Accurate identification of three cryptic species of rodents of the genus Calomys using RAPD-PCR and mitDNA RFLP markers

Calomys musculinus, Calomys laucha and Calomys venustus are cryptic species with overlapping distribution ranges. C. musculinus is the natural reservoir of Junin virus (Arenaviridae), the etiological agent of Argentine Hemorrhagic Fever. In epidemiological studies it is very important to unequivocally identify the species of individuals collected in the field in order to test virus infection. The purpose of this work was to describe molecular markers allowing a prompt and clear characterization of individuals of the three species. We studied the D-loop region of mitochondrial DNA by restriction fragment length polymorphism (RFLP). This region was amplified by PCR and the product was digested with nine restriction endonucleases (RE). Eight recognize 4 bp restriction sites (Taqα I, Tsp509 I, Aci I, Rsa I, Alu I, Nla III, Hae III and Mse I) and one recognized a 6 bp sequence (Ase I). Two of them (Aci I and Hae III) did not distinguish any of the three species. Alu I did not discriminate between C. musculinus and C. laucha, but clearly distinguished both from C. venustus. Taq I did not distinguish C. laucha from C. venustus, but differentiated both species from C. musculinus. Mse I distinguished the three species, but some of the polymorphisms of C. musculinus are very similar to C. laucha's restriction pattern. The enzyme Nla III distinguished the three species, but it is highly polymorphic within species. The enzymes Tsp509 I, Rsa I and Ase I clearly discriminated the three species, and patterns obtained with the three of them are recommended for reliable identification of individuals collected in the field. The same DNA samples were used to obtain Random Amplified Polymorphic DNAs (RAPDs) patterns. Several bands produced with primers A02, A06, A08, A09, B09 and OPA02 are species specific and could also be used for identification.     

Environmental determinants of the small mammal assemblage in an agroecosystem of central Argentina: The role of Calomys musculinus

Agricultural intensification in the central region of Argentina has been accompanied by rodent assemblage changes, involving an increase in Calomys species densities. In particular, Calomys musculinus, the main reservoir of Junín virus (etiological agent of the Argentine Haemorrhagic Fever, AHF) selects field borders over crop-fields. Borders represent relatively-stable habitats within agroecosystems, otherwise highly human modified landscapes. In this article, we assessed the effect of environmental variables on rodent species occurrence and assemblage in the field borders of a pampean agroecosystem. Small rodent occurrence was examined for variation according to vegetation species composition, structure and productivity, using canonical ordination techniques. In a total of 72 crop-field borders of Córdoba province, in spring, summer and autumn (2005-2006), we captured 1041 rodents of 8 different species. Akodon azarae, Calomys venustus and C. musculinus were the dominant species of the assemblage, with the first two associated one another, while C. musculinus tended to be related to different environmental variables. We showed that rodent species associations were mediated by resources that changed seasonally, such as vegetation cover. Also, the strength and “sign” of the association between species seemed to be a consequence of different habitat preferences and activity patterns. Finally, the intra-annual population cycle seemed to influence the relation between species and habitat structure and, possibly, inter-specific competition for resources. Therefore, we suggest that less generalist species of the rodent assemblage, well adapted to linear habitats, may limit C. musculinus occurrence throughout the agroecosystem by habitat and spatial competition. Any action aimed to control AHF, and therefore C. musculinus population density, should exclude negative effects on coexisting species.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

A review of hybridization in marine angelfishes (Perciformes: Pomacanthidae)

Synopsis Although hybridization of terrestrial and freshwater organisms has been well-studied, very little work has focused on hybridization among coral reef fish species. In the present paper, eleven examples of probable hybrids between marine angelfishes (Pomacanthidae) are reviewed. Evidence is presented which strongly suggests that the nominal speciesApolemichthys armitagei is invalid and that specimens previously identified as this species represent hybrids betweenA. trimaculatus andA. xanthurus. Of the remaining ten probable pomacanthid hybrids, five are inCentropyge (C. eibli x C. flavissimus, C. eibli x C. vrolikii, C. flavissimus x C. vrolikii, C. loricu0lus x C. potteri, andC. multifasciatus x C. venustus); one inHolacanthus (H. bermudensis x H. ciliaris), and four inPomacanthus (P. arcuatus x P. paru, P. chrysurus x P. maculosus, P. maculosus x P. semicirculatus, andP. sexstriatus x P. xanthometapon). An additional five examples of possible pomacanthid hybrids are described, two inCentropyge, two inChaetodontoplus and one inPomacanthus. Examination of hybrids may provide clues on reproductive behavior, dispersal capabilities, and phylogenetic relationships of species. More studies on hybridization in coral reef fish species, particularly those involving molecular techniques, are needed.     

Population genetic responses of two minnow species (Cyprinidae) to seasonal stream intermittency

Two species of minnows, Cyprinella lutrensis (Notropis) and C. umbratilis (Cyprinidae) were sampled from Denton and Hickory Creeks, respectively, in northcentral Texas during periods of continuous flow and intermittency from 1983 to 1986. Allozyme variability was assessed for 14 structural loci in each species. Average heterozygosity (H) in C. lutrensis ranged from 0.10 to 0.16, and proportion of polymorphic loci (P) ranged from 0.44 to 0.78, while H and P in C. umbratilis ranged from 0.00 to 0.08 and 0.00 to 0.44, respectively. Heterozygote deficiencies were observed during flowing and intermittent conditions for both species, accounting for deviations from Hardy-Weinberg equilibrium. Temporal heterogeneity, measured by F_ST, was higher in C. lutrensis (0.403) than in C. umbratilis (0.121). Allele frequencies fluctuated significantly in C. lutrensis but not in C. umbratilis. Genetic similarities between populations of C. lutrensis from flowing and intermittent conditions were highest between intermittent and subsequent flowing events. It is likely that intermittency resulted in a bottleneck and genetic drift, restructuring the genome of these fish as colonizers for subsequent flowing populations. These effects were less pronounced in C. umbratilis. Alternate effects of intermittency on the genomes of the species reflect differences in genetic strategies of a generalist, C. lutrensis, and a relative specialist, C. umbratilis.     

Genetic identification of three species of the genus Clarias using allozyme and mitochondrial DNA markers

Genetic variability in three clariid species, Clarias batrachus, C. gariepinus and C. macrocephalus, were investigated by allozyme electrophoresis and mitochondrial DNA (ND 5/6) RFLP markers. The catfish species C. batrachus and C. macrocephalus are native to the Indian subcontinent and Southeast Asia, respectively, while the African catfish C. gariepinus was introduced to Asian countries for culture. Sixteen gene loci from 12 enzyme systems were analyzed. Fixed allelic differences were evident between pairs of species at least at four loci. In mtDNA RFLP analysis, eight composite haplotypes were observed and each species was characterized by a set of haplotypes. The UPGMA phylogenetic tree revealed three distinct clusters: C. batrachus; C. gariepinus (India and Thailand); and C. macrocephalus.     

Spatial variation in abundance of the junin virus hosts in endemic and nonendemic Argentine haemorrhagic fever zones

Abstract Argentine haemorrhagic fever (AHF) is caused by Junin (JUN) virus, which is hosted by the drylands vesper mouse (Calomys musculinus). In this work we monitored population abundance of C. musculinus and rodent assemblages for 3 years in and outside the AHF endemic zones (northern Buenos Aires, southern Córdoba and Santa Fe Provinces, Argentina). The study area was divided into endemic and nonendemic zones. In the endemic zone epidemic sites were recognized, characterized by recent emergence and maintenance of AHF cases, and also historical sites, characterized by decreased incidence or disappearance of AHF human cases. In the nonendemic zone AHF has never been recognized. Although differences were statistically significant only during some periods, population abundance of C. musculinus was usually lower in the nonendemic sites. The pattern and magnitude of seasonal fluctuations in C. musculinus populations were also distinct in the nonendemic sites as compared to endemic sites. The relative abundance of C. musculinus in rodent assemblage was lower in nonendemic sites than in the endemic sites. The lower population densities and dampened seasonal dynamics may be at least partly responsible for the absence of AHF cases in the nonendemic zone. It is suggested that the balance between intra and interspecific interactions might be the cause of the pattern of incidence and prevalence of pathogens in the host species.     

Protein variation,taxonomy and differentiation in five species of marmosets (genusCallithrix Erxleben, 1777)

The electrophoretic patterns of 15 protein systems codified for 20 genetic loci were investigated using horizontal electrophoresis. A total of 150 blood samples, from five species of the genusCallithrix were analyzed. Polymorphic variation was observed in 10 out 20 loci analyzed. The genotypic distributions are in Hardy-Weinberg equilibrium. The average heterozygosity (H) varied from 1% to 5%, similar to those observed for other Neotropical primates. The genetic distance coefficients revealed a phylogenetic separation of these species into two groups: (1) “argentata” (C. humeralifer andC. emiliae); (2) “jacchus” (C. jacchus, C. penicillata, andC. geoffroyi). This arrangement is according to the taxonomic arrangement proposed byHershkovitz (1977),de Vivo (1988), andMittermeier et al. (1988). The results in each group are compatible with the subspecies values recorded for the Platyrrhini. These values showed that:C. humeralifer andC. emiliae are subspecies ofC. argentata;C. jacchus, C. penicillata, andC. geoffroyi are subspecies ofC. jacchus. These results also suggest thatC. j. geoffroyi is the “jacchus” group taxon, most similar genetically to the “argentata” group.     

Assessment of habitat quality for four small mammal species of the Monte Desert, Argentina

In the temperate desert of Argentina, the combined action of climatic and anthropogenic factors has contributed to the formation of a highly heterogeneous landscape. In the central region of the Monte desert, four small mammal species (Eligmodontia typus, Calomys musculinus, Akodon molinae and Graomys griseoflavus) coexist and show different habitat uses in response to spatial variability. Three main habitat types are present in the region: mesquite forest, the creosotebush community and sand dunes. These habitat types are present also in the surrounding grazing area.The objective of this study was to determine habitat quality for these species in a protected area (Reserve MaB Ñacuñán) and in the adjacent grazed area. For each species we estimated demographic parameters that are highly correlated to fitness in each habitat, and for both treatments (protected and grazed).We found that the protected area offered a higher quality habitat than the grazed area for all species, but principally A. molinae and G. griseoflavus. At a local scale, we found that A. molinae and C. musculinus clearly showed higher fitness in the more complex habitats as the creosotebush community and the mesquite forest. In contrast, for E. typus, open and simplest patches, such as sand dunes, were optimal for its survival and reproduction.     

Chromosome rearrangements in Pectinidae (Bivalvia: Pteriomorphia) implied based on chromosomal localization of histone H3 gene in four scallops

Chromosomal structural rearrangement in four scallops, Chlamys farreri (n = 19), Patinopecten yessoensis (n = 19), Chlamys nobilis (n = 16) and Argopecten irradians (n = 16), was studied by fluorescence in situ hybridization using histone H3 gene probes. The results show that histone H3 gene sites differ strikingly with regard to number, location, and intensity among, or even within these species. For example, two histone H3 gene loci were detected on the metaphase chromosomes of P. yessoensis, while one locus was found in the others. In P. yessoensis, differing intensities of hybridization signals were detected between homologues 5 and 11, and within homologue 11. These data suggest that the histone H3 gene is a qualified chromosome marker for the preliminary understanding of the historical chromosomal reconstructing of the Pectinidae family. The variable distribution patterns of the histone H3 gene suggest that gene duplication/diminution as well as chromosome rearrangements by inversion and translocation may have played important roles in the genomic evolution of Pectinidae. We also compiled our present results with former published data regarding the chromosome mapping of rDNAs in species of the Pectinidae family. Such comparative chromosomal mapping should improve our understanding of historical chromosomal reconstructions of modern-day scallops.     

Eight polymorphic microsatellite loci for bottlenose dolphin and other cetacean species

We developed eight polymorphic microsatellite loci that can be used to elucidate population structure and aid management of bottlenose dolphins, Tursiops truncatus, in the northwest Atlantic. These loci averaged nine alleles, with no evidence for null alleles. Their cross‐species utility was examined in Cephalorhynchus commersonii, Delphinus delphis, Delphinapterus leucas, Eschrictius robustus, Globicephala macrorhynchus, Lagenorhynchus obliquidens, Orcinus orca, Steno bredanensis and Stenella clymene. On average, 75% of the loci were polymorphic in these species (range = 50–88%). Our results indicate that these loci will be useful for elucidating population structure of bottlenose dolphin as well as of other cetacean species.     

Chromosomal polymorphism and small karyotypic differentiation in a group of Ctenomys species from Central Argentina (Rodentia: Octodontidae)

Cytogenetic analysis was performed in six nominal taxa of the genus Ctenomys with a diploid number of 2n=47/48. The studied species were: C. australis (2n=48, FN=76); C. mendocinus (2n=47/48, FN=68/75/76); C. porteousi (2n=47/48, FN=71/72/73); C. azarae (2n=47, FN=71); C. sp. (chasiquensis) (2n=47/48); and C. talarum (2n=48, FN=80). The first three species shared the whole complement, C. talarum shares with them 19 arms from a total of 43 (44%). In all species analyzed constitutive heterochromatin was detected in most short arms, and in several centromeres. Polymorphisms for several pairs involving the heterochromatic short arms together with a complex polymorphism of pair A1 were found in C. azarae, C. sp., C. mendocinus and C. porteousi. Intraindividual variation found in one specimen of C. porteousi involving heterochromatic arms is discussed.These results lead us to propose the inclusion of all species except C. talarum, within a complex called the mendocinus-group. A new case of conservatism for chromosomal number in the genus Ctenomys is found in this group.     

Territorial behaviour in corn mice, <Emphasis Type="Italic">Calomys musculinus</Emphasis> (Muridae: Sigmodontinae), with regard to mating system

We studied territorial behaviour of Calomys musculinus, a promiscuous-polygynous species, in order to test the predictions that during the breeding period, home-range-owner females are more aggressive than home-range intruders and that males do not display territorial behaviour. At the core of the breeding season, we conducted 136 intrasexual and 66 intersexual encounters. The study was carried out in four 0.25-ha enclosures, each containing one independent population. We studied territorial behaviour utilising focal animal sampling to record behaviour. Encounters were conducted in a circular opaque arena (COA) near the home-range’s activity mathematical centre (AMC). Amicable behaviour was not observed in any of the 78 trials among females. The rates of aggressive and submissive behaviour per female–female encounters varied in relation to whether they were home-range owners or intruders, the former being aggressive, the latter submissive. So we could link these drives to the territorial behaviour of C. musculinus. This result fits well with the already known territoriality of C. musculinus during the breeding season. In contrast, the 58 intrasexual male encounters did not show direct aggressive interactions between them. The rates of the different behaviours between males did not vary in relation to whether they were home-range owners or intruders. In our study, which took place during the breeding period, C. musculinus males were nonterritorial, and they never exhibited aggressiveness towards females.     

马尾松和黄山松两个核基因位点的群体遗传多样性和种间分化

利用两个核基因座位C3H和GI, 对重叠分布于中国东南部的两个松属(Pinus)物种马尾松(P. massoniana)和黄山松(P. hwangshanensis)的22个群体88个个体进行了遗传多样性和种间分化模式研究。在这两个核基因座位上, 两种植物都表现出较低的核苷酸多样性水平(马尾松π_sil = 0.001 71; 黄山松π_sil = 0.003 40), 但是马尾松要显著低于黄山松; 在种内分化水平上, 马尾松的种内遗传分化也明显低于黄山松(马尾松F_ST = 0.059; 黄山松F_ST = 0.339)。这可能是由于黄山松的海拔分布高于马尾松, 而高海拔分布使黄山松的分布区域更加片段化, 促使其形成较高的种内遗传多样性和遗传分化。分子变异分析(AMOVA)发现, 两物种基于两个核基因座位的种间差异为48.86%, 而GI基因座位上的种间差异明显高于C3H座位(GI: 77.24%, C3H: 20.48%), 同时, 基因谱系显示两物种的共享单倍型仅在C3H座位上存在。结合这两个基因的功能, 推测GI基因可能在物种形成过程中受到了一定的选择压力, 因为GI基因参与调控植物的开花时间, 而C3H与木质素表达水平的调控有关。不同的选择压力使得GI的进化速度相对较快, 从而加速了黄山松和马尾松的物种分化。     

Ecological preference between generalist and specialist rodents: spatial and environmental correlates of phenotypic variation

Different ecological preferences among species may result in differences in response to similar environmental variation. To test this hypothesis, we assessed the patterns of skull and mandible size and shape variation in three Sigmodontinae mice from agroecosystems of central Argentina with increasing degree of specialization: Calomys musculinus, Akodon azarae and Oxymycterus rufus. Spatial patterns in size and shape were analysed after controlling for allometry and sexual dimorphism using a total of 697 specimens. We then evaluated the covariation between shape, climatic and environmental variables and assessed the contribution of distinct climatic and environmental variables to phenotypic variability. Oxymycterus rufus displayed a marked spatial structure, and there was a high correlation between shape, climatic and environmental variables in this species. Climatic and environmental variables had a moderate effect on the phenotype of A. azarae, and were not correlated with morphological variation in C. musculinus. Our study highlights the difference in phenotypic responses to spatial and environmental gradients across coexisting species, specialist species displaying a more marked spatial structure in morphology than generalist species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 180–203.     

Genetic Divergence of Far-Eastern Dace Species Belonging to the Genus Tribolodon (Pisces, Cyprinidae) and Closely Related Taxa

Based on a biochemical–genetic approach, heterozygosity and divergence of structural genes at 30 enzyme loci were analyzed in six dace species. In addition, intra- and interspecific divergence of gene expression was analyzed based on a sample of 12 to 15 loci. Mean heterozygosities per individual varied as follows: Tribolodon species, H _obs = 0.007 ± 0.007 and H _exp = 0.007 ± 0.007; T. ezoe, H _obs = 0.045 ± 0.016 and H _exp = 0.067 ± 0.029. Several variants of genetic distances were estimated. Standard Nei's distances (D _N) varied from 0.145 to 0.284 in four dace species studied. As related to Tribolodon dace species, the following genetic distances were obtained for two members of other genera:Pseudaspius leptocephalus, D _N = 0.269; Leuciscus waleckii, D _N = 0.769. Based on the distance matrices, different clustering algorithms were realized. The main feature shared by different dendrograms was a separate position of the cluster joining Far-Eastern dace species, to whichP. leptocephalus and L. waleckii are successively added. Among the species studied, the proportion of loci similar by expression (E) varied from 87 to 100%. The greatest difference was found between landlocked and landlocked ecotypes of T. hakonensis, E = 67%. The following conclusions can be made: (1) Four studied species of the genus Tribolodon are rather well genetically differentiated. Diagnostic loci are available. (2) A nominal dace species, T. species, should be considered the fourth unambigous species of this genus, which is confirmed by its recent zoological acceptance of this species. (3) The origin and divergence of dace species belonging to the genus Tribolodon are relatively late (1 to 3 Myr ago) historical events. (4) Taxonomically, the genus Tribolodon belong to the tribe Pseudaspinini together with P. leptocephalus, which is confirmed by genetic data. (5) Data on heterozygosity and the divergence of structural and regulatory elements of genome, along with the proposed scheme of speciation types, suggest the following speciation modes for the species studied: for four species, adaptive divergence and for two species, genetic transformation.     

黄土高原典型草原优势种植物及其根际土壤化学计量对降雨变化的响应

为探讨黄土高原典型草原优势种植物及其根际土壤化学计量对不同降水量的响应,以宁夏固原云雾山封育20年典型草原为研究对象,利用遮雨棚技术模拟了3个不同的降水梯度(自然降水量的50%、100%和150%),测定了优势种长芒草(Stipa bungeana)、星毛委陵菜(Potentilla acaulis)和白莲蒿(Artemisia sacrorum)的地上、地下全碳(TC)、全氮(TN)、全磷(TP)含量及其根际土壤有机碳(RSOC)、全氮、全磷含量,分析了各个优势种及其根际土壤化学计量及其之间的相关性。结果表明：(1)3个优势种地上、地下总C含量、地上N含量均在50%降水下较高,星毛委陵菜地上和地下P含量及地上、地下总P含量均在100%降水处理下最高。(2)50%降水与150%降水处理相比较,长芒草地上C/N、C/P及白莲蒿、星毛委陵菜地下C/P均有显著差异(P<0.05),150%降水处理下优势种N/P显著高于50%降水处理(P<0.05)。(3) 3个优势种根际土壤C、N含量高于黄土高原土壤C、N含量且150%降水处理下根际土壤C含量显著高于50%降水处理(P<0...     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

Utilization of food resources by small and medium‐sized mammals in the Monte Desert biome,Argentina

Theoretical models of species coexistence between desert mammals have generally been based on a combination of food and microhabitat selection by granivorous rodents. Although these models are applicable in various deserts of the world, they cannot explain resource use by mammals in Neotropical deserts. The present study examines diet composition in a mammal assemblage in the Monte desert, Argentina. The results show that two main strategies are used by these mammals: medium‐sized species (hystricognath rodents: Dolichotis patagonum, Lagostomus maximus, Microcavia australis and Galea musteloides; and an exotic lagomorph: Lepus europaeus) are herbivores, whereas small‐sized species (a marsupial: Thylamys pusillus; and sigmodontine rodents: Graomys griseoflavus, Akodon molinae, Calomys musculinus, Eligmodontia typus) are omnivorous. Small mammals also show a tendency towards granivory (C. musculinus), insectivory (A. molinae and T. pusillus) and folivory (G. griseoflavus).     

Genetic differentiation of the Iberian tree frogs Hyla arborea molleri and Hyla meridionalis (Amphibia: Anura)

Genetic differentiation of Hyla arborea molleri and H. meridionalis was studied through allozyme electrophoresis and through essays of artificial hybridization. Samples were taken at several localities in Portugal, where these species occur either in sympatry or allopatry, and at one locality in France. Of the 21 presumptive loci analyzed, 6 were monoallelic, while 9 loci were discriminative between both species. A relatively high level of genetic differentiation was found between the two species (NEI's D = 0.725), contrasting with their morphological similarity. Remarkably low levels of genetic variability were found within species, with greater differentiation found among samples of H. meridionalis than of H. a. molleri. Although the capacity for interspecific hybridization was low, hybrid embryos resulted from both reciprocal crosses, but with much greater success in the cross ♂H. a. molleri x δH. meridionalis. Hybrids showed greater similarity with the male parent, and showed heterozygote allozymic phenotypes for discriminative loci. Results are compared with those of similar studies on the same and different species of Hyla, and differentiation of H. a. molleri and H. meridionalis is discussed in relation to their taxonomic status and origin.