation rates in Lacertids

All about being old and shooting hairs: clade age and urticating hair explain the patterns of diversification in tarantulas.

The extreme asymmetry of species richness distribution across the tree of life has always intrigued evolutionary biologists. Two competing explanations have been proposed to explain this pattern-the clade age hypothesis and diversification rate variation. While these two scenarios may not be mutually exclusive, to what extent time and diversification rates interact to explain species richness patterns remains understudied. Here, we investigate the relative influence of these two scenarios using tarantulas (Family: Theraphosidae) as a model. Tarantulas represent a speciose group of spiders found worldwide but exceptionally diverse in South America. These spiders show two distinct patterns of microhabitat use (ground-dwelling or arboreal) and defense strategies (presence or absence of urticating hairs). Using various trait-independent and dependent diversification models, we test the clade age hypothesis, the role of microhabitat, antipredator defense strategy, and geography in influencing diversification rates. Our results suggest that clade age is the primary predictor of species richness distribution across the tarantula subfamilies. However, the presence of urticating hair probably disrupted this pattern in some clades by increasing the net diversification rates, not by increasing the speciation rate but by reducing the extinction rate.

Commonly used Bayesian diversification methods lead to biologically meaningful differences in branch-specific rates on empirical phylogenies.

Identifying along which lineages shifts in diversification rates occur is a central goal of comparative phylogenetics; these shifts may coincide with key evolutionary events such as the development of novel morphological characters, the acquisition of adaptive traits, polyploidization or other structural genomic changes, or dispersal to a new habitat and subsequent increase in environmental niche space. However, while multiple methods now exist to estimate diversification rates and identify shifts using phylogenetic topologies, the appropriate use and accuracy of these methods are hotly debated. Here we test whether five Bayesian methods-Bayesian Analysis of Macroevolutionary Mixtures (BAMM), two implementations of the Lineage-Specific Birth-Death-Shift model (LSBDS and PESTO), the approximate Multi-Type Birth-Death model (MTBD; implemented in BEAST2), and the Cladogenetic Diversification Rate Shift model (ClaDS2)-produce comparable results. We apply each of these methods to a set of 65 empirical time-calibrated phylogenies and compare inferences of speciation rate, extinction rate, and net diversification rate. We find that the five methods often infer different speciation, extinction, and net-diversification rates. Consequently, these different estimates may lead to different interpretations of the macroevolutionary dynamics. The different estimates can be attributed to fundamental differences among the compared models. Therefore, the inference of shifts in diversification rates is strongly method dependent. We advise biologists to apply multiple methods to test the robustness of the conclusions or to carefully select the method based on the validity of the underlying model assumptions to their particular empirical system.