The largest reservoir of mitochondrial introns is a relic of an ancestral split gene

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms (ref. 1-4). Large introns (0.1 to 5 kbp) are frequent in mitochondrial genomes of plant and fungi (ref. 1,5) but scarce in Metazoa, despite these organisms are grouped with fungi among Opisthokonts. Introns are classified in two main groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism (ref. 5,6). Most of the group I introns carry a "Homing Endonuclease Gene" (ref. 7-9) encoding a DNA endonuclease acting in the transfer and site specific integration "homing") and allowing the intron spreading and gain after lateral transfer even between species from different kingdoms (ref. 10,11). Opposite to this "late intron" paradigm, the "early intron" theory indicates that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss (ref. 12,13).

Here we report the sequence of the cox1 gene of the button mushroom _Agaricus bisporus_, the most worldwide cultivated mushroom. This gene is both the longest mitochondrial gene (29,902 nt) and the largest Group I intron reservoir reported to date. An analysis of the group I introns available in _cox1_ genes shows that they are ancestral mobile genetic elements, whose frequent events of loss (according to the "late theory") and gain by lateral transfer ("early theory") must be combined to explain their wide and patchy distribution extending on several kingdoms. This allows the conciliation of the "early" and "late intron" paradigms, which are still matters of much debate (ref. 14,15). The overview of the intron distribution indicates that they evolve towards elimination. In such a landscape of eroded and lost intron sequences, the _A. bisporus_ largest intron reservoir, by its singular dynamics of intron keeping and catching, constitutes the most fitted relic of an early split gene

The Agaricus bisporus cox1 Gene: The Longest Mitochondrial Gene and the Largest Reservoir of Mitochondrial Group I Introns

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a “Homing Endonuclease Gene” (heg) encoding a DNA endonuclease acting in transfer and site-specific integration (“homing”) and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain) is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt) and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote

HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors

Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species

Réplication de l'ADN mitochondrial (identification d'une seconde activité ADN polymérase dans la mitochondrie de S.cerevisiae et Contribution à l'étude du réplisome mitochondrial)

Au cours de la croissance des levures, la cellule doit dupliquer sont génome nucléaire et mitochondrial, le processus de réplication est bien moins étudié dans les mitochondries. Néanmoins, si de multiples ADN polymérases sont impliquées dans les processus de réplication et de réparation dans le noyau, il est considéré jusqu à aujourd hui qu une seule ADN polymérase est impliquée dans ces processus dans la mitochondrie. Des résultats récents mettent en exergue le fait que la situation est bien plus compliquée qu il n y apparait au départ. Pour élucider le processus de réplication dans la mitochondrie de levure, j ai focalisé mon intérêt à tenter de purifier et de caractériser le complexe de réplication. Ce travail était important à développer étant donné la découverte au laboratoire d une seconde ADN polymérase supplémentaire à la polymérase gamma, dans les mitochondries de levure. Une première partie de ma thèse a été de m investir afin d obtenir suffisamment de protéines dans le but d une identification par spectrométrie de masse, compte tenu de la faible proportion des ADN polymérases dans la cellule et en particulier dans la mitochondrie. Nous avons démontré que cette polymérase est codée par le gène unique POL1. Par des techniques d ultracentrifugation et d analyse biochimiques, j ai réussi à isoler et caractériser un complexe de réplication mitochondrial. Des techniques d exclusion chromatographiques ont permis d attribuer une masse native à ce complexe. Sa composition a été étudiée grâce à des colonnes ioniques et hydrophobes, une autre méthode d analyse repose sur l utilisation de colonnes d affinité afin de reconstituer in-vitro les interactions existant entre plusieurs protéines présumées impliquées. Ainsi, un réseau d interactions impliquant les deux ADN polymérases mitochondriales avec cinq autres protéines a été reconstitué. La masse native de différentes formes stables de ce complexe se situent à 500 kDa ou au-delà de 1 MDa.During yeast growth, cells must duplicate their nuclear and mitochondrial DNA. The replication process involved is less studied in mitochondria. Nevertheless, if multiple DNA polymerases are implicated in the nuclear replication and repair mechanisms, until now it is believed that only one DNA polymerase is involved in these processes in mitochondria. Recent results pointed out that the situation is more complicated than preliminary believed. To elucidate the replication process in yeast mitochondria I focused my interest in attempts to purify and characterize the replication complexes. This work was important to develop in accord with the discovery in the laboratory of a second DNA polymerase in addition to the polymerase gamma in yeast mitochondria. One first part of my thesis was to hardly purify enough of this enzyme to be allowed to identify it by mass spectrometry as the DNA polymerase alpha, encoded by the unique POL1 gene. By ultracentrifugation and biochemical techniques, I succeeded to purify the complex. Exclusion chromatographies were managed to elucidate the native mass of this complex. In addition ionic and hydrophobic chromatographic columns were carried out to determine its composition. Another way to study the complex was the reconstitution in vitro of the interactions happening with some usual suspect proteins with the help of chromatographic affinity columns. I reconstituted partly an interactions model network, including the two mitochondrial DNA polymerases and 5 others proteins implicated in replication. I determined the mass of different stable forms of the isolated complexes, around 500 kDa and over 1 MDaBORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

Caractérisation et identification de deux ADN polymérases mitochondriales de la levure Saccharomyces cerevisiae

Nous avons purifié deux ADN polymérases à partir des mitochondries de S. cerevisiae. Leur caractérisation montre que l'une des deux est l'ADN pol g. Ces résultats ont été confirmés par deux stratégies qui font appel à la technique de délétion de gènes et spectométrie de masse. Les résultats obtenus ont confirmé les résultats précédents. Ces techniques étant fiables, nous les avons utilisées pour identifier l'autre ADN pol. Les résultats obtenus par ces deux stratégies, complétés par ceux d'une troisième technique qui consiste à marquer l'ADN polymérase par la GFP, indiquent que la deuxième activité est due à l'ADN polymérase 2. Cependant, cette ADN polymérase est trouvée sous deux formes. Nous avons trois activités ADN polymérase dont deux sont issues de l'ADN polymérase 2.La présence de l'ADN polymérase 2 dans les mitochondries est confortée par la présence de Dpb2p dans les extraits mitochondriaux, une sous-unité du complexe de l'ADN pol2 qui semble posséder une séquence d'adressage mitochondrial, mais aussi par la présence d'une séquence d'adressage mitochondriale située côté N-terminal de l'ADN polymérase 2, et capable d'amener la GFP dans la mitochondrie.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity

International audienc

Hyalinobatrachium mesai

<i>Hyalinobatrachium mesai</i> <p>Barrio-Amorós and Brewer-Carías, 2008: 18.</p> <p> <b>Type locality.</b> Southern slope of Sarisariñama-tepui (04°25’ N, 64°7’ W; 420 m), Bolívar, Venezuela.</p> <p> <b>Diagnosis.</b> (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin shagreened in life and preservative; (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium transparent, visceral and hepatic peritonea white, all other peritonea presumably transparent (not dissected); (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III 2 1/3 – 2+ IV; (10) webbing formula of toes I 1 – 2 2/3 II 1 – 2 1/2 III 1 – 2 IV 2 – 1 V; (11) low and enameled ulnar and tarsal folds; (12) nuptial pad inconspicuous, prepollex not evident from external view; (13) Finger I longer Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum light green with big irregular darker green patches, black dots, and minute melanophores, bones green; (16) coloration in preservative: cream with big irregular white patches and black dots; (17) iris white with black flecks; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes unknown; (19) advertisement call composed by one to two notes, each lasting 0.075 s, dominant frequency of 4414.5 Hz, one male observed to call from the upper side of a leave; (20) fighting behavior unknown; (21) egg clutches unknown, parental care unknown; (22) tadpole unknown; (23) adult size 20.0 mm in one male, unknown in females.</p> <p> <b>Comparisons.</b> This species can only be differentiated from <i>Hyalinobatrachium iaspidiense</i> by the presence of white bones in the later (versus green in <i>H</i>. <i>mesai</i>). All other characters compared are identical (see below for more details).</p> <p> <b>Remarks.</b> This species is only known from the holotype, an adult male. Although the original description (Barrio-Amorós & Brewer-Carías, 2008) listed seven differences between <i>Hyalinobatrachium iaspidiense</i> and <i>H</i>. <i>mesai</i> the authors did not directly compare it with material of <i>H</i>. <i>iaspidiense</i> but rather follow descriptions in the literature. We have identified descriptive lapses in their report of character states of <i>H</i>. <i>iaspidiense</i> and their comparison with those of <i>H</i>. <i>mesai</i> (in parentheses character states used by the authors): both species show low and enameled ulnar and tarsal folds (<i>H</i>. <i>iaspidiense</i> has folds but not <i>H</i>. <i>mesai</i>); as in most species of glassfrogs <i>H</i>. <i>iaspidiense</i> has a thenar tubercle, although low and difficult to appreciate (thenar tubercle absent in <i>H</i>. <i>iaspidiense</i> but present in <i>H</i>. <i>mesai</i>); the ventral skin of <i>H</i>. <i>iaspidiense</i> is granular and not smooth, we did not observe differences with <i>H</i>. <i>iaspidiense</i> when we examined the holotype of <i>H</i>. <i>mesai</i> (ventral skin smooth in <i>H</i>. <i>iaspidiense</i> but areolate in <i>H</i>. <i>mesai</i>); the <i>canthus rostralis</i> of both species is similar and we did not find differences that would suggest considering them different character states (well defined in <i>H</i>. <i>iaspidiense</i> not well defined in <i>H</i>. <i>mesai</i>); webbing formula of toes is basically identical (toes approximately two-thirds webbed in <i>H</i>. <i>mesai</i> versus three-fourths in <i>H</i>. <i>mesai</i>); we did not find differences in the shape of the choanae, which is approximately oval in both species (choanae trilobate in <i>H</i>. <i>iaspidiense</i> and oval in <i>H</i>. <i>mesai</i>). Furthermore, the re-analysis of the recorded call revealed that it is identical to that of <i>H. iaspidienses</i> (Fig. 2 E–G). The only consisted divergent character is color of bones in life specimens, which is white in <i>H</i>. <i>iaspidiense</i> but green in <i>H</i>. <i>mesai</i>. Although there is no record in the literature of intraspecific polymorphism of bone colors in <i>Hyalinobatrachium</i>, the fact that <i>H</i>. <i>mesai</i> is just known from a single specimen for which there are no ventral photographs in live (color of bones is lost in preservative) does not allow us to evaluate the validity of this character. Unfortunately, we could not amplify DNA from a tissue sample of the specimen. Thus and till more data are available, we consider <i>H</i>. <i>mesai</i> as a valid species but its status is pending revaluation.</p> <p> <b>Ecology and distribution.</b> Only known from the type locality in the Venezuelan GS.</p>Published as part of <i>Castroviejo-Fisher, Santiago, Vilà, Carles, Ayarzagüena, José, Blanc, Michel & Ernst, Raffael, 2011, Species diversity of Hyalinobatrachium glassfrogs (Amphibia: Centrolenidae) from the Guiana Shield, with the description of two new species, pp. 1-55 in Zootaxa 3132</i> on pages 26-27, DOI: <a href="http://zenodo.org/record/200895">10.5281/zenodo.200895</a&gt

Hyalinobatrachium iaspidiense

<i>Hyalinobatrachium iaspidiense</i> <p>(Fig. 5)</p> <p> <i>Centrolenella iaspidiensis</i> Ayarzagüena, 1992: 23.</p> <p> <i>Centrolene iaspidiensis</i> Duellman, 1993: 50.</p> <p> <i>Hyalinobatrachium iaspidiense</i> Myers and Donnelly, 1997: 16. <i>Hyalinobatrachium nouraguensis</i> Lescure and Marty, 2000: 74, <b>synonym</b>. <i>Hyalinobatrachium nouraguense</i> Kok and Castroviejo-Fisher, 2008: 48.</p> <p> <b>Type locality.</b> Quebrada de Jaspe, San Ignacio de Yuraní, (04°55’N, 61°05’W; 800–1000 m) Bolívar, Venezuela.</p> <p> <b>Diagnosis.</b> (1) Dentigerous processes on vomer and vomerine teeth absent; (2) snout truncate in dorsal and lateral view; (3) tympanum covered by skin, not visible through skin; (4) dorsal skin from smooth to shagreened in life and preservative, (5) presence of small cloacal enameled warts; (6) parietal peritoneum transparent, pericardium transparent, visceral and hepatic peritonea white, all other peritonea transparent; (7) liver bulbous; (8) humeral spine absent; (9) webbing formula of fingers III (2– – 2) – (2– – 2+) IV; (10) webbing formula of toes I (1 – 1+) – (2+ – 2 1/3) II (1 – 1 1/3) – (2+ – 2 1/4) III (1 – 1+) – (2 1/2 – 2 3/4) IV (2+ – 2 1/4) – (1+ – 1 1/4) V; (11) enameled ulnar and tarsal folds; (12) nuptial pad Type-V composed by a group of packed glands and situated in the medial, dorso-lateral internal side of Finger I, glands not present in other fingers, prepollex not evident from external view; (13) Finger I longer than Finger II; (14) eye diameter larger than width of disc on Finger III; (15) coloration in life: dorsum light green with big irregular darker green patches, black dots, and minute melanophores, bones white; (16) coloration in preservative: cream with big irregular white patches and black dots; (17) iris yellow with dark grey flecks; (18) minute melanophores not extending throughout fingers and toes except base of Finger IV and Toe V; in life, tip of fingers and toes white; (19) advertisement call composed by a single pulsed note lasting 0.05– 0.10 s, dominant frequency of 4220.5–5000.5 Hz, males call from the underside of leaves; (20) fighting behavior unknown; (21) egg clutches deposited on the underside of leaves, males often on the same leaf than eggs; (22) tadpole not described; (23) medium size adult males, SVL = 18.7–22.2 (20.3 ± 0.8, N = 25) mm and one female 22.0 mm.</p> <p> <b>Comparisons.</b> The following unique combination of phenotypic characters differentiates <i>Hyalinobatrachium iaspidiense</i> from all other species in the genus: snout truncated in dorsal and lateral views, tympanic membrane and annulus not appreciable in life, pericardium transparent, hand webbing formula III (2- -2) – (2–-2+) IV, dorsal coloration in life light green with big irregular darker green patches and black dots, dorsal coloration in preservative cream with big irregular white patches and black dots, iris coloration in life yellowish, clearing out towards eyelids, with dark flecks, coloration of bones in life white, coloration of hands and feet in life white, and a pulsed single note advertisement call without frequency or amplitude modulation, lasting 0.05– 0.10 s and with dominant frequency of 4220.5–5000.5 Hz.</p> <p> Morphological, bioacoustic and genetic evidences allowing the differentiation between <i>Hyalinobatrachium iaspidiense</i> and all other species of <i>Hyalinobatrachium</i> from the GS are summarized in Figures 2, 3, 5 and Tables 1, 3.</p> <p> <b>Remarks.</b> To address the question of whether or not there are morphological differences between <i>Hyalinobatrachium iaspidiense</i> and <i>H. nouraguense</i>, we compared the type material of both species plus additional specimens from Guyana (N = 8), French Guiana (N = 4), Suriname (N = 11), and Venezuela (N = 2). We could not find any difference in the studied characters. Furthermore, we found descriptive lapses in both Lescure and Marty (2000) and Cisneros-Heredia and McDiarmid (2007). These are not preservation artifacts as stated by Yáñez- Muñoz <i>et al</i>. (2009). Both works described the pericardium of <i>H</i>. <i>nouraguense</i> as white (versus transparent in <i>H</i>. <i>iaspidiense</i>); however, all the specimens examined (including the type series of <i>H</i>. <i>nouraguense</i> and other material from French Guiana) have a transparent pericardium (Fig. 5 D). The morphological characters, therefore, do not allow us to separate two species.</p> <p> We also compared the morphology of those specimens with that of the holotype of <i>Hyalinobatrachium mesai</i> (the only known specimen for this species). The only consistent difference was that <i>H</i>. <i>mesai</i> has green bones in life and those of <i>H</i>. <i>iaspidiense</i> and <i>H</i>. <i>nouraguense</i> are white (see also the Remarks section for <i>H. mesai</i>).</p> <p> Our bioacoustic analyses show, in concordance with the morphological data, that there are no divergent features between the calls of <i>H</i>. <i>iaspidiense</i>, <i>H</i>. <i>mesai</i> and <i>H</i>. <i>nouraguense</i>. The description of the call of <i>H</i>. <i>mesai</i> by Barrio-Amorós and Brewer-Carías (2008) indicates that it is longer than that of <i>H</i>. <i>iaspidiense</i> and <i>H</i>. <i>nouraguense</i>. However, the audiospectogram pictured in their publication has too little resolution to be properly compared. Our own analysis indicates that there are no differences in the calls between <i>H</i>. <i>iaspidiense</i> and <i>H</i>. <i>mesai</i>.</p> <p> Cocroft <i>et al.</i> (2001) report an unidentified species of <i>Hyalinobatrachium</i> from the Amazonian foothills of the Peruvian Andes in Manu National Park. They provide a photograph and the advertisement call of a single male. The specimen fully corresponds to the description here provided for <i>H</i>. <i>iaspidiense</i> and the parameters and structure of its advertisement call falls within the variability of <i>H</i>. <i>iaspidiense</i>. Rivera and Knell (2006) report a photograph of a specimen from the Amazonian lowlands of Tapiche, Loreto, Peru, which shares all of its morphological characters with <i>H</i>. <i>iaspidiense</i>. Photographs of six specimens collected from Río Ituxi, Amazonas, Brazil fully agree with the morphological characteristics of <i>H</i>. <i>iaspidiense</i> (J.P. Caldwell, unpublished data). The sequences obtained from tissue samples of these Brazilian specimens included in our study further support their identification (Fig. 3) and corroborate the morphological analysis. Accordingly, we assign all these specimens to <i>H</i>. <i>iaspidiense</i>, extending its distribution from its most western record in Brazil and Peru.</p> <p> <b>Biology and tadpole.</b> Very little information is available. Position of calling males and advertisement call are described in Señaris and Ayarzagüena (2005) and Lescure and Marty (2000). Here we provide new information from the known locality in Guyana (Ernst <i>et al.</i> 2005, 2006). Calling males were always found on the underside of leaves, usually facing leaf axils. Up to three males were observed calling from the same tree usually during and after rain. Individuals of <i>Hyalinobatrachium iaspidiense</i> occupied lower forest strata (≈ 1.0–2.0 m) in vegetation overhanging fast flowing segments of the creek, as compared to the sympatric <i>H</i>. <i>mondolfii</i>, whose calling sites were located in higher strata (≈ 4.0–6.0 m). Calling males of <i>H</i>. <i>iaspidiense</i> were found near by (≤ 5 cm) clutches containing 22 (in all cases recorded, N = 4) relatively large white eggs. A clutch containing 22 semi-developed tadpoles (Gosner 1960; stages 21–22) attached to a leaf overhanging a medium sized black water creek was collected on March 15, 2004 (tadpoles deposited at SMNS under field number MABT0104, no collection number assigned), and transferred to a plastic aquarium. The leaf was fastened to the lid of the container, which was filled with 10 cm of creek water. Eleven tadpoles had dropped into the container the following day, nine additional tadpoles followed on March 17. It took an additional day before all remaining tadpoles had left the clutch. The tadpoles of <i>H</i>. <i>iaspidiense</i> are typical exotroph, lotic, fossorial tadpoles (eco-morphological guild after Altig & Johnston 1989) with an elongate, vermiform habitus (Fig. 6 B). Compared to the larvae of the sympatric <i>H</i>. <i>mondolfii</i>, tadpoles of <i>H</i>. <i>iaspidiense</i> do not exhibit a clearly ovoid body. Rather, the anterior half is markedly triangular in shape, narrowing towards the base of the tail. Tadpoles of <i>H</i>. <i>iaspidiense</i> are always much darker than those of <i>H</i>. <i>mondolfii</i> (Fig. 6).</p> <p> <b>Ecology and distribution.</b> <i>Hyalinobatrachium iaspidiense</i> inhabits the lowland and upland forests of eastern Guiana Shield (50–1000 m) and western Amazon. It has always been found associated with streams. It is known from Brazil (Cordeiro-Duarte <i>et al.</i> 2002; Yáñez-Muñoz <i>et al.</i> 2009; Avila-Pires <i>et al.</i> 2010; this work), Ecuador (Yáñez-Muñoz <i>et al.</i> 2009; Guayasamin & North 2009), French Guiana (Lescure & Marty 2000; this work), Guyana (Ernst <i>et al.</i> 2005), Peru (Yáñez-Muñoz <i>et al.</i> 2009; this work), Suriname (Kok & Castroviejo-Fisher 2008), Venezuela (Ayarzagüena 1992; Señaris & Ayarzagüena 2005; this work), and expected to occur in the Amazon areas between the Ecuadorian and Peruvian localities and the GS.</p>Published as part of <i>Castroviejo-Fisher, Santiago, Vilà, Carles, Ayarzagüena, José, Blanc, Michel & Ernst, Raffael, 2011, Species diversity of Hyalinobatrachium glassfrogs (Amphibia: Centrolenidae) from the Guiana Shield, with the description of two new species, pp. 1-55 in Zootaxa 3132</i> on pages 20-23, DOI: <a href="http://zenodo.org/record/200895">10.5281/zenodo.200895</a&gt