Transcription Factors Evolve Faster Than Their Structural Gene Targets in the Flavonoid Pigment Pathway.

Dissecting the relationship between gene function and substitution rates is key to understanding genome-wide patterns of molecular evolution. Biochemical pathways provide powerful systems for investigating this relationship because the functional role of each gene is often well characterized. Here, we investigate the evolution of the flavonoid pigment pathway in the colorful Petunieae clade of the tomato family (Solanaceae). This pathway is broadly conserved in plants, both in terms of its structural elements and its MYB, basic helix-loop-helix, and WD40 transcriptional regulators, and its function has been extensively studied, particularly in model species of petunia. We built a phylotranscriptomic data set for 69 species of Petunieae to infer patterns of molecular evolution across pathway genes and across lineages. We found that transcription factors exhibit faster rates of molecular evolution (dN/dS) than their targets, with the highly specialized MYB genes evolving fastest. Using the largest comparative data set to date, we recovered little support for the hypothesis that upstream enzymes evolve slower than those occupying more downstream positions, although expression levels do predict molecular evolutionary rates. Although shifts in floral pigmentation were only weakly related to changes affecting coding regions, we found a strong relationship with the presence/absence patterns of MYB transcripts. Intensely pigmented species express all three main MYB anthocyanin activators in petals, whereas pale or white species express few or none. Our findings reinforce the notion that pathway regulators have a dynamic history, involving higher rates of molecular evolution than structural components, along with frequent changes in expression during color transitions.

LeafMachine: Using machine learning to automate leaf trait extraction from digitized herbarium specimens.

PREMISE: Obtaining phenotypic data from herbarium specimens can provide important insights into plant evolution and ecology but requires significant manual effort and time. Here, we present LeafMachine, an application designed to autonomously measure leaves from digitized herbarium specimens or leaf images using an ensemble of machine learning algorithms. METHODS AND RESULTS: We trained LeafMachine on 2685 randomly sampled specimens from 138 herbaria and evaluated its performance on specimens spanning 20 diverse families and varying widely in resolution, quality, and layout. LeafMachine successfully extracted at least one leaf measurement from 82.0% and 60.8% of high- and low-resolution images, respectively. Of the unmeasured specimens, only 0.9% and 2.1% of high- and low-resolution images, respectively, were visually judged to have measurable leaves. CONCLUSIONS: This flexible autonomous tool has the potential to vastly increase available trait information from herbarium specimens, and inform a multitude of evolutionary and ecological studies.

Transforming mentorship in STEM by training scientists to be better leaders.

Effective mentoring is a key component of academic and career success that contributes to overall measures of productivity. Mentoring relationships also play an important role in mental health and in recruiting and retaining students from groups underrepresented in STEM fields. Despite these clear and measurable benefits, faculty generally do not receive mentorship training, and feedback mechanisms and assessment to improve mentoring in academia are limited. Ineffective mentoring can negatively impact students, faculty, departments, and institutions via decreased productivity, increased stress, and the loss of valuable research products and talented personnel. Thus, there are clear incentives to invest in and implement formal training to improve mentorship in STEM fields. Here, we outline the unique challenges of mentoring in academia and present results from a survey of STEM scientists that support both the need and desire for more formal mentorship training. Using survey results and the primary literature, we identify common behaviors of effective mentors and outline a set of mentorship best practices. We argue that these best practices, as well as the key qualities of flexibility, communication, and trust, are skills that can be taught to prospective and current faculty. We present a model and resources for mentorship training based on our research, which we successfully implemented at the University of Colorado, Boulder, with graduate students and postdocs. We conclude that such training is an important and cost-effective step toward improving mentorship in STEM fields.

Stepwise evolution of floral pigmentation predicted by biochemical pathway structure.

Developmental pathways play a major role in influencing the distribution of naturally occurring phenotypes. For example, pathway structure and regulation could make some phenotypes inaccessible or restrict the routes through which phenotypes evolve. In this study, we examine floral anthocyanin pigments across the Solanaceae family and test whether patterns of phenotypic variation are consistent with predicted constraints based on the structure of the flavonoid biosynthetic pathway. We find that anthocyanin evolution occurs in a stepwise manner whereby transitions between the production of red mono hydroxylated pelargonidin pigments and blue trihydroxylated delphinidin pigments first passes through an intermediate step of producing purple dihydroxylated cyanidin pigments. Although the transitions between these three pigment types differ in frequency, we infer that these shifts are often reversible, suggesting that the functionality of the underlying biochemical pathway is generally conserved. Furthermore, our study finds that some pigment combinations are never observed, pointing to additional constraints on naturally occurring phenotypes. Overall, our findings provide insights into how the structure of an angiosperm-wide biochemical pathway has shaped macroevolutionary variation in floral pigmentation.

Why are red flowers so rare? Testing the macroevolutionary causes of tippiness.

Traits that have arisen multiple times yet still remain rare present a curious paradox. A number of these rare traits show a distinct tippy pattern, where they appear widely dispersed across a phylogeny, are associated with short branches and differ between recently diverged sister species. This phylogenetic pattern has classically been attributed to the trait being an evolutionary dead end, where the trait arises due to some short-term evolutionary advantage, but it ultimately leads species to extinction. While the higher extinction rate associated with a dead end trait could produce such a tippy pattern, a similar pattern could appear if lineages with the trait speciated slower than other lineages, or if the trait was lost more often that it was gained. In this study, we quantify the degree of tippiness of red flowers in the tomato family, Solanaceae, and investigate the macroevolutionary processes that could explain the sparse phylogenetic distribution of this trait. Using a suite of metrics, we confirm that red-flowered lineages are significantly overdispersed across the tree and form smaller clades than expected under a null model. Next, we fit 22 alternative models using HiSSE (Hidden State Speciation and Extinction), which accommodates asymmetries in speciation, extinction and transition rates that depend on observed and unobserved (hidden) character states. Results of the model fitting indicated significant variation in diversification rates across the family, which is best explained by the inclusion of hidden states. Our best fitting model differs between the maximum clade credibility tree and when incorporating phylogenetic uncertainty, suggesting that the extreme tippiness and rarity of red Solanaceae flowers makes it difficult to distinguish among different underlying processes. However, both of the best models strongly support a bias towards the loss of red flowers. The best fitting HiSSE model when incorporating phylogenetic uncertainty lends some support to the hypothesis that lineages with red flowers exhibit reduced diversification rates due to elevated extinction rates. Future studies employing simulations or targeting population-level processes may allow us to determine whether red flowers in Solanaceae or other angiosperms clades are rare and tippy due to a combination of processes, or asymmetrical transitions alone.

Adaptive signal coloration maintained in the face of gene flow in a Hispaniolan Anolis Lizard.

BACKGROUND: Studies of geographic variation can provide insight into the evolutionary processes involved in the early stages of biological diversification. In particular, multiple, replicated cases of geographic trait divergence present a powerful approach to study how patterns of introgression and adaptive divergence can vary with geographic space and time. In this study, we conduct replicated, fine-scaled molecular genetic analyses of striking geographic dewlap color variation of a Hispaniolan Anolis lizard, Anolis distichus, to investigate whether adaptive trait divergence is consistently associated with speciation, whereby genetic divergence is observed with neutral markers, or whether locally adapted traits are maintained in the face of continued gene flow. RESULTS: We find instances where shifts in adaptive dewlap coloration across short geographic distances are associated with reproductive isolation as well as maintained in the face of gene flow, suggesting the importance of both processes in maintaining geographic dewlap variation. CONCLUSION: Our study suggests that adaptive dewlap color differences are maintained under strong divergent natural selection, but this divergence does not necessarily lead to anole speciation.

Discovery of a Giant Chameleon-Like Lizard (Anolis) on Hispaniola and Its Significance to Understanding Replicated Adaptive Radiations.

We report a new chameleon-like Anolis species from Hispaniola that is ecomorphologically similar to congeners found only on Cuba. Lizards from both clades possess short limbs and a short tail and utilize relatively narrow perches, leading us to recognize a novel example of ecomorphological matching among islands in the well-known Greater Antillean anole radiation. This discovery supports the hypothesis that the assembly of island faunas can be substantially deterministic and highlights the continued potential for basic discovery to reveal new insights in well-studied groups. Restricted to a threatened band of midelevation transitional forest near the border of the Dominican Republic and Haiti, this new species appears to be highly endangered.

How to make a red flower: the combinatorial effect of pigments.

Red flowers have evolved repeatedly across angiosperms and are frequently examined in an ecological context. However, less is known about the biochemical basis of red colouration in different taxa. In this study, we examine the spectral properties, anthocyanin composition and carotenoid expression of red flowers in the tomato family, Solanaceae, which have evolved independently multiple times across the group. Our study demonstrates that Solanaceae typically make red flowers either by the sole production of red anthocyanins or, more commonly, by the dual production of purple or blue anthocyanins and orange carotenoids. In using carotenoids to modify the effect of purple and/or blue anthocyanins, these Solanaceae species have converged on the same floral hue as those solely producing red anthocyanins, even when considering the visual system of pollinators. The use of blue anthocyanins in red flowers appears to differ from other groups, and suggests that the genetic changes underlying evolutionary shifts to red flowers may not be as predictable as previously suggested.

Widespread flower color convergence in Solanaceae via alternate biochemical pathways.

Phenotypic convergence is rampant throughout the tree of life. While recent studies have made significant progress in ascertaining the proximate mechanisms underlying convergent phenotypes, less is known about the frequency and predictability with which convergent phenotypes arise via the same or multiple pathways at the macroevolutionary scale. We investigated the proximate causes and evolutionary patterns of red flower color in the tomato family, Solanaceae, using large-scale data mining and new sequence data to reconstruct a megaphylogeny of 1341 species. We then combined spectral and anatomical data to assess how many times red flowers have evolved, the relative contribution of different pathways to independent origins of red, and whether the underlying pathway is predicted by phylogenetic relatedness. We estimated at least 30 relatively recent origins of red flowers using anthocyanins, carotenoids, or a dual production of both pigments, with significant phylogenetic signal in the use of anthocyanins and dual production, indicating that closely related red-flowered species tend to employ the same mechanism for coloration. Our study is the first to test whether developmental pathways exhibit phylogenetic signal and implies that historical contingency strongly influences the evolution of new phenotypes.

The role of heritable and dietary factors in the sexual signal of a Hispaniolan Anolis lizard, Anolis distichus.

The diversity of sexual signals is astounding, and divergence in these traits is believed to be associated with the early stages of speciation. An increasing number of studies also suggest a role for natural selection in driving signal divergence for effective transmission in heterogeneous environments. Both speciation and adaptive divergence, however, are contingent on the sexual signal being heritable, yet this often remains assumed and untested. It is particularly critical that the heritability of carotenoid-based sexual signals is investigated because such traits may instead be phenotypically plastic indicators of an individual's quality that exhibit no or little heritable variation. We present the first study to investigate the relative contribution of genetic and environmental factors to the striking diversity of dewlap color and pattern in Anolis lizards. Using a breeding experiment with Anolis distichus populations exhibiting different dewlap phenotypes, we raise F1 offspring in a common garden experiment to assess whether dewlap color is inherited. We follow this with carotenoid supplementation to investigate the influence of dietary pigments to dewlap color variation. We find significant differences in several aspects of dewlap color and pattern to persist to the F1 generation (fathers: N = 19; F1 males: N = 50; P < 0.01) with no change in dewlap phenotype with carotenoid supplementation (N = 52; P > 0.05). These results strongly support that genetic differences underlie dewlap color variation, thereby satisfying a key requirement of natural selection. Our findings provide an important stepping-stone to understanding the evolution of an incredibly diverse signal important for sexual selection and species recognition.

Phylogenetic analyses of novel squamate adenovirus sequences in wild-caught Anolis lizards.

Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity.

Correlation between Anolis lizard dewlap phenotype and environmental variation indicates adaptive divergence of a signal important to sexual selection and species recognition.

Although the importance of signals involved in species recognition and sexual selection to speciation is widely recognized, the processes that underlie signal divergence are still a matter of debate. Several possible processes have been hypothesized, including genetic drift, arbitrary sexual selection, and adaptation to local signaling environments. We use comparative analyses to investigate whether the remarkable geographic variation of dewlap phenotype in a Hispaniolan trunk Anolis lizard (A. distichus) is a result of adaptive signal divergence to heterogeneous environments. We recover a repeated pattern of divergence in A. distichus dewlap color, pattern, and size with environmental variation across Hispaniola. These results are aligned with ecological models of signal divergence and provide strong evidence for dewlap adaptation to local signaling environments. We also find that A. distichus dewlaps vary with the environment in a different manner to other previously studied anoles, thus expanding upon previous predictions on the direction dewlaps will diverge in perceptual color space in response to the environment.

Assessment of xylazine for euthanasia of anoles (Anolis carolinensis and Anolis distichus).

Intracoelomic (IC) injection of xylazine was evaluated as a chemical euthanasia method for Anolis lizards (Anolis carolinensis or Anolis distichus). Lizards were allocated into 5 groups of 10 animals each. Each group was euthanized by one of these methods: 10 mg xylazine (100 mg/mL) IC; 10 mg xylazine and 0.5 mg acepromazine (10 mg/mL) IC; 10 mg xylazine IC followed by intracardiac injection of 0.1 mEq KCl (2 mEq/mL) once heart beats were no longer discernable by Doppler; 500 mg/kg 1% NaCO(3)-buffered MS222 solution IC followed by IC injection of 0.1 mL unbuffered 50% (v/v) MS222 solution (experimental groups); and 1.95 mg sodium pentobarbital, diluted 1:10 in sterile water (38.9 mg/mL) given IC (control group). Compared with those given sodium pentobarbital or MS222, lizards euthanized by using xylazine showed prolonged persistence of purposeful movement after cardiac arrest. Therefore, xylazine is not an acceptable alternative euthanasia agent for use in anoles.

Genetic differentiation among populations of a Hispaniolan trunk anole that exhibit geographical variation in dewlap colour.

Long neglected by classic island biogeographical theory, speciation within and among islands is increasingly recognized as a major contributor to insular diversity. Although the factors responsible for island speciation remain poorly understood, this process appears critically dependent on geographical variation and speciation in allopatry or parapatry. Here, we investigate geographical variation and speciation in a complex of Hispaniolan trunk anoles (Anolis distichus), where populations with strikingly distinct dewlap colours and patterns correspond with deeply divergent mtDNA structure. Using a multilocus, population-level analysis, we investigate whether these phenotypically and mitochondrially distinct populations exhibit the type of nuclear differentiation expected among species or incipient species. Along a transect that extends across a recently recessed marine barrier, our results are consistent with the persistence of an abrupt phenotypic and mitochondrial transition following secondary contact, in spite of little or no evidence for a reduction in nuclear gene flow. Along a second transect extending across a steep environmental gradient, our phenotypic and microsatellite data suggest a sharp genetic break with little or no admixture, whereas mtDNA recovers a signature of extensive unidirectional introgression. Together, these results are consistent with previous studies of Lesser Antillean anoles, suggesting that allopatric divergence alone is insufficient for speciation, whereas reduced gene flow and partial reproductive isolation may accumulate in the presence of ecological gradients.