The phylogenetic distribution of the ampulla ureter and ampulla urogenital/uriniferous papilla in the Serpentes

The ampulla ureter and ampulla urogenital/uriniferous papilla represent differing morphologies of the caudal urogenital ducts in snakes. The ampulla ureter is an enlarged portion of the caudal extremity of the ureter that communicates the cranial regions of the ureter and the ductus deferens/Wolffian duct to the urodaeum. The ampulla urogenital/uriniferous papilla is an enlarged pouch, distinct from the ureter, which communicates the ureter and ductus deferens/Wolffian duct to the urodaeum. Although functional differences of these two structures are unknown, the ampulla urogenital/uriniferous papilla may have evolved for urine storage in males and females, and secondarily evolved a reproductive function in males. The most parsimonious optimization of the ampulla ureter and ampulla urogenital/uriniferous papilla indicates that the ampulla ureter is the ancestral state in snakes. Examining the presence or absence of the ampulla ureter and ampulla urogenital/uriniferous papilla in snakes on conflicting caenophidian phylogenies results in two hypotheses for the evolution of these variant morphologies: (1) The ampulla urogenital/uriniferous papilla evolved from the ampulla ureter independently in the Colubroidea and Elapoidea with subsequent losses of the ampulla urogenital/uriniferous papilla in the Elapoidea and (2) a single transition from the ampulla ureter to the ampulla urogenital/uriniferous papilla on the branch leading to the Colubroidea + Elapoidea with subsequent losses of the ampulla urogenital/uriniferous papilla in the Elapoidea and Colubroidea. The presence of the ampullae urogenital/uriniferous papilla in only the Colubroidea and Elapoidea highlights the affinity of these two taxonomic groups, a relationship that is strongly supported in published cladograms produced with molecular datasets.     

Comparative histology of the vaginal–cloacal region in Squamata and its phylogenetic implications

We described the histology and morphology of the vaginal–cloacal region in 18 species from 12 Squamata families. This comparative study revealed a wide variation in the cloacal morphology. Fifteen morphological characters were considered to be primary homology hypotheses and were optimized over the topology derived from the parsimony analysis of the available soft morphological evidence, including the characters described in this study. The synapomorphies optimized for Squamata are bifid urodaeum, common urodaeal cavity with similar histological features of the urodaeal horns, and presence of glands in the anterior urodaeum; for Scleroglossa the synapomorphy is the lateral position of the vaginal intrusion into the anterior urodaeal chamber, for Nyctisaura + Scincomorpha the synapomorphy is the presence of a bifid posterior urodaeum; and for Xantusidae + Annulata it is the presence of simple glands in the anterior urodaeum. The central position of the vaginal intrusion into the urodaeal chamber and the intraepithelial position of the glandular unit in the anterior urodaeum behave as autapomorphies for Iguanidae. This study contributes evidence that defines the relationships within Scleroglossa. Cloacal features provide interesting information that is useful as a source of morphological characters for phylogenetic studies in Squamata.     

Caudal oviduct coiling in a viperid snake,Crotalus durissus

Sperm storage is common in the oviducts of female snakes and results in the decoupling of mating from ovulation and fertilization. In the majority of female snakes examined, sperm storage occurs in receptacles of the infundibular regions of the oviducts. In pitvipers (Viperida, Crotalinae), the storage of sperm was described in the caudal regions of the oviducts (utero-vaginal junction) through a mechanism termed uterine muscular twisting (UMT). Uterine muscular twisting was described as a twisting of the oviducts after copulation because of uterine contractions. The twisting remains until ovulation at which time the oviducts straighten and sperm migrate cranially to fertilize ovulated ova. Here, we demonstrate that the UMT is not formed by twisting (rotation around axis) of the oviducts of Crotalus durissus but rather coils formed by the inner layers of the oviducts at the utero-vaginal junction. Contrary to previous findings, coiling of the oviducts is present in females throughout the year, not only in the postcopulatory period; however, the degree of coiling is variable and may be linked to the seasonal reproductive cycle of C. durissus. We categorize the degree of coiling as pronounced coil, discreet coil or absent coil.     

Wing morphology,winter ecology,and fecundity selection: evidence for sex-dependence in barn swallows (<Emphasis Type="Italic">Hirundo rustica</Emphasis>)

Variation in wing morphology results from the combination of diverse selection pressures. Wing feather morphology within species varies with sex and ontogenetic effects, and also with ecological factors. Yet, the direction of causation for the wing morphology–ecology association remains to be elucidated. Under the ‘ecology-dependence’ hypothesis, wing morphology covaries with ecological conditions, because the latter affect feather molt. Alternatively, the ‘habitat choice’ hypothesis posits that individuals with different wing morphology choose different habitats because of the habitat-dependent advantages of a specific wing morphology. We tested these competing hypotheses in the migratory, aerially insectivorous barn swallow (Hirundo rustica). We quantified wing morphology (isometric size, pointedness, and convexity) on the same individuals during consecutive breeding seasons (i.e., before and after molt in sub-Saharan wintering areas) and located wintering areas using light-level geolocators. Wing pointedness of females but not males during 1 year negatively correlated with vegetation vigor (gauged by the Normalized Difference Vegetation Index; NDVI) in the African area where individuals spent the next winter. Partial least-squares path modelling showed that the association between wing morphology and NDVI was sex-dependent. Conversely, NDVI during wintering did not predict wing morphology in the next breeding season. Because wing morphology can have carry-over effects on subsequent performance, we investigated selection on wing traits and found strong positive fecundity selection on wing size of females. Our results suggest that female barn swallows choose their wintering habitat depending on their wing morphology. In addition, directional fecundity selection operates on females, suggesting sex-dependence of current selection on the flight apparatus.     

The role of ecology,neutral processes and antagonistic coevolution in an apparent sexual arms race

Some of the strongest examples of a sexual ‘arms race’ come from observations of correlated evolution in sexually antagonistic traits among populations. However, it remains unclear whether these cases truly represent sexually antagonistic coevolution; alternatively, ecological or neutral processes might also drive correlated evolution. To investigate these alternatives, we evaluated the contributions of intersex genetic correlations, ecological context, neutral genetic divergence and sexual coevolution in the correlated evolution of antagonistic traits among populations of Gerris incognitus water striders. We could not detect intersex genetic correlations for these sexually antagonistic traits. Ecological variation was related to population variation in the key female antagonistic trait (spine length, a defence against males), as well as body size. Nevertheless, population covariation between sexually antagonistic traits remained substantial and significant even after accounting for all of these processes. Our results therefore provide strong evidence for a contemporary sexual arms race.     

Recent rapid speciation and ecomorph divergence in Indo‐Australian sea snakes

The viviparous sea snakes (Hydrophiinae) are a young radiation of at least 62 species that display spectacular morphological diversity and high levels of local sympatry. To shed light on the mechanisms underlying sea snake diversification, we investigated recent speciation and eco‐morphological differentiation in a clade of four nominal species with overlapping ranges in Southeast Asia and Australia. Analyses of morphology and stomach contents identified the presence of two distinct ecomorphs: a ‘macrocephalic’ ecomorph that reaches >2 m in length, has a large head and feeds on crevice‐dwelling eels and gobies; and a ‘microcephalic’ ecomorph that rarely exceeds 1 m in length, has a small head and narrow fore‐body and hunts snake eels in burrows. Mitochondrial sequences show a lack of reciprocal monophyly between ecomorphs and among putative species. However, individual assignment based on newly developed microsatellites separated co‐distributed specimens into four significantly differentiated clusters corresponding to morphological species designations, indicating limited recent gene flow and progress towards speciation. A coalescent species tree (based on mitochondrial and nuclear sequences) and isolation‐migration model (mitochondrial and microsatellite markers) suggest between one and three transitions between ecomorphs within the last approximately 1.2 million to approximately 840 000 years. In particular, the macrocephalic ‘eastern’ population of Hydrophis cyanocinctus and microcephalic H. melanocephalus appear to have diverged very recently and rapidly, resulting in major phenotypic differences and restriction of gene flow in sympatry. These results highlight the viviparous sea snakes as a promising system for speciation studies in the marine environment.     

Geographic variation in mimetic precision among different species of coral snake mimics

Batesian mimicry is widespread, but whether and why different species of mimics vary geographically in resemblance to their model is unclear. We characterized geographic variation in mimetic precision among four Batesian mimics of coral snakes. Each mimic occurs where its model is abundant (i.e. in ‘deep sympatry’), rare (i.e. at the sympatry/allopatry boundary or ‘edge sympatry’) and absent (i.e. in allopatry). Geographic variation in mimetic precision was qualitatively different among these mimics. In one mimic, the most precise individuals occurred in edge sympatry; in another, they occurred in deep sympatry; in the third, they occurred in allopatry; and in the fourth, precise mimics were not concentrated anywhere throughout their range. Mimicry was less precise in allopatry than in sympatry in only two mimics. We present several nonmutually exclusive hypotheses for these patterns. Generally, examining geographic variation in mimetic precision – within and among different mimics – offers novel insights into the causes and consequences of mimicry.     

Hidden support from unpromising data sets strongly unites snakes with anguimorph ‘lizards’

A combined analysis of nuclear, mitochondrial and morphological data robustly resolves snakes as the sister taxon to anguimorph ‘lizards’. Analysed in isolation, nuclear DNA (nDNA) produces a trichotomy between snakes, iguanians and anguimorphs, mitochondrial DNA (mtDNA) is largely uninformative at deeper levels, and morphology tends to nest snakes deep within anguimorphs or with various legless squamate groups. When analysed simultaneously, the nuclear signal is sufficiently strong that mtDNA and morphology are constrained to choose between alternative resolutions of the iguanian–anguimorph–snake trichotomy (generated by the nDNA) – and both support the snake–anguimorph solution. Combined analyses of fast‐evolving or idiosyncratically evolving markers (mtNDA, morphology) with conservative traits (e.g. nuclear genes) might be the best way to resolve ancient, closely spaced divergences. Fast or idiosyncratic markers potentially provide the most information about short, ancient internodes, but can converge on spurious trees if analysed in isolation. However, if constrained to only choosing between plausible trees, such data can contribute unique and valuable phylogenetic signal that resolves such problematic divergences.     

Bergmann's and Rensch's rules and the spur‐thighed tortoise (Testudo graeca)

Body size is an ecologically important variable in animals. The geographical size variation of most snakes and some lizards counters Bergmann's rule in that, among related taxa, the larger ones live at warmer latitudes. However, exceptions notwithstanding, and despite being ectothermic, turtles as a group tend to obey Bergmann's rule. We examined this idea in Testudo graeca, ranging from Morocco to Romania and to Iran with disputed systematics, both at the global scale (using literature) and within the focal area of Israel (using museum specimens). Both globally and locally, carapace length correlated with latitude, in accordance with Bergmann's rule. The scant data on reproduction fully support the hypothesis that Bergmann's rule enables larger clutches where the climate would limit repeated clutches. The sexual size dimorphism (SSD) was approached using two methodologies: (1) ‘conventional’, using globally literature data and locally museum samples and (2) ‘innovated’, using photographs of copulating tortoises from Israel and Turkey. By each methodology, SSD emerged as being male biased in the larger‐bodied populations and female biased in the smaller‐bodied populations, obeying Rensch's rule. Some observations support the hypothesis that the evolution of large males serves intermale combating. Finally, Rensch's rule was found to apply separately within Anatolia and within the Levant, possibly indicating that these populations are separate.     

Morphology and evolution of the snake cornea

To investigate whether the thickness of the cornea in snakes correlates with overall anatomy, habitat or daily activity pattern, we measured corneal thickness using optical coherence tomography scanning in 44 species from 14 families (214 specimens) in the collection at the Natural History Museum (Denmark). Specifically, we analyzed whether the thickness of the cornea varies among species in absolute terms and relative to morphometrics, such as body length, spectacle diameter, and spectacle thickness. Furthermore, we examined whether corneal thickness reflects adaptation to different habitats and/or daily activity patterns. The snakes were defined as arboreal (n = 8), terrestrial (n = 22), fossorial (n = 7), and aquatic (n = 7); 14 species were classified as diurnal and 30 as nocturnal. We reveal that the interspecific variation in corneal thickness is largely explained by differences in body size, but find a tendency towards thicker corneas in diurnal (313 ± 227 μm) compared to nocturnal species (205 ± 169 μm). Furthermore, arboreal snakes had the thickest corneas and fossorial snakes the thinnest. Our study shows that body length, habitat, and daily activity pattern could explain the interspecific variation in corneal morphology among snakes. This study provides a quantitative analysis of the evolution of the corneal morphology in snakes, and it presents baseline values of corneal thickness of multiple snake species. We speculate that the cornea likely plays a role in snake vision, despite the fact that results from previous studies suggest that the cornea in snakes is not relevant for vision (Sivak, Vision Research, 1977, 17, 293–298).     

Snake Population Venomics: Proteomics-Based Analyses of Individual Variation Reveals Significant Gene Regulation Effects on Venom Protein Expression in <Emphasis Type="Italic">Sistrurus</Emphasis> Rattlesnakes

Studies of the molecular basis of adaptations seek to understand the relative importance of structural changes in proteins versus gene regulation effects as determinants of phenotype. Amino acid substitutions in gene coding sequences are well documented as causes of variation in snake venom proteins, whereas the importance of gene regulation effects on venom protein abundance and composition is less well known. Here, we use a proteomics-based approach to infer the effects of gene regulation on protein expression by comparing the relative abundance of specific, known venom proteins among different individuals in each of two species of Sistrurus rattlesnakes. Variation in the presence or absence, and in the relative amounts, of proteins was high in both species across all major protein families. Based on our empirical criteria for inferring regulatory effects (presence-absence of specific proteins and/or more than threefold variation in abundance) between 51% and 83% of S. catenatus individuals and between 40% and 63% of S. miliarius individuals showed evidence for gene regulation across the four most abundant proteins (disintegrins, phospholipase A₂’s, serine proteinases, and snake venom metalloproteases). Thus, the effects of gene regulation should be considered an important cause of variation in the composition of whole venoms at the intraspecific level. They also suggest the need for testing the adaptive hypothesis for venom plasticity in relation to prey consumed by adult snakes. Finally, the venom variability reported may have an impact in the treatment of bite victims, highlighting the necessity of using pooled venoms as a substrate for antivenom production.     

Plasticity of the mate choice mind: courtship evokes choice‐like brain responses in females from a coercive mating system

Female mate choice is fundamental to sexual selection, and determining molecular underpinnings of female preference variation is important for understanding mating character evolution. Previously it was shown that whole‐brain expression of a synaptic plasticity marker, neuroserpin, positively correlates with mating bias in the female choice poeciliid, Xiphophorus nigrensis, when exposed to conspecific courting males, whereas this relationship is reversed in Gambusia affinis, a mate coercive poeciliid with no courting males. Here we explore whether species‐level differences in female behavioral and brain molecular responses represent ‘canalized’ or ‘plastic’ traits. We expose female G. affinis to conspecific males and females, as well as coercive and courting male Poecilia latipinna, for preference assays followed by whole‐brain gene expression analyses of neuroserpin, egr‐1 and early B. We find positive correlations between gene expression and female preference strength during exposure to courting heterospecific males, but a reversed pattern following exposure to coercive heterospecific males. This suggests that the neuromolecular processes associated with female preference behavior are plastic and responsive to different male phenotypes (courting or coercive) rather than a canalized response linked to mating system. Further, we propose that female behavioral plasticity may involve learning because female association patterns shifted with experience. Compared to younger females, we found larger, more experienced females spend less time near coercive males but associate more with males in the presence of courters. We thus suggest a conserved learning‐based neuromolecular process underlying the diversity of female mate preference across the mate choice and coercion‐driven mating systems.     

Predatory Attacks to the Head vs. Body Modify Behavioral Responses of Garter Snakes

An animal's response to predatory attack may depend upon which part of its body is the focus of that attack, because of differential vulnerability to injury. Many avian and mammalian predators direct attacks preferentially toward the prey's head, so simulated attacks that do not have this focus may elicit non‐natural responses. We ‘pecked’ 152 free‐ranging adult male garter snakes (Thamnophis sirtalis parietalis) in Manitoba either on the head or the midbody, and recorded their responses. The snakes’ antipredator tactics were affected not only by body size (larger snakes performed threat displays more often) and body temperature (hotter snakes were more likely to flee), but also by location of the attack. Pecks to the head generally resulted in snakes coiling and hiding their heads, often simultaneously elevating and wriggling their tails in an apparent distraction display. In contrast, pecks to the midbody stimulated either escape responses, or (in snakes that did not flee) open‐mouthed threat displays. More generally, antipredator tactics may respond in flexible ways to details of the predator–prey encounter (including attributes of the habitat as well as the morphology and behavior of both participants) and hence, experimental studies need to carefully simulate such details. The part of the body under attack may be an important factor in this respect.     

Phenotypically plastic responses to predation threat in the mangrove rivulus fish (<Emphasis Type="Italic">Kryptolebias marmoratus</Emphasis>): behavior and morphology

Early life environments have important effects on phenotype development, but it can be difficult to disentangle the relative influences of genotype and environment on phenotypic variation within and among populations. Mangrove rivulus fish (Kryptolebias marmoratus) reproduce by self-fertilization and can generate isogenic lineages, which provides opportunities to resolve how the environment shapes the phenotype independent of genetic variation. Rivulus’ ecology is not well understood, but mangrove water snakes (Nerodia clarkii compressicauda) are thought to be a major predator. To test developmental responses to predator-related cues, four rivulus lineages (two that naturally co-exist with snakes; two that do not) were exposed to one of three treatments for 30 days post-hatching: cues from snakes that were fasted, fed rivulus, or fed heterospecifics. One week after exposure, fear and boldness responses were quantified. Individuals were photographed at 2 and 6 months of age for body size, growth, and body shape analysis. Animals that have historically encountered snakes were more risk averse and had wider heads than animals that historically have not encountered snakes. Rivulus exposed to cues from snakes fed conspecifics or heterospecifics grew faster than those exposed to fasted snake cues. Body shape was more streamlined in animals exposed to cues from snakes fed conspecifics, which may facilitate increased jumping performance as a way to escape aquatic predators. Our results suggest that rivulus exhibit phenotypic plasticity in response to cues associated with predator threat and that historical effects from selection or other evolutionary processes also are important determinants of behavioral and morphological variation.     

Seasonal studies on the alginate and its biochemical composition I: Sargassum polycystum (Fucales), Phaeophyceae

Investigations were made on the brown seaweed Sargassum polycystum C. Agardh collected from Rameswaram Coast, Tamil Nadu. The alginates extracted from ‘leaf’, ‘stem’ and entire thallus of S. polycystum were investigated for their viscosity and chemical constituents, namely β‐D‐mannuronic acid (M‐block), α‐L‐guluronic acid (G‐block) and alternating sequences of β‐D‐mannuronic acid and α‐L‐guluronic acid (MG‐block) for six different seasons between August 1998 and November 1999. Significant seasonal variation (P< 0.05) was observed with high yield of alginate in February. The alginate extracted from the ‘leaf’ region showed a maximum yield whereas the ‘stem’ region exhibited maximum viscosity. The amount of G‐block was found to be more than M‐ and MG‐blocks in all the samples tested. The amount of G‐block was high in ‘stem’ followed by leaf and entire thallus. A positive correlation was recorded between viscosity and G‐block. Among the three alginates, the ratio of M/G was low in the ‘stem’ followed by ‘leaf’ and entire thallus.     

Seasonal variation in hormonal responses of timber rattlesnakes (<Emphasis Type="Italic">Crotalus horridus</Emphasis>) to reproductive and environmental stressors

Data addressing adrenocortical modulation across taxonomic groups are limited, especially with regard to how female reproductive condition influences the sensitivity of the hypothalamus–pituitary–adrenal axis. We investigated seasonal and reproductive variation in basal and stress-induced hormone profiles in a population of free-ranging timber rattlesnakes (Crotalus horridus) in north-central Pennsylvania during spring (i.e., May), summer (i.e., July), and early fall (i.e., September). Baseline corticosterone concentrations varied seasonally and were significantly lower during the summer sampling period in July. We observed a significant negative relationship between baseline corticosterone and testosterone in male snakes, while baseline corticosterone and estradiol tended to be positively correlated in females. Treatment of snakes with 1 h of capture stress significantly increased corticosterone across all seasons. However, there was a significant interaction between corticosterone responses to capture stress and season, suggesting that adrenocortical function is modulated seasonally. Because elevated corticosterone may be associated with reproduction, we asked whether hormonal stress responses vary with female reproductive condition. Although sample sizes are low, reproductive snakes had significantly higher baseline and stress-induced corticosterone concentrations than non-reproductive or post-parturient females. Further, despite similar baseline corticosterone concentrations between non-reproductive and post-parturient rattlesnakes, post-parturient females responded to capture stress with a significantly higher increase in corticosterone. Collectively, these data suggest that the sensitivity of the hypothalamus–pituitary–adrenal axis varies both seasonally and with changing reproductive states.     

Integrating phylogeny,environment and space to explore variation in macroecological traits of Viperidae and Elapidae (Squamata: Serpentes)

We used eigenvector mapping in space and phylogeny to investigate the relationships among space, phylogeny and environment on body size and range size variation across two groups of venomous snakes – Viperidae and Elapidae – from the New World. Data on species geographic range sizes, maximum body sizes and phylogenetic relationships were compiled from the available literature. The distributional data were also used to calculate the latitudinal and longitudinal midpoint and the environmental centroids for each species. The eigenvectors extracted from the pair wise spatial and phylogenetic distance matrices were integrated with environmental variables into a method of variation partitioning where the variation in each trait was quantitatively attributed to ‘pure’ and/or shared effects of phylogeny, environment and space. Our results showed that variation in body size was predominantly determined by phylogeny in both groups of snakes. For Viperidae, we found that pure ‘effects’ of phylogeny were the strongest, indicating that most of the body size evolution that was phylogenetically determined in this group occurred independently of environment and geographical proximity. Regarding range sizes, pure phylogenetic influences were very low in both groups, whereas the largest single fraction of explained variation corresponded to overlapped influences of the three sets of predictors, especially for Elapidae. Along with this, we found evidence that niche conservatism is an important processes underlying variation in body size and range size in both groups of snakes.     

From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences,morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera)

Tan, D. S. H., Ang, Y., Lim, G. S., Ismail, M. R. B. & Meier, R. (2010). From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). —Zoologica Scripta, 39, 51–61. The increased availability of DNA sequences has led to a surge of ‘cryptic species’ in the literature. These units are usually proposed based on finding genetically distinct lineages within species that were initially defined based on morphological characters. However, few authors attempt to confirm whether these ‘cryptic’ units are species and even fewer authors are explicit about which species concept is applied. Here, we use an example from Sepsidae (Diptera) to demonstrate how cryptic species can be validated by an iterative process involving several data sources and an evaluation of the data under different species concepts. A phylogeographic analysis based on 50 specimens for five species of the flavimana group revealed deep mitochondrial splits within Sepsis flavimana which was suggestive of a cryptic species. We resolve the initial conflict between DNA sequences and morphology by adding new morphological data as well as behavioural evidence and tests for reproductive isolation. One cryptic species is confirmed and Sepsis pyrrhosoma, a former synonym of S. flavimana, is here shown to be a valid species under most species concepts. We can thus document that the same data can lead to similar conclusions under conflicting concepts once different kinds of data are integrated.     

Associations between physical isolation and geographical variation within three species of Neotropical birds

We studied effects of physical isolation on geographical variation in mtDNA RFLP polymorphisms and a suite of morphological characters within three species of neotropical forest birds; the crimson-backed tanager Ramphocelus dimidiatus, the blue-gray tanager Thraupis episcopus, and the streaked saltator Saltator albicollis. Variation among populations within continuous habitat on the Isthmus of Panama was compared with that among island populations isolated for about 10000 years. Putative barriers to dispersal were influential, but apparent isolation effects varied by species, geographical scale, and whether molecular or morphological traits were being assessed. We found no geographical structuring among the contiguous, mainland sampling sites. Migration rates among the islands appeared sufficient to maintain homogeneity in mtDNA haplotype frequencies. In contrast, variation in external morphology among islands was significant within two of three species. For all species, we found significant variation in genetic and morphological traits between the island (collectively) and mainland populations. Interspecific variation in the effects of isolation was likely related to differential vagility. These data generally corroborate other studies reporting relatively great geographical structuring within tropical birds over short distances. Behaviourally based traits - low vagility and high ‘sensitivity’ to geographical barriers - may underlie extensive diversification within neotropical forest birds, but more extensive ecological and phylogeographic information are needed on a diverse sample of species.