Albumin evolution in West Indian frogs of the genus Eleutherodactylus (Leptodactylidae): Caribbean biogeography and a calibration of the albumin immunological clock

Antisera to serum albumins from five West Indian species of the frog genus Eleutheroductylus were prepared, and the reciprocal immunological distances (IDs) obtained were used to provide a time frame for the evolution of this group in the West Indies. One-way IDS were obtained to 25 additional species within the genus, with emphasis on those from the West Indies. These immunological data support both a recent classification of Eleutheroductylus based on an analysis of slow-evolving allozyme loci, and the monophyly of the 17 native Jamaican species as indicated by a more comprehensive electrophoretic study. This is in contrast to the results of morphological studies supporting multiple invasions of Jamaica by Eleutherodactylus . Within the subgenus Euhyas , IDS ranged from6–27 between Jamaican species, whereas between species on different islands the range was29–67. The subgenus Syrrhophus in southern North America was found to be the sister group to the subgenus Euhyus , a western Caribbean clade. Pelorius , a subgenus restricted to Hispaniola, was found to be the sister group of the subgenus Eleutheroductylus in the West Indies. The largest IDs obtained for West Indian species were those between the two major groups, the subgenera Eleutheroductylus and Euhyas .
The albumin immunological clock for Eleutheroductylus was calibrated with three events in the geologic history of the Caribbean: the breakup of the proto-Antilles (65-75 million years before present [mybp]), the emergence of Jamaica (20-30 mybp), and the uplift of the Blue Mountains in Jamaica (5-10 mybp). Immunological distances corresponding to those events yield a calibration of 1 ID=0.60 million years (my), the same as that previously obtained for other groups of amphibians and thus supports the use of albumin immunological distance as a molecular chronometer in the genus Eleutherodactylus     

Cenozoic biogeography and evolution in direct-developing frogs of Central America (Leptodactylidae: Eleutherodactylus) as inferred from a phylogenetic analysis of nuclear and mitochondrial genes

We report the first phylogenetic analysis of DNA sequence data for the Central American component of the genus Eleutherodactylus (Anura: Leptodactylidae: Eleutherodactylinae), one of the most ubiquitous, diverse, and abundant components of the Neotropical amphibian fauna. We obtained DNA sequence data from 55 specimens representing 45 species. Sampling was focused on Central America, but also included Bolivia, Brazil, Jamaica, and the USA. We sequenced 1460 contiguous base pairs (bp) of the mitochondrial genome containing ND2 and five neighboring tRNA genes, plus 1300 bp of the c-myc nuclear gene. The resulting phylogenetic inferences were broadly concordant between data sets and among analytical methods. The subgenus Craugastor is monophyletic and its initial radiation was potentially rapid and adaptive. Within Craugastor, the earliest splits separate three northern Central American species groups, milesi, augusti, and alfredi, from a clade comprising the rest of Craugastor. Within the latter clade, the rhodopis group as formerly recognized comprises three deeply divergent clades that do not form a monophyletic group; we therefore restrict the content of the rhodopis group to one of two northern clades, and use new names for the other northern (mexicanus group) and one southern clade (bransfordii group). The new rhodopis and bransfordii groups together form the sister taxon to a clade comprising the biporcatus, fitzingeri, mexicanus, and rugulosus groups. We used a Bayesian MCMC approach together with geological and biogeographic assumptions to estimate divergence times from the combined DNA sequence data. Our results corroborated three independent dispersal events for the origins of Central American Eleutherodactylus: (1) an ancestor of Craugastor entered northern Central America from South American in the early Paleocene, (2) an ancestor of the subgenus Syrrhophus entered northern Central America from the Caribbean at the end of the Eocene, and (3) a wave of independent dispersal events from South America coincided with formation of the Isthmus of Panama during the Pliocene. We elevate the subgenus Craugastor to the genus rank.     

Molecular phylogeny and biogeography of West Indian frogs of the genus Leptodactylus (Anura, Leptodactylidae)

Three endemic species of the aquatic-breeding frog genus Leptodactylus are recognized from the West Indies: Leptodactylus albilabris (Puerto Rico and the Virgin Islands), Leptodactylus dominicensis (Hispaniola), and Leptodactylus fallax (Lesser Antilles). DNA sequences were obtained from several mitochondrial genes to resolve taxonomic questions involving these species and to provide insights into their origin and distribution in the islands. We found low levels of sequence divergence between L. dominicensis and L. albilabris, supporting morphological evidence that the former species is a junior synonym of the latter species. Phylogenetic analysis supported previous species-group allocations, finding that L. albilabris is a member of the fuscus group and L. fallax is a member of the pentadactylus group. Molecular time estimates for the divergence of L. albilabris from its closest relative in South America (24-58 million years ago, Ma) and for L. fallax from its closest relative in South America (23-34Ma) indicate that they colonized the West Indies independently by over-water dispersal in the mid-Cenozoic. The absence of detectable sequence divergence between the two extant populations of L. fallax (Dominica and Montserrat), a species used for human food and now critically endangered, suggests that one or both arose by human introduction from an island or islands where that species originated. The relatively minor genetic differentiation of populations of L. albilabris can be explained by vicariance and dispersal in the Pleistocene and Holocene, although human introduction of some populations cannot be ruled out.     

Physiological basis of habitat partitioning in Jamaican Eleutherodactylus

Summary Rate of evaporation, resistance to drying, temperature selection, and critical thermal maximum were measured for Jamaican Eleutherodactylus acclimated to 20° C and a 12:12 L:D photoperiod. Two introduced species, E. planirostris and E. johnstonei, selected higher temperatures and had higher CTMs than two native species, E. cundalli and E. gossei. The introduced species lost water as rapidly as the native species, but tolerated 30–73% greater water loss before losing their righting response. The physiological differences are reflected in the microhabitat selection and activity patterns of the four species.     

Cytogenetic analysis of four species of the genus Alsodes (Anura: Leptodactylidae) with comments about the karyological evolution of the genus

A comparative cytogenetic analysis of Alsodes pehuenche, A. vanzolinii, A. verrucosus and A. aff. vittatus show that all four species share the same diploid number 2n = 26; the fundamental number is 50 in A. vanzolinii and 52 in A. aff. vittatus, A. pehuenche and A. verrucosus. The karyotypes of A. pehuenche and A. aff. vittatus are described for the first time; the C-band patterns, the NOR locations and Q-band patterns are also described for the first time for the four species. C-band patterns are species specific and useful to identify the taxa. The usefulness of the chromosomal data in taxonomy and systematics of Alsodes species is discussed. Transformation of euchromatin into heterochromatin and centric fissions and translocations are proposed as the main mechanisms that govern the chromosomal evolution of the frog genus Alsodes.     

Origins and introductions of the Caribbean frog, Eleutherodactylus johnstonei (Leptodactylidae): management and conservation concerns

Johnstone's Whistling Frog, Eleutherodactylus johnstonei, is a highly successful colonizer that has become widely distributed throughout the Caribbean region. It has been introduced both purposefully and unintentionally by humans, and it continues to expand its range locally and regionally. Its current distribution and recent expansion do not support the hypothesis that E. johnstonei is expanding into new habitats exclusively by outcompeting native species. Instead, its range expansion progresses mainly parallel to human expansion (habitat disturbance through land development) and extreme climatic events (habitat disturbance through hurricanes and volcanism). Once a habitat has been disturbed and E. johnstonei has arrived, any previously existing endemic Eleutherodactylus species tend not to be found again at their previous ranges or population densities. The most probable explanation for this is that the broader physiological tolerance of the ecological generalist E. johnstonei allows it to become permanently established in a disturbed biota, whereas ecologically specialized endemics are prevented from recolonizing such habitats. Invasion of E. johnstonei can result in a parapatric distribution with endemics (e.g. E. euphronides, E. shrevei) or in sympatry (e.g. E. martinicensis), and habitats include areas with widely divergent climatic conditions (e.g. xeric: Anguilla, Barbuda; mesic: Grenada, St Vincent). Management for this species includes prevention of further or repeat introductions, close monitoring of ranges, and preservation of native habitats to ensure survival of local endemics.     

A new species of rainfrog of the Eleutherodactylus milesi species group (Anura: Leptodactylidae) from Guatemala

A new rainfrog of the Eleutherodactylus milesi group is described from the Sierra de Xucaneb, Guatemala. It is compared to the other 11 upland species from southern Mexico, Guatemala and Honduras referred to the group. Within the milesi group the new species differs from the other five forms which also have toe fringes in size of the finger disks, the snout shape in profile and the amount of toe webbing and dorsal tuberculation. Difficulties in distinguishing among the character states for the tympanum (distinct, indistinct and hidden) and between toe ridges and fringes indicate that very subtle differences separate recognized species.     

Purine-nucleoside phosphorylase expression in Eleutherodactylus and Leptodactylus frogs

Purine-nucleoside phosphorylase expression as ascertained electrophoretically is discussed for seven species of Eleutherodactylus and one species of Leptodactylus frogs from Costa Rica. Eleutherodactylus angelicus, E. bransfordii, and E. podiciferus exhibit intraspecific variation unlike the samples of the other five species. In E. angelicus, heterozygotes form a four-banded pattern on starch gels consistent with a trimeric enzyme structure. Heterozygotes of E. bransfordii and E. podiciferus are characterized by a two-banded pattern as the heterotrimeric isozymes are apparently not expressed. Such differences in gene expression are discussed in terms of their potential phylogenetic and systematic application and significance.     

Isolation of simple and compound polymorphic tetranucleotide microsatellites for the neotropical leaflitter frog Eleutherodactylus ockendeni (Leptodactylidae)

Few studies of population structure and genetic diversity exist for frogs in the Amazon of South America, an area renowned for exceptionally high species richness. We isolated seven highly variable tetranucleotide microsatellite loci for the neotropical leaflitter frog, Eleutherodactylus ockendeni using an enrichment method. Three of the repeats are simple, three are compound and one is imperfect. We screened all loci with 175 individuals from one geographical area in the upper Napo of Ecuador and found high polymorphism in all loci (> 14 alleles/locus). These markers are suitable for population genetics studies of E. ockendeni and perhaps other leaflitter frogs of the same genus.     

Clutch effects explain heart rate variation in embryonic frogs (Cave Coqui, Eleutherodactylus cooki)

Few physiological studies to date have focused on whether variation among sibling groups during development can account for often large, intraspecific physiological variation. In this study, we measured heart rate in the direct-developing frog Eleutherodactylus cooki throughout its embryonic development and examined heart rate variation among egg clutches comprising from 10 to 40 eggs. Clutches were collected in the wild in Yubucoa, Puerto Rico, and individual eggs were maintained under equivalent conditions in the lab. Heart rate showed large increases during development, rising from about 40 beats min(-1) in the earliest stages to about 110 beats min(-1) at hatching. The effect of stage (averaged across clutches) was highly significant (P<0.001). However, repeated-measures MANOVA also revealed that there were highly significant effects on heart rate associated with both clutch (variation among clutches averaged across development; P<0.001) and clutch-stage interactions (differences among clutches in the developmental change in heart rate; P<0.0001). These effects and interactions reveal that throughout development, heart rate in siblings is much more similar than in nonsiblings and that sib groups follow different heart rate trajectories during their development. Collectively, these data indicate that "clutch effects" caused by genetic and/or maternal influences can strongly affect patterns of heart function during development within cave coqui populations. This phenomenon also occurs in bird eggs and armadillo neonates, suggesting that physiological variation attributable to clutch effects might be a widespread phenomenon in vertebrates.     

Immunological relationships of the South American frog genus Eupsophus (Leptodactylidae)

The agar-gel precipitin technique was used to examine the immunological relationships of the South American frog genus Eupsophus. This taxon shows similarities with the members of the Telmatobiini tribe and presents a characteristic precipitin band, here labelled as A.     

Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.

Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative karyotype analysis and chromosome evolution in the genus Aplastodiscus (Cophomantini, Hylinae, Hylidae)

ABSTRACT: BACKGROUND: The frogs of the Tribe Cophomantini present, in general, 2n?=?24 karyotype, but data on Aplastodiscus showed variation in diploid number from 2n?=?24 to 2n?=?18. Five species were karyotyped, one of them for the first time, using conventional and molecular cytogenetic techniques, with the aim to perform a comprehensive comparative analysis towards the understanding of chromosome evolution in light of the phylogeny. RESULTS: Aplastodiscus perviridis showed 2n?=?24, A. arildae and A. eugenioi, 2n?=?22, A. callipygius, 2n?=?20, and A. leucopygius, 2n?=?18. In the metaphase I cells of two species only bivalents occurred, whereas in A. arildae, A. callipygius, and A. leucopygius one tetravalent was also observed besides the bivalents. BrdU incorporation produced replication bands especially in the largest chromosomes, and a relatively good banding correspondence was noticed among some of them. Silver impregnation and FISH with an rDNA probe identified a single NOR pair: the 11 in A. perviridis and A. arildae; the 6 in A. eugenioi; and the 9 in A. callipygius and A. leucopygius. C-banding showed a predominantly centromeric distribution of the heterochromatin, and in one of the species distinct molecular composition was revealed by CMA3. The telomeric probe hybridised all chromosome ends and additionally disclosed the presence of telomere-like sequences in centromeric regions of three species. CONCLUSIONS: Based on the hypothesis of 2n?=?24 ancestral karyotype for Aplastodiscus, and considering the karyotype differences and similarities, two evolutionary pathways through fusion events were suggested. One of them corresponded to the reduction of 2n?=?24 to 22, and the other, the reduction of 2n?=?24 to 20, and subsequently to 18. Regarding the NOR, two conditions were recognised: plesiomorphy, represented by the homeologous small-sized NOR-bearing pairs, and derivation, represented by the NOR in a medium-sized pair. In spite of the apparent uniformity of C-banding patterns, heterogeneity in the molecular composition of some repetitive regions was revealed by CMA3 staining and by interstitial telomeric labelling. The meiotic tetravalent might be due to minute reciprocal translocations or to non-chiasmatic ectopic pairing between terminal repetitive sequences. The comparative cytogenetic analysis allowed to outline the chromosome evolution and contributed to enlighten the relationships within the genus Aplastodiscus.     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.     

Chromosome evolution in the genus Mikania (Compositae)

Karyotypic analysis of ten species of the genus Mikania was carried out using Feulgen staining. Species belonging to the following sections were analyzed: Section Thyrsigerae containing M. additicia (2n = 34), M. hemisphaerica, M. lanuginosa, and M. punctata (2n = 36), and Mikania sericea (2n = 42), which adds a new basic chromosome number (x = 21) to the genus and to the tribe Eupatorieae; Section Corymbosae with M. hastato-cordata (2n = 34) and M. involucrata and M. microptera with 2n = 36 chromosomes; Section Spicato-Racemosae with M. sessilifolia, with 2n = 108 chromosomes. One unidentified species with 2n = 34 chromosomes was also analyzed. All the species studied show one large pair of chromosomes with a secondary constriction in the middle region of the long arm. The morphology of this chromosome suggests that it can be considered as a cytological marker for the genus. Because of the distinctive inflorescence types found in the genus Mikania and the high frequency of species with x = 18, a correlation between morphological and chromosomal evolution is discussed. The present study suggests that the basic original chromosome number for the genus is x = 18, from which the others (x = 17, 19, 20, 21) have been derived by aneuploidy to form the observed aneuploid series.     

The role of chromosome rearrangements in the evolution of mole voles of the genus Ellobius (Rodentia,Mammalia)

Modern mole voles of the genus Ellobius are characterized by species-specific features of autosomes and sex chromosomes. Owing to the use of the Zoo-FISH method, the nomenclature of chromosomes was refined and nonhomologous Robertsonian translocations indistinguishable by G-staining were identified for Ellobius tancrei, which is a species with a wide chromosome variation of the Robertsonian type. The electron-microscopic analysis of synaptonemal complexes in F₁ hybrids of forms with 2n = 50 and 2n = 48 revealed the formation of a closed SC-pentavalent composed of three metacentrics with monobrachial homology and two acrocentrics. Segregation of chromosomes of such complex systems is impeded by disturbances in the nucleus architecture leading to the formation of unbalanced gametes and to a dramatic reduction in fertility of hybrids. Our data support the hypothesis that the formation of monobrachial homologous metacentric chromosomes can be considered as a way of chromosomal speciation.