Musculoskeletal morphogenesis supports the convergent evolution of bat laryngeal echolocation.

The order Chiroptera (bats) is the second largest group of mammals. One of the essential adaptations that have allowed bats to dominate the night skies is laryngeal echolocation, where bats emit ultrasonic pulses and listen to the returned echo to produce high-resolution 'images' of their surroundings. There are two possible scenarios for the evolutionary origin of laryngeal echolocation in bats: (1) a single origin in a common ancestor followed by the secondary loss in Pteropodidae, or (2) two convergent origins in Rhinolophoidea and Yangochiroptera. Although data from palaeontological, anatomical, developmental and genomic studies of auditory apparatuses exist, they remain inconclusive concerning the evolutionary origin of bat laryngeal echolocation. Here we compared musculoskeletal morphogenesis of the larynx in several chiropteran lineages and found distinct laryngeal modifications in two echolocating lineages, rhinolophoids and yangochiropterans. Our findings support the second scenario that rhinolophoids and yangochiropterans convergently evolved advanced laryngeal echolocation through anatomical modifications of the larynx for ultrasonic sound generation and refinement of the auditory apparatuses for more detailed sound perception.

Creating morphological diversity in reptilian temporal skull region: A review of potential developmental mechanisms.

Reptilian skull morphology is highly diverse and broadly categorized into three categories based on the number and position of the temporal fenestrations: anapsid, synapsid, and diapsid. According to recent phylogenetic analysis, temporal fenestrations evolved twice independently in amniotes, once in Synapsida and once in Diapsida. Although functional aspects underlying the evolution of tetrapod temporal fenestrations have been well investigated, few studies have investigated the developmental mechanisms responsible for differences in the pattern of temporal skull region. To determine what these mechanisms might be, we first examined how the five temporal bones develop by comparing embryonic cranial osteogenesis between representative extant reptilian species. The pattern of temporal skull region may depend on differences in temporal bone growth rate and growth direction during ontogeny. Next, we compared the histogenesis patterns and the expression of two key osteogenic genes, Runx2 and Msx2, in the temporal region of the representative reptilian embryos. Our comparative analyses suggest that the embryonic histological condition of the domain where temporal fenestrations would form predicts temporal skull morphology in adults and regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal skull region of reptiles.

Facial muscle modification associated with chiropteran noseleaf development: Insights into the developmental basis of a movable rostral appendage in mammals.

BACKGROUND: Mammal evolution has generated diverse craniofacial morphologies, including remarkable movable rostral appendages. However, the muscular and skeletal architecture providing the mobility of these appendages remains largely unexplored. Here, we focus on chiropteran noseleaves and compare the three-dimensional internal morphology of late-stage embryos between the greater horseshoe bat Rhinolophus ferrumequinum, which possesses a noseleaf, and the Asian bent-winged bat Miniopterus fuliginosus and Egyptian fruit bat Rousettus aegyptiacus, which do not. We also assess earlier stage cell proliferation within the rostrum to elucidate cellular mechanisms underlying noseleaf-associated morphological modifications. RESULTS: The musculus maxillolabialis inserted into proximal vibrissae follicles in M fuliginosus and R aegyptiacus embryos but instead inserted into the horseshoe plate in R ferrumequinum. This modification suggests that the M maxillolabialis has adapted to controlling the noseleaf rather than vibrissae in rhinolophid bats. Our cellular analysis showed higher cell proliferation within the maxillary and frontonasal processes of St. 14 embryos in R ferrumequinum compared to M fuliginosus and R aegyptiacus, suggesting that the spatial alteration of noseleaf-associated muscle is derived from changes in facial morphogenesis that occur by St. 14. CONCLUSION: This is the first study clarifying the morphological and cellular bases underlying the development of mammalian rostral appendages.

Characterization of the Adaptive Morphology of Japanese Stream Toad (Bufo torrenticola) Using Geometric Morphometrics.

The order Anura (frogs and toads) is a group of amphibians and contains over 6500 extant species living in a variety of environments. Each frog species evolved body form adaptive for living and breeding in their own habitats. In Japan, four taxa of Bufo are living: Western-Japanese common toad (Bufo japonicus japonicus), Eastern-Japanese common toad (B. japonicus formosus), Miyako toad (B. gargarizans miyakonis) and Japanese stream toad (B. torrenticola). The former three taxa breed in still water as many other species of Bufo do. In contrast, B. torrenticola breeds in running water such as streams in mountainous area. Corresponding to their breeding in a stream environment, both adult and larva of B. torrenticola acquired unique morphological characters. However, few study have explored differences in the body form between B. torrenticola and closely-related Bufo species quantitatively, remaining the details about the morphological adaptation to a stream environment in this toad species poorly understood. In this study, we quantitatively compared the shape of the foot and skull between the adult male of B. torrenticola and its close relative B. j. formosus using landmark-based geometric morphometrics. Our analyses revealed that B. torrenticola has relatively longer toe phalanges with relatively larger foot webs and relatively shorter metatarsals and a narrower and more streamlined skull, compared to closely-related B. j. formosus. These morphological characteristics are considered adaptive for their breeding in mountain torrents.

The oestrogen pathway underlies the evolution of exaggerated male cranial shapes in Anolis lizards.

Sexual dimorphisms vary widely among species. This variation must arise through sex-specific evolutionary modifications to developmental processes. Anolis lizards vary extensively in their expression of cranial dimorphism. Compared with other Anolis species, members of the carolinensis clade have evolved relatively high levels of cranial dimorphism; males of this clade have exceptionally long faces relative to conspecific females. Developmentally, this facial length dimorphism arises through an evolutionarily novel, clade-specific strategy. Our analyses herein reveal that sex-specific regulation of the oestrogen pathway underlies evolution of this exaggerated male phenotype, rather than the androgen or insulin growth factor pathways that have long been considered the primary regulators of male-biased dimorphism among vertebrates. Our results suggest greater intricacy in the genetic mechanisms that underlie sexual dimorphisms than previously appreciated.

Molecular and cellular changes associated with the evolution of novel jaw muscles in parrots.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfß2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

Turtle skull development unveils a molecular basis for amniote cranial diversity.

Amniote skulls display diverse architectural patterns including remarkable variations in the number of temporal arches surrounding the upper and lower temporal fenestrae. However, the cellular and molecular basis underlying this diversification remains elusive. Turtles are a useful model to understand skull diversity due to the presence of secondarily closed temporal fenestrae and different extents of temporal emarginations (marginal reduction of dermal bones). Here, we analyzed embryos of three turtle species with varying degrees of temporal emargination and identified shared widespread coexpression of upstream osteogenic genes Msx2 and Runx2 and species-specific expression of more downstream osteogenic genes Sp7 and Sparc in the head. Further analysis of representative amniote embryos revealed differential expression patterns of osteogenic genes in the temporal region, suggesting that the spatiotemporal regulation of Msx2, Runx2, and Sp7 distinguishes the temporal skull morphology among amniotes. Moreover, the presence of Msx2- and/or Runx2-positive temporal mesenchyme with osteogenic potential may have contributed to their extremely diverse cranial morphology in reptiles.

Mesenchymal and mechanical mechanisms of secondary cartilage induction.

Secondary cartilage occurs at articulations, sutures, and muscle attachments, and facilitates proper kinetic movement of the skeleton. Secondary cartilage requires mechanical stimulation for its induction and maintenance, and accordingly, its evolutionary presence or absence reflects species-specific variation in functional anatomy. Avians illustrate this point well. In conjunction with their distinct adult mode of feeding via levered straining, duck develop a pronounced secondary cartilage at the insertion (i.e., enthesis) of the mandibular adductor muscles on the lower jaw skeleton. An equivalent cartilage is absent in quail, which peck at their food. We hypothesized that species-specific pattern and a concomitant dissimilarity in the local mechanical environment promote secondary chondrogenesis in the mandibular adductor enthesis of duck versus quail. To test our hypothesis we employed two experimental approaches. First, we transplanted neural crest mesenchyme (NCM) from quail into duck, which produced chimeric "quck" with a jaw complex resembling that of quail, including an absence of enthesis secondary cartilage. Second, we modified the mechanical environment in embryonic duck by paralyzing skeletal muscles, and by blocking the ability of NCM to support mechanotransduction through stretch-activated ion channels. Paralysis inhibited secondary cartilage, as evidenced by changes in histology and expression of genes that affect chondrogenesis, including members of the FGF and BMP pathways. Ion channel inhibition did not alter enthesis secondary cartilage but caused bone to form in place of secondary cartilage at articulations. Thus, our study reveals that enthesis secondary cartilage forms through mechanisms that are distinct from those regulating other secondary cartilage. We conclude that by directing the musculoskeletal patterning and integration of the jaw complex, NCM modulates the mechanical forces and molecular signals necessary to control secondary cartilage formation during development and evolution.

Sex identification in embryos and adults of Darwin's finches.

Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.

Developmental origins of species-specific muscle pattern.

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.

The mitochondrial phylogeny of an ancient lineage of ray-finned fishes (Polypteridae) with implications for the evolution of body elongation, pelvic fin loss, and craniofacial morphology in Osteichthyes.

BACKGROUND: The family Polypteridae, commonly known as "bichirs", is a lineage that diverged early in the evolutionary history of Actinopterygii (ray-finned fish), but has been the subject of far less evolutionary study than other members of that clade. Uncovering patterns of morphological change within Polypteridae provides an important opportunity to evaluate if the mechanisms underlying morphological evolution are shared among actinoptyerygians, and in fact, perhaps the entire osteichthyan (bony fish and tetrapods) tree of life. However, the greatest impediment to elucidating these patterns is the lack of a well-resolved, highly-supported phylogenetic tree of Polypteridae. In fact, the interrelationships of polypterid species have never been subject to molecular phylogenetic analysis. Here, we infer the first molecular phylogeny of bichirs, including all 12 recognized species and multiple subspecies using Bayesian analyses of 16S and cyt-b mtDNA. We use this mitochondrial phylogeny, ancestral state reconstruction, and geometric morphometrics to test whether patterns of morphological evolution, including the evolution of body elongation, pelvic fin reduction, and craniofacial morphology, are shared throughout the osteichthyan tree of life. RESULTS: Our molecular phylogeny reveals 1) a basal divergence between Erpetoichthys and Polypterus, 2) polyphyly of P. endlicheri and P. palmas, and thus 3) the current taxonomy of Polypteridae masks its underlying genetic diversity. Ancestral state reconstructions suggest that pelvic fins were lost independently in Erpetoichthys, and unambiguously estimate multiple independent derivations of body elongation and shortening. Our mitochondrial phylogeny suggested species that have lower jaw protrusion and up-righted orbit are closely related to each other, indicating a single transformation of craniofacial morphology. CONCLUSION: The mitochondrial phylogeny of polypterid fish provides a strongly-supported phylogenetic framework for future comparative evolutionary, physiological, ecological, and genetic analyses. Indeed, ancestral reconstruction and geometric morphometric analyses revealed that the patterns of morphological evolution in Polypteridae are similar to those seen in other osteichthyans, thus implying the underlying genetic and developmental mechanisms responsible for those patterns were established early in the evolutionary history of Osteichthyes. We propose developmental and genetic mechanisms to be tested under the light of this new phylogenetic framework.

Development of the pseudothumb in frogs.

Frogs have highly conserved hand and foot morphology, possessing four fingers and five toes. As an exception, two Japanese ranid frog species, the Otton frog Babina subaspera and the dagger frog Babina holsti, possess a unique thumb-like structure (the pseudothumb) in the forelimb, giving an appearance of a total of five fingers on the hand. To obtain insights into the developmental mechanisms that generate this novel character, we investigated the hand morphogenesis of the Otton frog. The unique morphological pattern of the pseudothumb was already established in juveniles. Surprisingly, the bud-like structure, which is similar to the area of inductive activity (e.g. feather buds in birds and the carapacial ridge in turtles), was detected over the site where the future prepollex develops in larvae. By contrast, this bud-like structure was not found in larvae of other ranid species. We discuss possible scenarios that would favour the evolution of this very unusual trait in frogs.

Developmental mechanisms underlying webbed foot morphological diversity in waterbirds.

The webbed feet of waterbirds are morphologically diverse and classified into four types: the palmate foot, semipalmate foot, totipalmate foot, and lobate foot. To understand the developmental mechanisms underlying this morphological diversity, we conducted a series of comparative analyses. Ancestral state reconstruction based on phylogeny assumed that the lobate feet possessed by the common coot and little grebe arose independently, perhaps through distinct developmental mechanisms. Gremlin1, which encodes a bone morphogenetic protein (BMP) antagonist and inhibits interdigital cell death (ICD) in the foot plate of avian embryos, remained expressed in the interdigital tissues of webbed feet in the duck, common coot, little grebe, and great cormorant. Differences in Gremlin1 expression pattern and proliferating cell distribution pattern in the toe tissues of the common coot and little grebe support the convergent evolution of lobate feet. In the totipalmate-footed great cormorant, Gremlin1 was expressed in all interdigital tissues at St. 31, but its expression disappeared except along the toes by St. 33. The webbing of the cormorant's totipalmate foot and duck's palmate foot may have risen from distinct developmental mechanisms.

Normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, with special reference to nose leaf formation.

The order Chiroptera (bats) is the second largest group of mammals, composed of more than 1,300 species. Although powered flight and echolocation in bats have attracted many biologists, diversity in bat facial morphology has been almost neglected. Some bat species have a "nose leaf," a leaf-like epithelial appendage around their nostrils. The nose leaf appears to have been acquired at least three times independently in bat evolution, and its morphology is highly diverse among bats species. Internal tissue morphology of nose-leaves has been investigated through histological analyses of late-stage fetuses of some bat species possessing the nose leaf. However, the proximate factors that bring about chiropteran nose-leaves have not been identified. As an initial step to address the question above, we describe the normal embryonic development of the greater horseshoe bat Rhinolophus ferrumequinum, and examine development of the tissues associated with their nose leaf during embryogenesis through histological analyses. We found that the nose leaf of R. ferrumequinum is formed through two phases. First, the primordium of the nose leaf appears as two tissue bulges aligned top and bottom on the face at embryonic stages 15-16. Second, the sub-regions of the nose leaf are differentiated through ingrowth as well as outgrowth of the epithelium at stage 17. In embryogenesis of Carollia perspicillata, a phyllostomid species with a nose leaf, the nose leaf primordium is formed as a small tissue bulge on the nostril at stage 17. This tissue bulge grows into a dorsally projected thin epithelial structure. Such differences in the nose leaf developmental process between chiropteran lineages may suggest that distinct developmental mechanisms have been employed in each lineage's nose leaf evolution.

The evolution of female mole ovotestes evidences high plasticity of mammalian gonad development.

Previous studies of the reproductive biology and genetics of European moles (Talpa spp.) showed that all females of these species have ovotestes (gonads with testicular and ovarian tissue) instead of normal ovaries, a unique specialization among mammals. Females are fertile as their ovarian tissue is fully functional. Testicular tissue is abnormal and sterile, but produces high levels of testosterone. This phenomenon also characterizes other talpid species from Europe and North America. To study the origin of this singular reproductive specialization, we examined the gonads of several female specimens belonging to two critical taxa. Although large Japanese moles (Mogera wogura) posses ovotestes, greater Japanese shrew moles (Urotrichus talpoides) are characterized by normal ovaries. The results fit parsimoniously with a recent phylogenetic study that places Urotrichus relatively basal in the talpid tree and separate from the American shrew mole. Parsimony reconstruction on alternative phylogenetic hypotheses clearly indicates that reversal(s) must have occurred and suggests that a relatively simple genetic mechanism must be associated with the evolution of female hermaphroditism in moles.

Creating diversity in mammalian facial morphology: a review of potential developmental mechanisms.

Mammals (class Mammalia) have evolved diverse craniofacial morphology to adapt to a wide range of ecological niches. However, the genetic and developmental mechanisms underlying the diversification of mammalian craniofacial morphology remain largely unknown. In this paper, we focus on the facial length and orofacial clefts of mammals and deduce potential mechanisms that produced diversity in mammalian facial morphology. Small-scale changes in facial morphology from the common ancestor, such as slight changes in facial length and the evolution of the midline cleft in some lineages of bats, could be attributed to heterochrony in facial bone ossification. In contrast, large-scale changes of facial morphology from the common ancestor, such as a truncated, widened face as well as the evolution of the bilateral cleft possessed by some bat species, could be brought about by changes in growth and patterning of the facial primordium (the facial processes) at the early stages of embryogenesis.

Allometry and integration do not strongly constrain beak shape evolution in large-billed (Corvus macrorhynchos) and carrion crows (Corvus corone).

A recent geometric morphometric study on certain landbird lineages revealed that a major part of the variation in beak shape is accounted for by skull size and cranial shape. The study interpreted this result as evidence for the presence of strong evolutionary constraints that severely prevented beak shape from evolving substantially away from predictions of allometry and morphological integration. However, there is another overlooked but similarly plausible explanation for this result: The reason why beak shape does not depart much from predictions might simply be that selection pressures favoring such changes in shape are themselves rare. Here, to evaluate the intensity of evolutionary constraints on avian beak shape more appropriately, we selected large-billed (Corvus macrorhynchos) and carrion crows (Corvus corone) as study objects. These landbird species seem to experience selection pressures favoring a departure from an allometric trajectory. A landmark-based geometric morphometric approach using three-dimensional reconstructions of CT scan images revealed that only 45.4% of the total shape variation was explained by allometry and beak-braincase integration. This suggests that when a selection pressure acts in a different direction to allometry and integration, avian beak shape can react to it and evolve flexibly. As traditionally considered, evolutionary constraints on avian beak shape might not be all that strong.

Cranial shape evolution in adaptive radiations of birds: comparative morphometrics of Darwin's finches and Hawaiian honeycreepers.

Adaptive radiation is the rapid evolution of morphologically and ecologically diverse species from a single ancestor. The two classic examples of adaptive radiation are Darwin's finches and the Hawaiian honeycreepers, which evolved remarkable levels of adaptive cranial morphological variation. To gain new insights into the nature of their diversification, we performed comparative three-dimensional geometric morphometric analyses based on X-ray microcomputed tomography (µCT) scanning of dried cranial skeletons. We show that cranial shapes in both Hawaiian honeycreepers and Coerebinae (Darwin's finches and their close relatives) are much more diverse than in their respective outgroups, but Hawaiian honeycreepers as a group display the highest diversity and disparity of all other bird groups studied. We also report a significant contribution of allometry to skull shape variation, and distinct patterns of evolutionary change in skull morphology in the two lineages of songbirds that underwent adaptive radiation on oceanic islands. These findings help to better understand the nature of adaptive radiations in general and provide a foundation for future investigations on the developmental and molecular mechanisms underlying diversification of these morphologically distinguished groups of birds.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Cranial neural crest cell migration in cockatiel Nymphicus hollandicus (Aves: Psittaciformes).

Parrots have developed unique jaw muscles in their evolutionary history. The M. pseudomasseter, which completely covers the lateral side of the jugal bar, is regarded as a jaw muscle unique to parrots. In a previous study, I presented a hypothesis on the relevance of modifications in the regulation of cranial neural crest cell (NCC) development to the generation of this novel jaw muscle based on histological analyses (Tokita [2004] J Morphol 259:69-81). In the present study, I investigated distribution and migration patterns of cranial neural crest cells (NCCs) through parrot embryogenesis with immunohistochemical techniques to further understand the role of cranial NCCs in the evolution of the M. pseudomasseter, and to provide new information on the relative plasticity in cranial NCC migration at early stages of avian development. The basic nature of cranial NCC development was mostly conserved between chick and parrot. In both, cranial NCCs migrated from the dorsal tip of the neural tube in a ventral direction. Three major populations were identified in their cranial NCCs. Migration pathways of these cells were almost identical between chick and parrot. The principal difference was seen in the relative timing of cranial NCC migration. In the parrot, cranial NCC migration into the first pharyngeal arch was more advanced than in the chick at early stages of development. Such a temporal shift in cranial NCC migration might influence architectural patterning of parrot jaw muscles that generates new muscle like M. pseudomasseter.

Normal embryonic development of the Japanese pipistrelle, Pipistrellus abramus.

The normal embryonic development of the Japanese pipistrelle, Pipistrellus abramus was described. A total of 13 stages between the early pharyngula and the embryo just before birth were established, based on external morphological features of 33 embryonic specimens. To aid embryological comparison between chiropteran lineages, the stage numbers used in the Carollia perspicillata embryonic staging system (Cretekos et al., 2005) were applied to each embryonic stage of P. abramus. The size (crown-rump length, CRL) of P. abramus embryos became larger throughout ontogeny at a relatively constant rate. When the CRL of embryos was compared in each morphologically corresponding embryonic stage, the CRL of P. abramus was always smaller than that of C. perspicillata. The overall nature of organogenesis was almost similar in both P. abramus and C. perspicillata. Species-specific differences in bats were observed at later stages of embryogenesis, notably in the craniofacial and tail regions.