Unconfirmed Candidate Species Research Articles

Frogs of the genus Platypelis from the Sorata massif in northern Madagascar: description of a new species and reports of range extensions

We describe a new species of arboreal microhylid frog, genusPlatypelis, from northeastern Madagascar and report the expansion of distribution ranges of two other species.Platypelis laetussp. nov.is small to medium-sized (24.3–25.6 mm snout-vent length) compared to otherPlatypelis, exhibits a greenish colored throat and was found in bamboo forest of the Sorata Massif. Its advertisement call consists of a single short tonal note repeated at regular intervals in long call series. Based on DNA sequences of a fragment of the mitochondrial 16S rRNA gene, the new species was placed in a clade withPlatypelis olgaefrom the Tsaratanana Massif, and with two other, unconfirmed candidate species from the Sorata Massif and from Andravory, herein namedPlatypelissp. Ca12 and Ca13. Molecular divergences among these lineages were substantial, amounting to 7.6‒8.1% uncorrected 16S p-distance to the closest nominal species,P. olgae, from which the new species is also distinguished by a lack of allele sharing in the nuclear RAG-1 gene. We also provide new records ofPlatypelis alticolaandP. tsaratananaensisfrom the Sorata Massif, supported by molecular analysis. This confirms a wider distribution of these two species that previously were considered to be endemic to the Tsaratanana Massif. However, their populations in Sorata were characterized by a certain degree of genetic differentiation from Tsaratanana populations suggesting they require more detailed taxonomic assessment.

High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae)

For many years, Paramacrobiotus richtersi was reported to consist of populations with different chromosome numbers and reproductive modes. To clarify the relationships among different populations, the type locality of the species (Clare Island, Ireland) and several Italian localities were sampled. Populations were investigated with an integrated approach, using morphological (LM, CLSM, SEM), morphometric, karyological, and molecular (18S rRNA, cox1 genes) data. Paramacrobiotus richtersi was redescribed and a neotype designed from the Irish bisexual population. Animals of all populations had very similar qualitative and quantitative characters, apart from the absence of males and the presence of triploidy in some of them, whereas some differences were recorded in the egg shell. All populations examined had the same 18S haplotype, while 21 haplotypes were found in the cox1 gene. In four cases, those qualitative characters were correlated with clear molecular (cox1) differences (genetic distance 14.6–21.8%). The integrative approach, which considered the morphological differences in the eggs, the reproductive biology and the wide genetic distances among putative species, led to the description of four new species (Paramacrobiotus arduus sp. n., Paramacrobiotus celsus sp. n., Paramacrobiotus depressus sp. n., Paramacrobiotus spatialis sp. n.) and two Unconfirmed Candidate Species (UCS) within the P. richtersi complex. Paramacrobiotus fairbanksi, the only ascertained parthenogenetic, triploid species, was redescribed and showed a wide distribution (Italy, Spain, Poland, Alaska), while the amphimictic species showed limited distributions. The difference in distribution between apomictic and amphimictic populations can be explained by the difference in the dispersal potentials associated with these two types of reproduction.

A Pan-Amazonian species delimitation: high species diversity within the genus Amazophrynella (Anura: Bufonidae).

Amphibians are probably the most vulnerable group to climate change and climate-change associate diseases. This ongoing biodiversity crisis makes it thus imperative to improve the taxonomy of anurans in biodiverse but understudied areas such as Amazonia. In this study, we applied robust integrative taxonomic methods combining genetic (mitochondrial 16S, 12S and COI genes), morphological and environmental data to delimit species of the genus Amazophrynella (Anura: Bufonidae) sampled from throughout their pan-Amazonian distribution. Our study confirms the hypothesis that the species diversity of the genus is grossly underestimated. Our analyses suggest the existence of eighteen linages of which seven are nominal species, three Deep Conspecific Lineages, one Unconfirmed Candidate Species, three Uncategorized Lineages, and four Confirmed Candidate Species and described herein. We also propose a phylogenetic hypothesis for the genus and discuss its implications for historical biogeography of this Amazonian group.

Body doubles: an integrative taxonomic approach reveals new sibling species of land planarians

Records of cryptic species have continued to emerge in the scientific literature, often revealed by the use of molecular phylogenetic analyses in an integrative taxonomic approach. This study addresses a group of four striped flatworms from the genus Pasipha Ogren & Kawakatsu, showing a pale median stripe on a dark dorsal surface. Based on morphological and molecular analyses from the cytochrome c oxidase subunit I gene (COI), we establish that we are dealing with sibling species that are closely related to P. brevilineata Leal-Zanchet, Rossi & Alvarenga, 2012, a recently described species with a similar colour pattern. Thus, we describe three of the studied flatworms as new species and propose one new unconfirmed candidate species based on molecular data. In addition, sequence analysis revealed 40 nucleotide autapomorphies supporting the species studied herein. Considering anatomical and histological features, the three new species are differentiated from their congeners mainly by details of the copulatory apparatus, such as the occurrence of an epithelium of pseudostratified appearance lining the female atrium and the shape and position of the proximal portion of the prostatic vesicle.

A new frog species of the subgenus Asperomantis (Anura, Mantellidae, Gephyromantis) from the Bealanana District of northern Madagascar

A recent study on a group of rough-skinned Gephyromantis frogs from Madagascar (Anura: Mantellidae: Mantellinae) established a new subgenus, Asperomantis, with five described species and one undescribed candidate species. Based on newly collected material from the Bealanana District, we address the taxonomy of this candidate species, and reveal that it consists of two populations with low genetic and morphological divergence but considerable bioacoustic differences that are obvious to the human ear. As a result, we describe some of the specimens formerly assigned to Gephyromantis sp. Ca28 as G. angano sp. n. and assign the remaining specimens from a locality between Bealanana and Antsohihy to a new Unconfirmed Candidate Species, G. sp. Ca29. Gephyromantis angano sp. n. is a small species that strongly resembles G. asper and G. ceratophrys, but it differs from these and all other Gephyromantis species by a unique, clinking advertisement call. The new species may be highly threatened by habitat fragmentation, but at present we recommend it be treated as Data Deficient until more data are available to assess its distribution. We discuss the curious relationship between G. angano sp. n. and G. sp. Ca29, which we suspect may represent a case of incipient speciation. We also identify two additional new Unconfirmed Candidate Species of Gephyromantis based on sequence data from other specimens collected during our surveys in the Bealanana District.

A Biodiversity Hotspot Heats Up: Nine New Species of Suckermouth Catfishes (Mochokidae:Chiloglanis) from Upper Guinean Forest Streams in West Africa

The widespread species, Chiloglanis occidentalis, is recorded in flowing waters from Senegal to Ghana. Recent studies of this species within the Upper Guinean Forests revealed genetic divergence among the populations sampled, suggesting the presence of several unconfirmed candidate species. A detailed study of these populations revealed morphological variation congruent with the molecular results. Nine new species are described herein: Chiloglanis camarabounyi, new species, Chiloglanis kolente, new species, Chiloglanis kabaensis, new species, Chiloglanis dialloi, new species, Chiloglanis loffabrevum, new species, Chiloglanis longibarbis, new species, Chiloglanis pezoldi, new species, Chiloglanis nzerekore, new species, and Chiloglanis tweddlei, new species. These new species, each endemic to specific river basins, are diagnosed through a combination of meristic and morphometric characters. Chiloglanis waterloti, from the Senegal and Niger river basins, is elevated from synonymy with Chiloglanis occidental...

Systematics of the Dendropsophus leucophyllatus species complex (Anura: Hylidae): Cryptic diversity and the description of two new species.

Genetic data in studies of systematics of Amazonian amphibians frequently reveal that purportedly widespread single species in reality comprise species complexes. This means that real species richness may be significantly higher than current estimates. Here we combine genetic, morphological, and bioacoustic data to assess the phylogenetic relationships and species boundaries of two Amazonian species of the Dendropsophus leucophyllatus species group: D. leucophyllatus and D. triangulum. Our results uncovered the existence of five confirmed and four unconfirmed candidate species. Among the confirmed candidate species, three have available names: Dendropsophus leucophyllatus, Dendropsophus triangulum, and Dendropsophus reticulatus, this last being removed from the synonymy of D. triangulum. A neotype of D. leucophyllatus is designated. We describe the remaining two confirmed candidate species, one from Bolivia and another from Peru. All confirmed candidate species are morphologically distinct and have much smaller geographic ranges than those previously reported for D. leucophyllatus and D. triangulum sensu lato. Dendropsophus leucophyllatus sensu stricto occurs in the Guianan region. Dendropsophus reticulatus comb. nov. corresponds to populations in the Amazon basin of Brazil, Ecuador, and Peru previously referred to as D. triangulum. Dendropsophus triangulum sensu stricto is the most widely distributed species; it occurs in Amazonian Ecuador, Peru and Brazil, reaching the state of Pará. We provide accounts for all described species including an assessment of their conservation status.

High Species Richness of Scinax Treefrogs (Hylidae) in a Threatened Amazonian Landscape Revealed by an Integrative Approach.

Rising habitat loss is one of the main drivers of the global amphibian decline. Nevertheless, knowledge of amphibian diversity needed for effective habitat protection is still highly inadequate in remote tropical regions, the greater part of the Amazonia. In this study we integrated molecular, morphological and bioacoustic evidence to evaluate the species richness of the treefrogs genus Scinax over a 1000 km transect across rainforest of the Purus-Madeira interfluve, and along the east bank of the upper Madeira river, Brazilian Amazonia. Analysis revealed that 82% of the regional species richness of Scinax is still undescribed; two nominal species, seven confirmed candidate species, two unconfirmed candidate species, and one deep conspecific lineage were detected in the study area. DNA barcoding based analysis of the 16s rRNA gene indicates possible existence of three discrete species groups within the genus Scinax, in addition to the already-known S. rostratus species Group. Quantifying and characterizing the number of undescribed Scinax taxa on a regional scale, we provide a framework for future taxonomic study in Amazonia. These findings indicate that the level to which Amazonian anura species richness has been underestimated is far greater than expected. Consequently, special attention should be paid both to taxonomic studies and protection of the still-neglected Amazonian Scinax treefrogs.

Hidden diversity in the Podarcis tauricus (Sauria, Lacertidae) species subgroup in the light of multilocus phylogeny and species delimitation

The monophyletic species subgroup of Podarcis tauricus is distributed in the western and southern parts of the Balkans, and includes four species with unresolved and unstudied inter- and intra-specific phylogenetic relationships. Using sequence data from two mitochondrial and three nuclear genes and applying several phylogenetic methods and species delimitation approaches to an extensive dataset, we have reconstructed the phylogeny of the Podarcis wall lizards in the Balkans, and re-investigated the taxonomic status of the P. tauricus species subgroup. Multilocus analyses revealed that the aforementioned subgroup consists of five major clades, with P. melisellensis as its most basal taxon. Monophyly of P. tauricus sensu stricto is not supported, with one of the subspecies (P. t. ionicus) displaying great genetic diversity (hidden diversity or cryptic species). It comprises five, geographically distinct, subclades with genetic distances on the species level. Species delimitation approaches revealed nine species within the P. tauricus species subgroup (P. melisellensis, P. gaigeae, P. milensis, and six in the P. tauricus complex), underlining the necessity of taxonomic re-evaluation. We thus synonymize some previously recognized subspecies in this subgroup, elevate P. t. tauricus and P. g. gaigeae to the species level and suggest a distinct Albanian-Greek clade, provisionally named as the P. ionicus species complex. The latter clade comprises five unconfirmed candidate species that call for comprehensive studies in the future.

The taxonomy of the Tarentola mauritanica species complex (Gekkota: Phyllodactylidae): Bayesian species delimitation supports six candidate species.

The lack of morphological diagnosable characters typical of cryptic species, poses a particular problem to taxonomists. This is especially true when taxa are closely related, sharing considerable amounts of ancestral polymorphism. Phylogenetic studies on the Moorish gecko species-complex, Tarentola mauritanica, uncovered extremely high levels of mtDNA diversity with six identified clades, including one from the Canary Islands identified as T. angustimentalis. Because of the conserved morphology of this species and its paraphyletic status with respect to T. angustimentalis, it was suggested that T. mauritanica is a cryptic species complex. Nevertheless, none of the nuclear loci used were reciprocally monophyletic regarding the mitochondrial lineages due to retention of ancestral polymorphism. In this study, we added three new intron markers to the already available dataset and used additional tools, namely phylogenetic gene trees, species tree and species limits within a Bayesian coalescent framework to confirm the support of the main lineages. Bayesian clustering analysis supports all six mtDNA lineages as independent groups, despite showing signs of ancestral polymorphism or possibly gene flow between the Maghreb/South Iberia and Central Morocco clades. The species tree recovered two major groups; one clustering taxa from Europe and Northern Maghreb and another one encompassing the lineages from Central/Southern Morocco, Central Morocco and Canary Islands, indicating that the ancestor of T. angustimentalis came from the Central/Southern Morocco region. Finally, Bayesian coalescent species delimitation analysis supports all six mitochondrial clades as "unconfirmed candidate species", pending morphological data to define them.

DNA Barcoding Survey of Anurans across the Eastern Cordillera of Colombia and the Impact of the Andes on Cryptic Diversity

Colombia hosts the second highest amphibian species diversity on Earth, yet its fauna remains poorly studied, especially using molecular genetic techniques. We present the results of the first wide-scale DNA barcoding survey of anurans of Colombia, focusing on a transect across the Eastern Cordillera. We surveyed 10 sites between the Magdalena Valley to the west and the eastern foothills of the Eastern Cordillera, sequencing portions of the mitochondrial 16S ribosomal RNA and cytochrome oxidase subunit 1 (CO1) genes for 235 individuals from 52 nominal species. We applied two barcode algorithms, Automatic Barcode Gap Discovery and Refined Single Linkage Analysis, to estimate the number of clusters or “unconfirmed candidate species” supported by DNA barcode data. Our survey included ~7% of the anuran species known from Colombia. While barcoding algorithms differed slightly in the number of clusters identified, between three and ten nominal species may be obscuring candidate species (in some cases, more than one cryptic species per nominal species). Our data suggest that the high elevations of the Eastern Cordillera and the low elevations of the Chicamocha canyon acted as geographic barriers in at least seven nominal species, promoting strong genetic divergences between populations associated with the Eastern Cordillera.

Revision of Madagascar's Dwarf Lemurs (Cheirogaleidae:Cheirogaleus): Designation of Species, Candidate Species Status and Geographic Boundaries Based on Molecular and Morphological Data

The genus Cheirogaleus, the dwarf lemurs, is a radiation of strepsirrhine primates endemic to the island of Madagascar. The dwarf lemurs are taxonomically grouped in the family Cheirogaleidae (Infraorder: Lemuriformes) along with the genera Microcebus, Mirza, Allocebus, and Phaner. The taxonomic history of the genus Cheirogaleus has been controversial since its inception due to a paucity of evidence in support of some proposed species. In this study, we addressed this issue by expanding the geographic breadth of samples by 91 individuals and built upon existing mitochondrial (cytb and COII) and nuclear (FIBA and vWF) DNA datasets to better resolve the phylogeny of Cheirogaleus. The mitochondrial gene fragments D-loop and PAST as well as the CFTR-PAIRB nuclear loci were also sequenced. In agreement with previous genetic studies, numerous deep divergences were resolved in the C. major, C. minor and C. medius lineages. Four of these lineages were segregated as new species, seven were identified as confirmed candidate species, and four were designated as unconfirmed candidate species based on comparative mitochondrial DNA sequence data gleaned from the literature or this study. Additionally, C. thomasi was resurrected. Given the widespread distribution of the genus Cheirogaleus throughout Madagascar, the methodology employed in this study combined all available lines of evidence to standardize investigative procedures in a genus with limited access to type material and a lack of comprehensive sampling across its total distribution. Our results highlighted lineages that likely represent new species and identified localities that may harbor an as-yet undescribed cryptic species diversity pending further field and laboratory work.

Integrative delineation of species of Mediterranean freshwater planarians (Platyhelminthes: Tricladida: Dugesiidae)

The paper presents an integrative taxonomic study on dugesiid freshwater flatworms from the north-eastern Mediterranean region by applying both morphological and molecular criteria in the formulation of stable species hypotheses. The morphological information obtained for the specimens was used in a traditional way by comparing the organismal traits of the various populations and candidate species with those of known species, as documented in the taxonomic literature and as revealed by examination of histological sections of museum specimens. In the molecular species delimitation the General Mixed Yule-Coalescent method (GMYC) was used. Results of this study (1) supported the presence of 13 Dugesia species in the Hellenic area (including D. sicula Lepori, 1948, a pan-Mediterranean species), (2) culminated in the description of four new Dugesia species, (3) suggested the presence of two Confirmed Candidate Species, (4) pointed to 12 GMYC-delimited units in Greece and two in Slovakia as Unconfirmed Candidate Species and (5) revealed the presence of an entirely new genus, represented by two newly described species and a third Unconfirmed Candidate Species. Our results revealed a high diversity of dugesiid species in this relatively small region. It is concluded that the morphological features used by taxonomists in comparative studies of dugesiid flatworms generally result in reliable identifications and delineations of species taxa, except in the case of cryptic species. © 2013 The Linnean Society of London

Phylogeny and phylogeography of the Malagasy leaf-tailed geckos in the Uroplatus ebenaui group

Leaf-tailed geckos, genus Uroplatus, are one of the most prominent endemic reptile groups from Madagascar, but the species diversity and diversification of this taxonomic group are not completely understood. Here, we present a phylogenetic reconstruction of the small-sized Uroplatus which are included in the Uroplatus ebenaui group, focusing on the most widespread species of these geckos, Uroplatus phantasticus, which occupies a large mid-altitude distribution range in the eastern rainforests of Madagascar. Our phylogeny is based on DNA sequences of four mitochondrial genes (12S rRNA, 16S rRNA, COI and ND4), with a total of 2 312 base pairs. Partitioned Bayesian analysis confirmed U. malama from south-eastern Madagascar as the most basal representative of the group, followed by U. ebenaui from northern lowlands, and clades containing various undescribed candidate species as well as U. finiavana from Montagne d'Ambre in the north. Within our main targeted species U. phantasticus, the northernmost population from Zahamena (here considered as unconfirmed candidate species) forms the most basal lineage, while the southernmost populations studied from Marolambo, Ranomafana and Kianjavato form a nested clade, suggesting the species has expanded its range southwards undergoing repeated events of isolation and differentiation of lineages. Within the Ranomafana–Kianjavato area, we identified three different lineages, of which the two from Ranomafana are differentiated by 28 mutational steps in a fragment of the ND4 gene and occur on opposing banks of the Namorona River. This result suggests that this relatively small river currently limits dispersal and gene flow in these geckos. However, further downstream the Kianjavato population is more closely related to the population on the opposite side of the Namorona in Ranomafana, indicating that this genetic isolation is not absolute, and the river probably represents only a secondary barrier to gene flow in U. phantasticus.

Integrated Operational Taxonomic Units (IOTUs) in Echolocating Bats: A Bridge between Molecular and Traditional Taxonomy

BackgroundNowadays, molecular techniques are widespread tools for the identification of biological entities. However, until very few years ago, their application to taxonomy provoked intense debates between traditional and molecular taxonomists. To prevent every kind of disagreement, it is essential to standardize taxonomic definitions. Along these lines, we introduced the concept of Integrated Operational Taxonomic Unit (IOTU). IOTUs come from the concept of Operational Taxonomic Unit (OTU) and paralleled the Molecular Operational Taxonomic Unit (MOTU). The latter is largely used as a standard in many molecular-based works (even if not always explicitly formalized). However, while MOTUs are assigned solely on molecular variation criteria, IOTUs are identified from patterns of molecular variation that are supported by at least one more taxonomic characteristic.Methodology/Principal FindingsWe tested the use of IOTUs on the widest DNA barcoding dataset of Italian echolocating bats species ever assembled (i.e. 31 species, 209 samples). We identified 31 molecular entities, 26 of which corresponded to the morphologically assigned species, two MOTUs and three IOTUs. Interestingly, we found three IOTUs in Myotis nattereri, one of which is a newly described lineage found only in central and southern Italy. In addition, we found a level of molecular variability within four vespertilionid species deserving further analyses. According to our scheme two of them (i.e. M. bechsteinii and Plecotus auritus) should be ranked as unconfirmed candidate species (UCS).Conclusions/SignificanceFrom a systematic point of view, IOTUs are more informative than the general concept of OTUs and the more recent MOTUs. According to information content, IOTUs are closer to species, although it is important to underline that IOTUs are not species. Overall, the use of a more precise panel of taxonomic entities increases the clarity in the systematic field and has the potential to fill the gaps between modern and traditional taxonomy.

Description of two tadpoles of Malagasy treefrogs, Spinomantis sp. aff. peraccae and Spinomantis tavaratra (Anura: Mantellidae)

The aim of this study is to present morphological descriptions of the tadpoles of two species of Malagasy treefrogs of the genus Spinomantis (Anura: Mantellidae). The first of these, Spinomantis sp. aff. peraccae, from Ambohitantely is an unconfirmed candidate species (not included in Vieites et al. 2009) of which no adult specimen was collected so far but which is distinguished by a substantial genetic divergence of 6% to S. peraccae, Genbank accession AY848415, from Antoetra in the 16S rRNA gene. This candidate species is thus far taxonomically undescribed, but describing its tadpole contributes to the understanding of larval diversity in the genus Spinomantis and might prove to be important for a future revision of the S. peraccae complex, once that the larvae of S. peraccae and other related candidate species from northern Madagascar become known.

Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory

Amphibians are in decline worldwide. However, their patterns of diversity, especially in the tropics, are not well understood, mainly because of incomplete information on taxonomy and distribution. We assess morphological, bioacoustic, and genetic variation of Madagascar's amphibians, one of the first near-complete taxon samplings from a biodiversity hotspot. Based on DNA sequences of 2,850 specimens sampled from over 170 localities, our analyses reveal an extreme proportion of amphibian diversity, projecting an almost 2-fold increase in species numbers from the currently described 244 species to a minimum of 373 and up to 465. This diversity is widespread geographically and across most major phylogenetic lineages except in a few previously well-studied genera, and is not restricted to morphologically cryptic clades. We classify the genealogical lineages in confirmed and unconfirmed candidate species or deeply divergent conspecific lineages based on concordance of genetic divergences with other characters. This integrative approach may be widely applicable to improve estimates of organismal diversity. Our results suggest that in Madagascar the spatial pattern of amphibian richness and endemism must be revisited, and current habitat destruction may be affecting more species than previously thought, in amphibians as well as in other animal groups. This case study suggests that worldwide tropical amphibian diversity is probably underestimated at an unprecedented level and stresses the need for integrated taxonomic surveys as a basis for prioritizing conservation efforts within biodiversity hotspots.