The reproductive pattern of male dusky salamanders (genus Desmognathus) is neither associated nor dissociated

Many seasonally breeding vertebrate species have an associated reproductive pattern: mating behavior, gonadal activity, and peak circulating androgen levels occur simultaneously. In these species, androgens influence the expression of male mating behavior. Other species have a dissociated reproductive pattern: mating behavior occurs at a different time than peak gonadal activity. In such species, it is hypothesized that mating behavior is not dependent on androgen levels [Crews, D., 1984. Gamete production, sex hormone secretion, and mating behavior uncoupled. Horm. Behav. 18, 22-28]. The salamander Desmognathus ochrophaeus mates in the spring and fall while spermatogenesis occurs during the summer, suggesting that it has a dissociated reproductive pattern and that androgens do not mediate mating behavior. To assess whether mating behavior is regulated by gonadal androgens, we castrated males to reduce endogenous androgens and implanted testosterone propionate (TP) to restore androgen levels. Castrated males mated significantly less than did control males. Castrated males given TP mated as much as control males. Compared to controls, circulating androgen levels (both testosterone (T) and dihydrotestosterone (DHT)) were reduced in castrated males and elevated in castrated males given TP implants. We also found that plasma corticosterone (CORT) levels were strongly and positively correlated with T levels. Together, these data indicate that, although spermatogenesis is dissociated in time from mating behavior, androgens are associated with the expression of mating. Thus, the associated-dissociated dichotomy does not adequately describe the reproductive pattern of D. ochrophaeus. We discuss the limitations of the associated-dissociated framework in clarifying hormone-behavior relationships in reptiles and amphibians.     

Population differentiation in plethodontid salamanders: divergence of allozymes and sexual compatibility among populations of Desmognathus imitator and D. ochrophaeus (Gaudata: Plethodontidae)

Plethodontid salamanders of the genus Desmognathus exhibit varying levels of genetic differentiation among and within both allopatric and sympatric taxa. This provides excellent opportunities to study population differentiation and speciation. Two morphologically similar species in this genus, D. imitator and D. ochrophaeus, are genetically well-differentiated from one another and occur in sympatry with no evidence of hybridization and introgression. We report that the degree of sexual isolation between these two species is very high, regardless of whether the populations under comparison are allopatric or sympatric with one another. Neither reinforcement nor reproductive character displacement are required to explain the evolution of sexual incompatibility in sympatry. Sexual behaviour apparently diverges while populations are allopatric with one another. Preliminary study indicates that D. imitator consists of populations among which there may be significant sexual isolation in the absence of detectable genetic differentiation.     

Hydric constraints upon foraging in a terrestrial salamander,Desmognathus ochrophaeus (Amphibia: Plethodontidae)

Summary The time until salamanders voluntarily abandoned foraging (the water time limit) and the amount of water lost when salamanders abandoned foraging (dehydration deficit) were determined for terrestrial plethodontid salamanders, Desmognathus ochrophaeus, foraging at various vapor pressure gradients in the laboratory. Salamander activity was correlated with the rate of water loss and was inversely related to the water time limit. Animals at 0.35–0.86 kPa vapor pressure gradients abandoned foraging and returned to moist retreats significantly sooner than animals in water-saturated air. The early retreat of animals in dry air was related in part to high rates of water loss and in part to the modest dehydration deficit (3.8%) at which animals abandoned foraging. Locomotor performance and foraging ability were unaffected by dehydration until dehydration deficits exceeded 12%. This suggests that salamanders in unsaturated air abandoned foraging at a low dehydration deficit to conserve and replenish water reserves rather than to avoid outright incapacitation or death. Present address: Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada     

Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930) salamanders (Caudata,Plethodontidae)

Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the “type” locality (Utinga, Belém, PA, Brazil). Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA₃ fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26). Nucleolar Organizer Regions, evidenced by CMA₃, were located distally on the long arm of pair 7 (7q). DAPI+ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.     

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae)

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.     

The courtship behaviour of the spotted dusky salamander, Desmognathus fuscus conanti (Amphibia: Caudata: Plethodontidae)

The courtship behaviour of the North American southern dusky salamander, Desmognathus fuscus conanti (Plethodontidae), is described for the first time. The male is the most active participant, as is typical for caudate amphibians, and much of his behaviour can be understood in terms of orientation to and stimulation (or persuasion) of the female. The latter is largely accomplished by the transfer of courtship pheromones from the male's mental (chin) gland. The orientation and persuasion phases of courtship appear necessary if sperm transfer by means of a spermatophore deposited upon the substratum is to be successful. The courtship of D. fuscus conanti is qualitatively identical to that of D. santeetlah , with which it is sexually compatible in the laboratory and with which it may hybridize in nature. However, a broader comparison of species in the genus Desmognathus reveals that the greatest interspecific differences occur during the orientation and persuasion phases of courtship. A complete understanding of the evolution of courtship behaviour in desmognathine salamanders awaits availability of ethograms for all other taxa and a robust, independent phytogeny for the entire subfamily.     

New Macroderoides (Digenea: Macroderoididae) from Florida gar, with molecular phylogeny of the genus

Macroderoides minutus n. sp. is described based on specimens collected from the Florida gar (Lepisosteus platyrhincus) from Orange Lake, Alachua County, Florida. The new species is the ninth recognized species of Macroderoides and is morphologically closest to Macroderoides flavus and Macroderoides typicus. It differs from both species in having a smaller body, ventral sucker larger than the oral sucker, substantially lower body length to width ratio, fewer and larger eggs, and other characters. A comparison of sequences of nuclear rDNA (partial 18S, complete ITS region, and partial 28S) of the new species, with sequences of 5 other Macroderoides, convincingly supports the status of M. minutus as a new species. Molecular phylogenetic analysis, including all North American Macroderoides species except for Macroderoides parvus, has demonstrated the presence of 2 distinct clades, 1 including Macroderoides spinifer + (Macroderoides minutus n. sp. + Macroderoides texanus) and the other including Macroderoides trilobatus + (M. typicus + M. flavus). The tree topology suggests that the similarity in body proportions among M. minutus n. sp., M. typicus + M. flavus is not a result of close phylogenetic relationship. It has also revealed for the first time the phylogenetic affinities of M. flavus, which is the only North American member of Macroderoides that switched from holostean fishes to teleosts.     

Appendicular skeletal morphology in minute salamanders,genus Thorius (amphibia: Plethodontidae): Growth regulation,adult size determination,and natural variation

Patterns of growth and variation of the appendicular skeleton were examined in Thorius, a speciose genus of minute terrestrial plethodontid salamanders from southern Mexico. Observations were based primarily on ontogenetic series of each of five species that collectively span the range of adult body size in the genus; samples of adults of each of seven additional species provided supplemental estimates of the full range of variation of limb skeletal morphology. Limbs are generally reduced, i.e., pedomorphic, in both overall size and development, and they are characterized by a pattern of extreme variation in the composition of the limb skeleton, especially mesopodial elements, both within and between species. Fifteen different combinations of fused carpal or tarsal elements are variably present in the genus, producing at least 18 different overall carpal or tarsal arrangements, many of which occur in no other plethodontid genus. As many as four carpal or tarsal arrangements were observed in single population samples of each of several; five tarsal arrangements were observed in one population of T. minutissimus. Left-right asymmetry of mesopodial arrangement in a given specimen is also common. In contrast, several unique, nonpedomorphic features of the limb skeleton, including ossification of the typically cartilaginous adult mesopodial elements and ontogenetic increase in the degree of ossification of long bones, are characteristic of all species and distinguish Thorius from most related genera. They form part of a mechanism of determinate skeletal growth that restricts skeletal growth after sexual maturity. Interspecific differences in the timing of the processes of appendicular skeletal maturation relative to body size are well correlated with interspecific differences in mean adult size and size at sexual maturity, suggesting that shifts in the timing of skeletal maturation provide a mechanism of achieving adult size differentiation among species. Processes of skeletal maturation that confer determinate skeletal growth in Thorius are analogous to those typical of most amniotes – both groups exhibit ontogenetic reduction and eventual disappearance of the complex of stratified layers of proliferating and maturing cartilage in long bone epiphyses – but, unlike most amniotes, Thorius lacks secondary ossification centers. Thus, the presence of secondary ossification centers cannot be used as a criterion for establishing determinate skeletal growth in all vertebrates.     

Time courses of thermal acclimation for critical thermal minima in the salamanders Desmognathus quadramaculatus, Desmognathus monticola, Desmognathus ochrophaeus, and Plethodon jordani

1. We examined the time courses of thermal acclimation for critical thermal minima (CTMin) in the salamanders Desmognathus quadramaculatus, D. monticola, D. ochrophaeus, and Plethodon jordani. 2. D. quadramaculatus showed no annual differences in their time courses, but we noted seasonal differences in the CTMin with these salamanders having lower CTMin in the spring. 3. Gain in CTMin (25 degrees C to 5 degrees C transfer) was faster than its corresponding loss (5 degrees C to 25 degrees C transfer) in D. quadramaculatus. 4. Comparison of all three Desmognathus species revealed no differences in the CTMin for the 5 degrees C to 25 degrees C time courses, but P. jordani had lower CTMin than corresponding D. quadramaculatus. 5. Our results indicate that the pattern of CTMin acclimation correlates with the constraints acting on salamanders in their natural environment.     

A molecular phylogeny of the hawkmoth genus Hyles (Lepidoptera: Sphingidae, Macroglossinae)

The hawkmoth genus Hyles is one of 15 genera in the subtribe Choerocampina of the subfamily Macroglossinae. Due to a remarkable uniformity, morphological characters usually used to identify and classify Lepidoptera at the species level cannot be used in this genus. Instead, we used DNA sequences comprising about 2300 bp derived from the mitochondrial genes COX I, COX II, and tRNA-leucine to elucidate the phylogeny of Hyles. The results corroborate the monophyly of Hyles but conflict with previous internal classifications of the genus based on morphology. Hyles seems to have evolved in the Neotropics during the Oligocene/Eocene epochs and the molecular data (which evolved clock-like) confirm the hypothesis that it is a very young genus that radiated on a global scale rather quickly. We hypothesize its sister group to be one of the genera Deilephila, Theretra or Xylophanes. The Nearctic may have been colonized rapidly by Hyles once the land bridge formed during the Pliocene, since within this same Epoch, the invasion of the Palaearctic appears to have proceeded from the East, via the Bering route. The colonization of Australia appears to have occurred rather early in Hyles radiation, although the route is not clear. We propose that the radiation of the Hyles euphorbiae-complex s. str. (HEC) occurred as recently as the Pliocene/Pleistocene boundary and that its roots can still be reconstructed in Asia. Hyles dahlii is closely related to the HEC, but a sister group relationship to the HEC s. str. cannot be corroborated unequivocally. HEC population ranges appear to have tracked climate oscillations during the Pleistocene Ice Ages, resulting in hybridization around the Mediterranean Sea as they repeatedly intermingled. Comparison of the phylogeny with food plant affiliations leads us to hypothesize that Euphorbia monophagy evolved at least two times independently within Hyles.     

Miniaturization and its effects on cranial morphology in plethodontid salamanders, genus Thorius (Amphibia: Plethodontidae). I. Osteological variation

Cranial skeletal morphology, ontogeny and variation are examined in five species of Thorius , a genus of diminutive plethodontid salamanders that are among the smallest, extant, tailed tetrapods. The skull of adull Thorius is characterized by: (1) limited development or absence of several ossified elements and dentition; (2) increased inter-and intraspecific variability; (3) novel morphological configurations of the braincase and jaw suspensorium. Posthatching cranial mineralization in all species of Thorius is truncated precociously with respect to that typical of larger and more generalized plethodontid genera, such as Pseudoeurycea. These features implicate paedomorphosis as a predominant mechanism responsible for the evolution of decreased size in Thorius from larger plethodontid ancestors. Interspecific differences in cranial morphology are evident; species may be characterized by greater or lesser degrees of truncated development. However, there is no consistent relationship between degree of paedomorphosis and mean adult body size in interspecific comparisons. Adult morphology of several individual elements represent potentially useful taxonomic characters for distinguishing species.
Reduction, increased variability, and morphological novelty are common to many lineages of dwarfed taxa. They represent a null hypothesis for examination of the developmental mechanisms and morphological consequences of miniaturization in other groups.     

A molecular phylogeny of the nightjars (Aves: Caprimulgidae) suggests extensive conservation of primitive morphological traits across multiple lineages

We report a molecular re-assessment of the classification of the nightjars which draws conclusions that are strongly at odds with the traditional, morphology-based classifications. We used maximum likelihood and Bayesian methods to compare the cytochrome b gene for 14 species from seven of the 15 genera of the Caprimulgidae and partial cytochrome b sequence data was available for a further seven species including three further genera. We found that within the Caprimulgidae there were four geographically isolated clades with bootstrap support greater than 70%. One of these clades contained just Chordeiles species, the remaining three clades each contained a mixture of genera including Caprimulgus sp. A clade of exclusively South American nightjars included the genera Caprimulgus, Uropsalis, Eleopthreptus and Hydropsalis. A clade of African and Eurasian birds included Caprimulgus and Macrodipteryx. Phalaenoptilus nuttallii and Caprimulgus vociferous formed a clade of North American birds. Two ecological factors appear to make morphological classification potentially misleading: first, the apparent retention of primitive anti-predator and foraging-related traits across genetically divergent groups; second, rapid divergence in other traits, especially those related to mating, which generate high levels of morphological divergence between species that are genetically very similar. The cytochrome b data suggests that the genus Caprimulgus is not monophyletic and is restricted to Africa and Eurasia and that Caprimulgus species from outside this area have been misclassified as a consequence of retention of primitive adaptations for crepuscular/nocturnal living. Some other genera also appear to have little support from the cytochrome b data.     

Integrative taxonomy and molecular phylogeny of genus aplysina (demospongiae: verongida) from mexican pacific

Integrative taxonomy provides a major approximation to species delimitation based on integration of different perspectives (e.g. morphology, biochemistry and DNA sequences). The aim of this study was to assess the relationships and boundaries among Eastern Pacific Aplysina species using morphological, biochemical and molecular data. For this, a collection of sponges of the genus Aplysina from the Mexican Pacific was studied on the basis of their morphological, chemical (chitin composition), and molecular markers (mitochondrial COI and nuclear ribosomal rDNA: ITS1-5.8-ITS2). Three morphological species were identified, two of which are new to science. A. clathrata sp. nov. is a yellow to yellow-reddish or -brownish sponge, characterized by external clathrate-like morphology; A. revillagigedi sp. nov. is a lemon yellow to green, cushion-shaped sometimes lobate sponge, characterized by conspicuous oscules, which are slightly elevated and usually linearly distributed on rims; and A. gerardogreeni a known species distributed along the Mexican Pacific coast. Chitin was identified as the main structural component within skeletons of the three species using FTIR, confirming that it is shared among Verongida sponges. Morphological differences were confirmed by DNA sequences from nuclear ITS1-5.8-ITS2. Mitochondrial COI sequences showed extremely low but diagnostic variability for Aplysina revillagigedi sp. nov., thus our results corroborate that COI has limited power for DNA-barcoding of sponges and should be complemented with other markers (e.g. rDNA). Phylogenetic analyses of Aplysina sequences from the Eastern Pacific and Caribbean, resolved two allopatric and reciprocally monophyletic groups for each region. Eastern Pacific species were grouped in general accordance with the taxonomic hypothesis based on morphological characters. An identification key of Eastern Pacific Aplysina species is presented. Our results constitute one of the first approximations to integrative taxonomy, phylogeny and evolutionary biogeography of Eastern Pacific marine sponges; an approach that will significantly contribute to our better understanding of their diversity and evolutionary history.     

A molecular phylogeny for the frog genus Limnodynastes (Anura: myobatrachidae)

Members of the genus Limnodynastes are a prominent and widespread feature of the Australian frog fauna. Yet despite their potential to be informative about biogeographic history and mechanisms of speciation, the relationships among these taxa are not well known. We investigated phylogenetic relationships within the genus Limnodynastes via sequencing of mitochondrial (mt)DNA from current members of the genus Limnodynastes and the monotypic genus Megistolotis. a 450-bp fragment of the 16S rRNA gene and a 370-bp fragment of the protein-coding gene ND4 were used to infer a molecular phylogeny. We revise traditional species groupings and now recognize four species groups within Limnodynastes: the L. ornatus group (L. ornatus and L. spenceri), the L. peronii group (L. peronii, L. tasmaniensis, L. fletcheri, the L. depressus), the L. salmini group (L. salmini, L. convexiusculus, and L. lignarius), and the L. dorsalis group (L. dorsalis, L. terraereginae, L. dumerilii and L. interioris). The L. ornatus species group forms a highly distinctive clade that is a sister group to the other Limnodynastes groups. Pending broader phylogenetic studies it could be removed from the genus Limnodynastes. Our results concur with previous suggestions that Megistolotis lignarius is nested within Limnodynastes, and we therefore reclassify this species as Limnodynastes lignarius. Furthermore, specimens identified as L. depressus form a mtDNA lineage distinct from other species in the genus, confirming the validity of the species. Specimens of species from the L. dorsalis group (L. dorsalis, L. dumerilii, L. interioris, and L. terraereginae) are closely related such that L. dumerilii is paraphyletic with two other species. Finally, our study provides broad support for previous phylogenies based on microcomplement fixation.     

Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny

The economically important genus Arachis (Fabaceae) comprises 80 species restricted to South America. One monograph on the genus divided it into nine sections and included an intuitive assessment of evolutionary relationships. There is no comprehensive phylogenetic study of the genus. To test the current systematic treatment of the genus, we reconstructed a phylogeny for Arachis using nuclear ITS and plastid trnT–trnF sequences from 46 species representing all nine sections. ITS cloning of the allotetraploid species of section Arachis indicated the presence of A and B genome alleles and chimeric sequences. Our study revealed that species from section Extranervosae were the first emerging lineage in the genus, followed by sections Triseminatae and Caulorrhizae, and two terminal major lineages, which we refer to as erectoides and arachis. The lineage erectoides comprises members of sections Erectoides, Heteranthae, Procumbentes, Rhizomatosae, and Trierectoides. Species in the arachis lineage form two major clades, arachis I (B and D genomes species and the aneuploids) and arachis II (A genome species). Our results substantiated the sectional treatment of Caulorrhizae, Extranervosae, and Triseminatae, but demonstrated that sections Erectoides, Procumbentes, and Trierectoides are not monophyletic. A detailed study of the genus Arachis with denser taxon sampling, additional genomic regions, plus information from morphology and cytogenetics is needed for comprehensive assessment of its systematics.     

Spinal nerves and their bearing on,salamander phylogeny

Examination of the vertebral columns of representatives of all families of salamanders revealed that, in contrast to the condition found in most other vertebrates, salamander spinal nerves often pass through foramina in the vertebrae. Two kinds of spinal nerve foramina were found: those in the anterior halves of vertebrae, and those in the posterior halves. In addition, many salamanders retain intervertebral nerves. However, within each family or, in a few cases, subfamily there is a characteristic pattern of spinal nerve-vertebral relationships. The first spinal nerve of all salamanders exits through a foramen in the anterior half of the atlas. All more posterior nerves are intervertebral in the families Cryptobranchidae, Hynobiidae and Proteidae. The posterior caudal nerves exit through the posterior halves of the caudal vertebrae in the family Amphiumidae, while in the subfamilies Dicamptodontinae and Rhyacotritoninae all post-sacral nerves exit through the posterior halves of the vertebrae. All but the first three nerves exit through posterior foramina in the family Plethodontidae and the subfamily Ambystomatinae, while all but the first two nerves pass through posterior foramina in the families Salamandridae and Sirenidae. Several fossil salamanders were also examined. These showed that the amphiumid and dicamptodontine-rhyacotritonine nerve patterns had evolved by the Late Cretaceous, and the sirenid pattern had probably evolved by that time. Other Cretaceous genera associated with the Ambystomatoidea still possessed the primitive intervertebral pattern. Using spinal nerve patterns and several other previously described morphological characters, a new hypothesis of the phylogeny of recent and fossil salamanders is presented and compared to earlier proposed phylogenies of the group. A new classification of salamander families is presented.     

Miniaturization and its effects on cranial morphology in plethodontid salamanders, genus Thorius (Amphibia, Plethodontidae): II. The fate of the brain and sense organs and their role in skull morphogenesis and evolution

Relative size and arrangement of the brain and paired sense organs are examined in three species of Thorius, a genus of minute, terrestrial salamanders that are among the smallest extant tailed tetrapods. Analogous measurements of representative species of three related genera of larger tropical (Pseudoeurycea, Chiropterotriton) and temperate (Plethodon) salamanders are used to identify changes in gross morphology of the brain and sense organs that have accompanied the evolution of decreased head size in Thorius and their relation to associated changes in skull morphology. In adult Thorius, relative size (area measured in frontal plane, and length) of the eyes, otic capsules, and brain each is greater than in adults of all of the larger genera; relative size of the nasal capsules is unchanged or slightly smaller. Interspecific scaling phenomena--negative allometry of otic capsule, eye and brain size, isometry or slight positive allometry of nasal capsule size, all with respect to skull length--also are characteristic of intraspecific (ontogenetic) comparisons in both T. narisovalis and Pseudoeurycea goebeli. Predominance of the brain and eyes in Thorius results in greater contact and overlap among these structures and the nasal capsules in the anterior portion of the head. This is associated with anterior displacement of both the eyes and nasal capsules, which now protrude anterior to the skull proper; a change in eye shape; and medial deformation of anterior braincase walls. Posteriorly, predominance of the otic capsules has effected a reorientation of the jaw suspensorium to a fully vertical position that is correlated with the novel presence of a posteriorly directed squamosal process and shift in origin of the quadropectoralis muscle. Many of these changes in cranial morphology may be explained simply as results of mechanical (physical) interactions among the skeletal, nervous, and sensory components during head development at reduced size. This provides further evidence of the role of nervous, sensory, and other "soft" tissues in cranial skeletal morphogenesis, and reinforces the need to consider these tissues in analyses of skull evolution.     

Phylogenomics,molecular evolution,and estimated ages of lineages from the deep phylogeny of Poaceae

The deeply diverging subfamilies of grasses: Anomochlooideae, Pharoideae, and Puelioideae, today inhabit tropical forest floors as sparsely distributed depauperate lineages. The BEP/PACMAD grasses, which make up the majority of the family, are the result of a more recent radiation. Species in the deeply diverging subfamilies were here investigated to better understand molecular evolutionary processes and ages of divergence. Complete chloroplast genomes (plastomes) of Pharus latifolius L., P. lappulaceus Aubl., and Puelia olyriformis (Franch.) Clayton were determined. Four plastome loci from seven species of the deep subfamilies were also sequenced. Phylogenetic and mutation analyses and divergence estimations were conducted on all sequences together with homologous sequences from other Poaceae. Mutation analyses surveyed insertion/deletion mutations across the plastomes, clarified a trend in the molecular evolution of the rpoC2 locus, and indicated unique pseudogenizations in the plastomes of Pharus and Puelia. Phylogenetic analyses largely confirmed earlier multi-gene phylogenies. Phylogenomic and divergence analyses produced estimated origins of the crown nodes of Anomochlooideae at 65–104 Ma, Pharoideae at 44–71 Ma, and Puelioideae at 62–96 Ma. The upper ends of our estimated ranges are in general agreement with previous estimates. However, the lower ends of our ranges are considerably older than previous estimates, reflecting the influence of the less commonly used oldest fossil calibration point. The deeply diverging subfamilies exhibited the accumulation of numerous substitution and indel mutations consistent with a long evolutionary history that predated the radiation of the BEP/PACMAD grasses. We hypothesize that relatively rapid warming and drying in Africa at 55–56.5 Ma may have acted as selective forces stimulating adaptive radiations of grasses from the African tropical forests into diverse habitats.