Integrative Taxonomic Approach Research Articles

Taxonomy and Melanism Patterns of Freshwater Leeches in the Genus Glossiphonia (Hirudinea: Glossiphoniidae) from Northeast Asia

Freshwater leeches belonging to the genus Glossiphonia Johnson, 1816 are fairly common benthic annelids in freshwater ecosystems throughout Eurasia, but the taxonomy of this group remains poorly resolved, and the species content of some local faunas is unsatisfactorily known. For example, it was thought that the only widespread species, Glossiphonia complanata (Linnaeus, 1758), dwells in Northeast Asia. Here, we revise the Northeast Asian Glossiphonia leeches with an integrative taxonomic approach. This revision is based on the largest DNA-sequence and morphological datasets collected to date. Two Glossiphonia species are recorded from the region. First, Glossiphonia koreaensissp. nov., a putative regional endemic species, was discovered in South Korea. Second, a valid name and complete description are provided for Glossiphonia mollissima Moore, 1898 (non-Grube, 1871). The latter name was wrongly applied through misidentification and, according to ICZN (Article 49), is unavailable. Hence, we propose a new name, Glossiphonia mooreisp. nov., in memory of Dr. J. Percy Moore, who discovered this leech. It is a prospective trans-Beringian species, ranging through Eastern Siberia, the Russian Far East, and Alaska (although Alaskan records are based on nonsequenced samples), showing a significant proportion of melanic individuals in samples north of the Arctic Circle. The latter pattern may reflect substrate-induced cryptic coloration (camouflage), but this hypothesis needs to be statistically checked in the future. Finally, a complete checklist of Glossiphonia species is presented.

Why several when one can unite them all? Integrative taxonomic revision of Indo-Pacific freshwater pipefish (Nerophinae)

Abstract The family Syngnathidae (seahorses, sea dragons and pipefishes) is a fascinating group. Species are mostly distributed in marine coastal waters, but about 30 species inhabit Indo-Pacific tropical island rivers. There are currently six freshwater genera of Nerophinae (trunk-brooders): Belonichthys, Coelonotus, Doryichthys, Lophocampus, Microphis and Oostethus. Unlike other Syngnathidae, freshwater pipefish have been little studied; the taxonomy of Nerophinae is unclear and only based on morphomeristic data. The purpose of our study is to undertake a revision of this group based on an integrative taxonomy approach. By combining molecular data from four mitochondrial markers (5415 bp), morphomeristic data with a character mapping study and ecological data, we now recognize a single genus as valid, Microphis, as opposed to the six previously accepted. Our results also reveal one new candidate species, Microphis nicoleae sp. nov., and the resurrection of two others: Microphis torrentius and Microphis vaillantii. This study shows the relevance of integrative taxonomy for higher taxa delineation, using data integration by congruence.

Hidden species diversity and mito-nuclear discordance within the Mediterranean cone snail, Lautoconus ventricosus

The Mediterranean cone snail, Lautoconus ventricosus, is currently considered a single species inhabiting the whole Mediterranean basin and the adjacent Atlantic coasts. Yet, no population genetic study has assessed its taxonomic status. Here, we collected 245 individuals from 75 localities throughout the Mediterranean Sea and used cox1 barcodes, complete mitochondrial genomes, and genome skims to test whether L. ventricosus represents a complex of cryptic species. The maximum likelihood phylogeny based on complete mitochondrial genomes recovered six main clades (hereby named blue, brown, green, orange, red, and violet) with sufficient sequence divergence to be considered putative species. On the other hand, phylogenomic analyses based on 437 nuclear genes only recovered four out of the six clades: blue and orange clades were thoroughly mixed and the brown one was not recovered. This mito-nuclear discordance revealed instances of incomplete lineage sorting and introgression, and may have caused important differences in the dating of main cladogenetic events. Species delimitation tests proposed the existence of at least three species: green, violet, and red + blue + orange (i.e., cyan). Green plus cyan (with sympatric distributions) and violet, had West and East Mediterranean distributions, respectively, mostly separated by the Siculo-Tunisian biogeographical barrier. Morphometric analyses of the shell using species hypotheses as factor and shell length as covariate showed that the discrimination power of the studied parameters was only 70.2%, reinforcing the cryptic nature of the uncovered species, and the importance of integrative taxonomic approaches considering morphology, ecology, biogeography, and mitochondrial and nuclear population genetic variation.

A third species of glassfrog in the genus Chimerella (Anura, Centrolenidae) from central Peru, discovered by an integrative taxonomic approach

We studied the taxonomic status of glassfrogs collected in Departamento Huánuco, central Peru, which in the field were tentatively allocated to Chimerella, one of the twelve genera currently recognized in the family Centrolenidae. Detailed analyses of their morphology, bioacoustics, and molecular genetics supported their generic allocation and provided evidence for them representing a divergent and unnamed evolutionary lineage within Chimerella. We herein describe this lineage as a new species, being mainly distinguished from the two other known congeners, C. corleone and C. mariaelenae, by details of colouration in life and preservative, substantial differences in advertisement call, and differentiation in mitochondrial markers (12S rRNA, 16S rRNA, cytochrome b) and a nuclear-encoded marker (Rag-1). The new species is the southernmost distributed species in the genus and was found in a swampy habitat at the bank of the Río Patay Rondos, a tributary of the Río Monzon, in rainforest at the Andean-Amazon foothills at 798 m above sea level. Aspects of species delimitation within Chimerella and related future research are briefly addressed and discussed.

The Genus Heterogynis Rambur, 1866 (Heterogynidae, Lepidoptera): Congruence of Molecular, Morphological and Morphometric Evidence Reveal New Species in Serbia

The Heterogynidae are a small family of moths consisting of a single genus Heterogynis and sixteen described species distributed in the Mediterranean region. A species new to science, Heterogynis serbica sp. nov., is described from the locality of Srebrenac, Mt. Kopaonik, Republic of Serbia, Balkan Peninsula, by applying an integrative taxonomic approach using morpho-anatomical characteristics, wing morphometics and DNA barcoding. Male genitalia, scanning electron micrographs of adult male head anatomy, abdominal tergites/sternites, cocoons and habitats of the closely related species H. serbica sp. nov. and H. zikici are discussed and illustrated. Photographs of adult males and females, cocoons, plants in which the cocoons were found and habitats are shown. Importantly, marked differences in genital structure and other morphological characters were noted. These differences were confirmed with forewing morphometrics and COI-based DNA barcoding results. Additionally, DNA barcodes for H. serbica sp. nov. and H. zikici were compared against previously available data for the genus to evaluate the phylogenetic relationships. We conclude that deep, previously unknown and unexpected intrageneric morphological diversity exists in the genus Heterogynis.

First Record of the Northern Wolffish Anarhichas denticulatus Krøyer, 1845 (Anarhichadidae: Zoarcoidei: Perciformes) in the Siberian Arctic: Further Evidence of Atlantification?

A single specimen of the northern wolffish Anarhichas denticulatus Krøyer, 1845, 393 mm in length, was documented for the first time in the Siberian Arctic (Laptev Sea, Russia). Species identification was confirmed by an integrative taxonomic approach that included examination of external morphology and DNA barcoding using the COI mtDNA gene. This species is widely distributed in the North Atlantic, but records in the Arctic Ocean are limited to the Canadian and US coasts. This record might represent a significant range extension of about 7500 km for the species and may be associated with the eastward transport of a pelagic juvenile specimen from the Northeastern Atlantic to the Laptev Sea by the North Atlantic current, consistent with the hypothesis of Atlantification of the Arctic Ocean. X-ray images of the Laptev Sea specimen and photographs showing ontogenetic variations of species’ coloration are provided for reference. The Laptev Sea specimen had a more elongated shape, longer preorbital distance, and longer pectoral, dorsal, and anal fins, as well as a larger eye and wider caudal fin compared to North Atlantic samples. The size differences are likely associated with conditions experienced as a juvenile during the pelagic stage of the lifecycle.

Revision of the Gondwanan genus Chilepyris Evans, 1964 (Hymenoptera: Bethylidae)

AbstractThe Gondwanan genus Chilepyris Evans, 1964 (Scleroderminae) is studied and revised. A new species from Oceanian region (New Caledonia) is described and illustrated, Chilepyris kanak sp. nov., and compared with the other two species of this genus, C. herbsti Evans, 1964 from Chile and C. platythelys Sorg & Walker, 1989 from New Zealand, based on an integrative taxonomic approach, combining morphological characters and molecular analyses. An emended diagnosis and a deep discussion about the morphological diagnostic characters of this genus and comments about the Gondwana distribution of Chilepyris are also presented. Additionally, a taxonomic key to the three species is provided.

Meeting in Liguria: Hybridisation between Apennine endemic Euphorbia barrelieri and western Mediterranean E. nicaeensis led to the allopolyploid origin of E. ligustica

The Mediterranean Basin is renowned for its extremely rich biota and is considered as one of the 25 Global Biodiversity Hotspots, but its diversity is not homogeneously distributed. Outstanding in the number of (endemic) species are the Ligurian Alps (Italy). At the foot of the Ligurian Alps, little above the Mediterranean Sea, a disjunct occurrence of Italian endemic Euphorbia barrelieri was reported. Using an array of integrative methods ranging from cytogenetic (chromosome number and relative genome size estimation), over phylogenetic approaches (plastid, ITS and RAD sequencing) to multivariate morphometrics we disentangled the origin of these populations that were shown to be tetraploid. We performed phylogenetic analyses of the nuclear ITS and plastid regions of a broad taxonomic sampling of Euphorbia sect. Pithyusa to identify possible species involved in the origin of the tetraploid populations and then applied various analyses of RADseq data to identify the putative parental species. Our results have shown that the Ligurian populations of E. barrelieri are of allotetraploid origin that involved E. barrelieri and western Mediterranean E. nicaeensis as parental species. We thus describe a new species, E. ligustica, and hypothesise that its adaptation to similar environments in which E. barrelieri occurs, triggered development of similar morphology, whereas its genetic composition appears to be closer to that of E. nicaeensis. Our study emphasises the importance of polyploidisation for plant diversification, highlights the value of the Ligurian Alps as a hotspot of biodiversity and endemism and underlines the importance of integrative taxonomic approaches in uncovering cryptic diversity.

Zooplankton studies in Colombian fresh and brackish water ecosystems: A review and future perspectives

Colombia is a global biodiversity hotspot, yet its zooplankton fauna remains poorly explored. In Colombian inland waters, 253 species of rotifers, 119 cladocerans and 82 copepods (not included benthic species) have been reported, in contrast with only 14 genera of protozoa. Most publications report on the Magdalena-Cauca basin, with the highest number of records for lowland areas (< 1000 m.a.s.l.). The least explored regions are the Chocó, Orinoco and Amazon basins. Colombian zooplankton checklists often include several taxa recognized as groups of cryptic species or ones described as cosmopolitan. Integrative taxonomic approaches are scarce, and diversity data need to be validated by molecular analyses and detailed morphological descriptions using optical and scanning electron microscopy. Historical, regional, and local environmental factors that explain the richness, distribution, and the abundance of zooplankton include: water bodies size, hydrological connectivity, trophic status; physical stability and, hydro-climatic patterns of variation. The analysis of ecosystem processes related to patterns in zooplankton communities highlights the importance of detritus as an alternative energy flow path in aquatic ecosystems at all altitude ranges. We propose to strengthen regional studies on functional groups, ecological redundancy, and adaptive traits to evaluate zooplankton responses to climatic, global and local stressors. Due to the great diversity of aquatic ecosystems in the extensive altitudinal gradient of Colombia, it is necessary to expand the coverage of ecological and taxonomic studies to understand zooplankton biodiversity and its role in ecosystem functioning.

Consilience Across Multiple, Independent Genomic Data Sets Reveals Species in a Complex with Limited Phenotypic Variation.

Species delimitation in the genomic era has focused predominantly on the application of multiple analytical methodologies to a single massive parallel sequencing (MPS) data set, rather than leveraging the unique but complementary insights provided by different classes of MPS data. In this study, we demonstrate how the use of two independent MPS data sets, a sequence capture data set and a single-nucleotide polymorphism (SNP) data set generated via genotyping-by-sequencing, enables the resolution of species in three complexes belonging to the grass genus Ehrharta, whose strong population structure and subtle morphological variation limit the effectiveness of traditional species delimitation approaches. Sequence capture data are used to construct a comprehensive phylogenetic tree of Ehrharta and to resolve population relationships within the focal clades, while SNP data are used to detect patterns of gene pool sharing across populations, using a novel approach that visualizes multiple values of K. Given that the two genomic data sets are independent, the strong congruence in the clusters they resolve provides powerful ratification of species boundaries in all three complexes studied. Our approach is also able to resolve a number of single-population species and a probable hybrid species, both of which would be difficult to detect and characterize using a single MPS data set. Overall, the data reveal the existence of 11 and five species in the E. setacea and E. rehmannii complexes, with the E. ramosa complex requiring further sampling before species limits are finalized. Despite phenotypic differentiation being generally subtle, true crypsis is limited to just a few species pairs and triplets. We conclude that, in the absence of strong morphological differentiation, the use of multiple, independent genomic data sets is necessary in order to provide the cross-data set corroboration that is foundational to an integrative taxonomic approach. [Species delimitation; genotyping-by-sequencing; population structure; integrative taxonomy; cryptic species; Ehrharta (Poaceae).].

More than meets the eye: characterizing the cryptic species complex and Symbiodiniaceae communities in the reef-dwelling nudibranch Pteraeolidia ‘semperi’ (Nudibranchia: Aeolidioidea) from Singapore

ABSTRACT The advent of molecular systematics has revolutionized our knowledge of biodiversity, revealing undiscovered cryptic lineages across the tree of life. Correspondingly, an increasing number of nudibranch species complexes have been found and described through integrative taxonomic approaches. Pteraeolidia ‘semperi’ (Nudibranchia: Cladobranchia: Aeolidioidea) presently represents a species complex, possessing ambiguous systematics and an underestimated diversity. Sequencing and phylogenetic reconstructions based on the cytochrome c oxidase subunit I marker from 48 samples of P. ‘semperi’ collected from Singapore's southern waters, as well as morphological analyses including scanning electron microscopy of the radula, suggest that P. ‘semperi’ comprises several distinct cryptic lineages. We further characterized the Symbiodiniaceae communities of P. ‘semperi’ from Singapore using metabarcoding of the internal transcribed spacer 2 gene. Pteraeolidia ‘semperi’ from Singapore was found to predominantly host Cladocopium and Durusdinium symbionts, and was significantly structured by biogeography. This study elucidates the present taxonomic diversity of P. ‘semperi’ in Singapore and contributes to the growing body of work on the diversity of algal symbionts associated with this enigmatic taxon.

Integrative taxonomy reveals two new narrowly-endemic crayfish species (Decapoda: Cambaridae) from the Yadkin River Basin in western North Carolina, USA.

Two new species of stream-dwelling crayfish, Cambarus lapidosus, the Stony Fork Crayfish, and Cambarus burchfielae, the Falls Crayfish, are described from the Yadkin River basin in western North Carolina, USA, using an integrative taxonomic approach consisting of morphological, genetic, and biogeographic data. Both species were previously considered to be members of the widely distributed Cambarus species C complex, which occurs throughout mid-Atlantic Slope river basins; however, they are in fact morphologically and genetically more similar to the Cambarus robustus species complex from interior basins in the south-central Appalachians, indicating Atlantic basin stream capture of an Interior basin faunal group has occurred in this region. Both new species described herein can be differentiated from these two complexes, and each other, by several morphological characteristics. Cambarus lapidosus and C. burchfielae are differentiated from C. species C by the absence of cervical spines and the presence of 1-2 subpalmar tubercles on the chelae in most specimens; both species are less-punctate across the areola than C. aff. robustus. Cambarus burchfielae is further differentiated from C. lapidosus, C. species C, and C. aff. robustus by the presence of a narrower and sparsely punctate areola, a single weak row of tubercles on the mesial margin of the palm, and the absence of tubercles on the dorsal surface of the dactyl. The newly described species are genetically sister taxa and together are most similar by genetic distance to undescribed members of the C. robustus species complex from the nearby (~30 km) Watauga River basin and most phylogenetically similar to C. aff. robustus from the adjacent New River basin; both are interior basin drainages. The newly-described species are endemic to the Blue Ridge Mountains of western North Carolina and have restricted distributions (<100 km2-150 km2) in small (<4th order) tributaries to the Yadkin River. Cambarus lapidosus is known only from the upper Stony Fork watershed and C. burchfielae is known only from the upper reaches of the adjacent Lewis Fork watershed; both species likely qualify for conservation status protections under narrow geographic range criteria.

First Study of Ascaris lumbricoides from the Semiwild Population of the Sumatran Orangutan Pongo abelii in the Context of Morphological Description and Molecular Phylogeny.

There is little evidence that the already described and accepted taxa of ascarids (Ascaris lumbricoides, A. suum, and A. ovis) infecting individuals of taxonomically distant groups (hominids, pigs, sheep, goats, and dogs) can be genetically or morphologically distinguished. However, despite described morphological differences, e.g., due to intraspecific variation, these are insufficient for species determination and may indicate differences amongst ascarids because of cross infections, hybrid production, and specific adaptations to hosts. Herein, the results of a molecular and morphological analysis of ascarids parasitising Sumatran orangutans (Pongo abelii Lesson, 1827) in native populations are presented. The research took place in the Bukit Lawang area, Indonesia, in 2009. Throughout the year, fresh faecal samples were collected regularly from 24 orangutans, and all were examined for the presence of nematode adults. Only five adult worms from two orangutan females were found during regular collection. Using the integrative taxonomic approach, the nematodes found were identified as A. lumbricoides. The significance of the find and its rarity is documented by the fact that this is the first confirmed finding of adult ascarids from an original orangutan site (not from a zoo) in more than 130 years (including the long-term study spanning the last 20 years focusing on orangutan parasites and natural antiparasitic drugs). More accurate morphometric parameters and genetic differences for the identification of ascarids were established. These parameters will be helpful for other findings in great apes and will also be suitable for further and precise determination of this parasite. The details distinguishing between male and female specimens are also stated and well defined. A comprehensive evaluation of the situation of Ascaris species parasitising orangutans, including a comparison with previously described orangutan parasite (i.e., A. satyri-species inquirenda), is discussed.

A New Spotted Species of the Cyrtodactylus (Geckoella) collegalensis (Beddome, 1870) (Reptilia: Squamata) Complex from Coastal Kerala, Southwestern India

We describe a new spotted species of ground-dwelling gecko of the genus Cyrtodactylus (Geckoella) from coastal Kerala, southwestern India, by using an integrative taxonomic approach. The new species is a member of the C. collegalensis species complex, with 10.0–13.5% uncorrected mitochondrial sequence divergence from the other six members of the C. collegalensis complex. The new species is also recognized in tree-based delimitation methods and can be morphologically diagnosed by a spotted dorsal pattern of four to six pairs of spots (occasionally fused into figure 8–shaped markings) from the banded species C. speciosus, C. rishivalleyensis, and C. yakhuna and from the spotted C. collegalensis, C. srilekhae, and C. varadgirii by the presence of a few scattered enlarged dorsal scales. This is the only Indian member of the C. collegalensis complex with a relatively broad distribution at low elevations, other than C. varadgirii from western India (distributed in Maharashtra, southern Gujarat, and western Madhya Pradesh). The sister species to the new species is the Sri Lankan C. yakhuna, which is a banded form from low elevations. The new species is the second gecko to have been described from Chengodumala and endemic to northern and central Kerala, highlighting the importance of this area, which is under increasing anthropogenic pressure. We also provide notes on the dorsal pholidosis of C. rishivalleyensis, which was overlooked in its original description.

Species of Ergasilus von Nordmann, 1832 (Copepoda: Ergasilidae) from cichlid fishes in Lake Tanganyika.

Ergasilus (von Nordmann, 1832) (Ergasilidae) is a species-rich group of parasitic copepods with a wide distribution in freshwater, marine and brackish environments. Up to now, 9 species of Ergasilus are known from cichlid fishes in Africa. In this study, 5 species, including 3 new, were collected from the gills of 12 cichlid species (11 genera: Bathybates, Ctenochromis, Eretmodus, Gnathochromis, Lamprologus, Neolamprologus, Ophthalmotilapia, Perissodus, Simochromis, Spathodus and Tanganicodus) of the northeastern shore of Lake Tanganyika in Burundi, namely E. macrodactylus (Sars, 1909), E. megacheir (Sars, 1909), E. caparti n. sp., E. parasarsi n. sp. and E. parvus n. sp. All species found were identified and described on the basis of adult female specimens using an integrative taxonomy approach mixing morphological characterization and molecular analyses of 2 ribosomal DNA markers (partial 18S and 28S rDNA sequences). An identification key for Ergasilus species from Lake Tanganyika is included. This study provides the first molecular data for Ergasilus species in Africa. The phylogenetic analyses suggest that the Ergasilus species parasitizing Lake Tanganyikan cichlids form a well-supported clade within the Ergasilidae. However, their phylogenetic relationships with other congeners still remain unclear due to a lack of molecular data for this diverse genus.

The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests

The distribution of the White-Lipped Frog Amnirana albolabris was long assumed to extend from eastern-central to western African rainforests. However, recent molecular phylogenetic analyses revealed that this taxon likely represents several undescribed species. Because the name-bearing types were collected in Gabon, the distantly related West African populations clearly represent an undescribed species that partly occurs in sympatry with Amnirana fonensis. Based on an integrative taxonomic approach, including molecular, morphological, and acoustic data, we describe the 'albolabris' populations from the Upper Guinean Forest Zone as a new species, and redescribe the morphologically similar A. fonensis on the bases of a larger series of genotyped individuals, including the first known females. We also provide new biological information for A. fonensis, including their advertisement call, habitat, and reproductive data. The new species is sister to A. fonensis and the two species differ by 5.8% in the mitochondrial 16S ribosomal RNA gene. Morphologically, males of the new species are smaller and have relatively smaller humeral glands. Females of the new species seem to have shorter legs than A. fonensis. In comparison to A. fonensis, the advertisement call of the new species has a higher dominant frequency and more pronounced frequency modulation. The two species differ in their distribution and habitat preferences, as revealed by environmental niche modelling. Whereas the new species is restricted to the Upper Guinean forests and thus is a true lowland-rainforest inhabitant, A. fonensis lives predominantly in Guinean montane forests in the forest-savanna mosaic zone.

Phylogenetic placement and reappraisal of Diorygma karnatakense including the new synonym, Diorygma dandeliense, from Maharashtra, India

AbstractThis study re-examined the status of species of Diorygma Eshw. known from the Western Ghats using an integrative taxonomy approach that includes morphological and chemical data, as well as multigene phylogenetic analyses. Prior to this work, the two species D. karnatakense and D. dandeliense were distinguished primarily on lirellae morphology (branching pattern) and the number of ascospores per ascus. Our study of the morphology, chemistry and molecular phylogeny (mtSSU, LSU and RPB2) of freshly collected samples and re-examination of type material suggests that both names should be synonymized. Consequently, D. karnatakense is accepted as the correct name, with D. dandeliense as a newly proposed synonym. Phylogenetically, D. karnatakense is allied to D. antillarum and D. hieroglyphicum.

Prevalence and ecological factors affecting the distribution of plant-parasitic nematodes in Prunus groves in Spain

A wide survey was conducted to study plant-parasitic nematodes (PPNs) associated with Prunus groves in Spain. This research aimed to determine the prevalence and distribution of PPNs in Prunus groves, as well as the influence of explanatory variables describing soil, climate and agricultural management in structuring the variation of PPNs community composition. A total of 218 sampling sites were surveyed and 84 PPN species belonging to 32 genera were identified based of an integrative taxonomic approach. PPN species considered as potential limiting factors in Prunus production, such as Meloidogyne arenaria, M. incognita, M. javanica, Pratylenchus penetrans and P. vulnus, were identified in this survey. Seven soil physico-chemical (C, Mg, N, Na, OM, P, pH and clay, loamy sand and sandy loam texture classes), four climate (Bio04, Bio05, Bio13 and Bio14) and four agricultural management variables (grove-use history less than 10 years, irrigation, apricot seedling rootstock, and Montclar rootstock) were identified as the most influential variables driving spatial patterns of PPNs communities. In particular, younger plantations showed higher values for species richness and diversity indices than groves cultivated for more than 20 years with Prunus spp. Our study increases the knowledge of the distribution and prevalence of PPNs associated with Prunus rhizosphere, as well as on the influence of explanatory variables driving the spatial structure PPNs communities, which has important implications for the successful design of sustainable management strategies in the future in this agricultural system.

A new glassfrog species of the genus Centrolene (Amphibia, Anura, Centrolenidae) from Cordillera del Cóndor, southern Ecuador.

Based on an integrative taxonomical approach, using molecular, morphological, and bioacoustics data, a new species of glassfrog of the genus Centrolene is described from Refugio de Vida Silvestre El Zarza, southern Ecuador. Centrolenezarzasp. nov. is a medium sized species, easily distinguished from all other glassfrogs by its unique combination of characters, such as a shagreen dorsum with elevated warts corresponding to white spots, an evident tympanum, half or more than half of the upper parietal peritoneum covered by iridophores, iridophores absent on all visceral peritonea, including the pericardium, a lobed liver lacking iridophores, males with small projecting humeral spines, the outer edges of forearms and tarsus with a row of enameled warts that often continue into the external edges of Finger IV and/or Toe V, and white or yellowish white iris with thick black reticulations. The new species is closely related to a currently undescribed species and superficially resembles C.condor, C.pipilata, C.solitaria, C.altitudinalis, and C.daidalea. The tadpole and advertisement and courtship calls are described, and the threats to the species survival, mainly represented by habitat loss and contamination due to mining activities, are briefly discussed.

Four new species of the genus Diploderma Hallowell, 1861 (Squamata, Agamidae) from China.

Four new species of Diploderma are described from Sichuan and Yunnan provinces, southwestern China, based on an integrative taxonomic approach, combining morphological and genetic data. The first new species from Danba County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.flaviceps, but it can be diagnosed from the latter by having a relatively much shorter tail and by a genetic distance of 4.4% in the ND2 gene; the second new species from Muli County, Sichuan Province, is phylogenetically closely related to D.daochengense, D.yongshengense, and D.yulongense, but it can be diagnosed from the latter three species by having a pale yellow gular spot and by genetic distances of 5.6-6.7% in the ND2 gene; the third new species from Jiulong County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.angustelinea, but it can be diagnosed from the latter by having a relatively much longer tail and by a genetic distance of 2.8% in the ND2 gene; and the last new species from Weixi County, Yunnan Province, is phylogenetically closely related to D.aorun, but it can be diagnosed from the latter by having a pale yellow gular spot and by a genetic distance of 2.9% in the ND2 gene. Our work brings the number of species within the genus Diploderma to 46.