Species-level Lineages Research Articles

Dispersal and speciation in purple swamphens (Rallidae:Porphyrio)

Dispersal, when accompanied by reduced gene flow and natural selection, influences speciation rates among groups of organisms. We used molecular phylogenetics, divergence time estimates, and population genetics to reconstruct the mode, pattern, and tempo of diversification within the wide-ranging purple swamphens (genus Porphyrio), with emphasis on the “supertramp” P. porphyrio. Our results suggest that the Porphyrio clade arose during the Middle Miocene in Africa, with a single colonization in the Americas and several other colonizations in Southeast Asia and the Indo-Pacific around 10 mya. We found that the widespread P. porphyrio is not monophyletic. Indeed, several subspecies and subspecies groups may represent species-level lineages. The P. p. melanotus lineage probably reached Australasia during the Pleistocene (600 kya), although some islands were colonized only in the past few hundred years. New Zealand, and some other islands, had previously been colonized (∼2.5 mya) by flying Porphyrio that evolved into flightless endemic species. Early and recent lineages are now sympatric. Widespread occupation of oceanic islands implies high dispersal and colonization rates, but gene flow probably occurs episodically and follows varying routes at different times. This pattern of colonization enables populations to differentiate and, ultimately, speciate.

Species delimitation in the lichenized fungal genus Vulpicida (Parmeliaceae, Ascomycota) using gene concatenation and coalescent-based species tree approaches.

• Species boundaries in many organism groups are still in a state of flux, and for empirical species delimitation, finding appropriate character sets and analytical tools are among the greatest challenges. In the lichenized fungal genus Vulpicida, six morphologically circumscribed species have been distinguished, but phenotypic characters partly overlap for three of these and intermediate forms occur. We used a combination of phylogenetic strategies to delimit the species in this genus.• Five DNA loci were sequenced and analyzed. Single-locus gene trees and a five-locus concatenated phylogeny were constructed to assess current Vulpicida species. Species boundaries were inferred from molecular data using two coalescent-based species delimitation methods (BP&P and Brownie) and from species trees reconstructed with three different algorithms (*BEAST, BEST, and STEM).• The two species restricted to North America, Vulpicida canadensis and V. viridis, are clearly distinct in all analyses. The four other traditionally accepted species form two strongly supported, closely related species-level lineages within the core group of the genus. On the basis of these results, we propose four instead of the current six species in the genus: V. canadensis, V. juniperinus, V. pinastri, and V. viridis, while V. tilesii and V. tubulosus are reduced to synonymy under V. juniperinus.• Coalescent species delimitation and tree inference give consistent results for fully distinct Vulpicida species but not for diverging populations. Even the inconsistent results were informative, revealing developing isolation despite a complex history of recombination and incomplete lineage sorting.

Echinostoma 'revolutum' (Digenea: Echinostomatidae) species complex revisited: species delimitation based on novel molecular and morphological data gathered in Europe.

BackgroundThe systematics of echinostomes within the so-called 'revolutum' group of the genus Echinostoma, which encompasses the type-species E. revolutum and a number of morphologically similar species, has long been controversial. Recent molecular studies indicate the existence of more species than previously considered valid, thus stressing the need for wider taxon sampling from natural host populations. This is especially true for Europe where morphological evidence indicates higher species diversity than previously thought, but where molecular data are virtually lacking. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in the investigation of a dataset with larger taxonomic and geographical coverage.MethodsMore than 20,000 freshwater snails belonging to 16 species were collected during 1998–2012 from various localities in eight countries in Europe. Snail screening provided representative larval isolates for five species of the 'revolutum' group, identified by their morphology. Adult isolates for four species recovered from natural and experimental infections were also identified. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for 74 and 16 isolates, respectively; these were analysed together with the sequences of Echinostoma spp. available on GenBank.ResultsDelineation of the European Echinostoma spp. was carried out based on molecular, morphological and ecological data. The large-scale screening revealed infections with five Echinostoma spp., including one new species: E. revolutum (sensu stricto), E. miyagawai, E. paraulum, E. bolschewense and Echinostoma n. sp. The newly-generated nad1 sequences from Europe fall into six distinct, well-supported, reciprocally monophyletic lineages corresponding to the species identifications based on morphology; this was corroborated by the 28S rDNA sequences. The analyses of the total nad1 dataset provided evidence for 12 monophyletic groups and five singletons, which represent seven described/named species and ten cryptic species-level lineages of Echinostoma.ConclusionWe conclude that nad1 should be the first choice for large-scale barcode-based identification of the species of the 'revolutum' group. Our study provides a comprehensive reference library for precisely identified isolates of the European species and highlights the importance of an integrative approach for species identification linking molecular, morphological and biological data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0520-8) contains supplementary material, which is available to authorized users.

Discordant phylogenies suggest repeated host shifts in the Fusarium–Euwallacea ambrosia beetle mutualism

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1–12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea–Fusarium mutualism near the Oligocene–Miocene boundary ∼19–24Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

Diplostomum von Nordmann, 1832 (Digenea: Diplostomidae) in the sub-Arctic: descriptions of the larval stages of six species discovered recently in Iceland

Frequent infections with Diplostomum spp. (Digenea: Diplostomidae) were found in the freshwater snail Radix peregra (Müller) and three fish species, the salmonids Salmo trutta fario L., Salvelinus alpinus (L.) and the gasterosteid Gasterosteus aculeatus L., collected in four lakes in south-western Iceland in 2012. Detailed analysis of the isolates integrating molecular, morphological and ecological data revealed that these belong to Diplostomum spathaceum (Rudolphi, 1819) and five putative new species (three infecting both snails and fish). This paper provides detailed descriptions of the metacercariae of the six species-level lineages of Diplostomum spp. and of the cercariae of three of the lineages discovered in Iceland with comments on the application of ITS1 rDNA for species distinction within Diplostomum von Nordmann, 1832 in the light of the novel data. We strongly suggest the use of molecular evidence based on cox1 gene sequences (in addition to ITS1-5.8S-ITS2 sequences) in association with detailed assessment of the morphology of the larval stages in future studies of Diplostomum spp. in fish and snails.

How do you solve a problem like Letharia? A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPs from multilocus sequence data.

The inclusion of molecular data is increasingly an integral part of studies assessing species boundaries. Analyses based on predefined groups may obscure patterns of differentiation, and population assignment tests provide an alternative for identifying population structure and barriers to gene flow. In this study, we apply population assignment tests implemented in the programs STRUCTURE and BAPS to single nucleotide polymorphisms from DNA sequence data generated for three previous studies of the lichenized fungal genus Letharia. Previous molecular work employing a gene genealogical approach circumscribed six species-level lineages within the genus, four putative lineages within the nominal taxon L. columbiana (Nutt.) J.W. Thomson and two sorediate lineages. We show that Bayesian clustering implemented in the program STRUCTURE was generally able to recover the same six putative Letharia lineages. Population assignments were largely consistent across a range of scenarios, including: extensive amounts of missing data, the exclusion of SNPs from variable markers, and inferences based on SNPs from as few as three gene regions. While our study provided additional evidence corroborating the six candidate Letharia species, the equivalence of these genetic clusters with species-level lineages is uncertain due, in part, to limited phylogenetic signal. Furthermore, both the BAPS analysis and the ad hoc ΔK statistic from results of the STRUCTURE analysis suggest that population structure can possibly be captured with fewer genetic groups. Our findings also suggest that uneven sampling across taxa may be responsible for the contrasting inferences of population substructure. Our results consistently supported two distinct sorediate groups, ‘L. lupina’ and L. vulpina, and subtle morphological differences support this distinction. Similarly, the putative apotheciate species ‘L. lucida’ was also consistently supported as a distinct genetic cluster. However, additional studies will be required to elucidate the relationships of other L. columbiana s.l. populations with the two sorediate genetic clusters.

Phylogenetic diversity of true morels (Morchella), the main edible non-timber product from native Patagonian forests of Argentina

Morchella species are edible fungi in high demand and therefore command high prices in world markets. Phenotypic-based identification at the species-level remains inadequate because of their complex life cycles, minor differences and plasticity of morphological characteristics between species, and the lack of agreement between scientific and common names. In Patagonia–Argentina, morels are associated with native forests of Austrocedrus chilensis (Cordilleran or Chilean cypress) and Nothofagus antarctica (ñire) and several exotic conifers that were introduced from western North America. Little is known about their taxonomy and phylogenetic relationships with other species in the genus. This work focused on the identification of collections of Morchella from Patagonia and their phylogenetic relationships with other species from the Northern Hemisphere. The comparison was made by analysis of DNA sequences obtained from four loci: the nuclear ribosomal internal transcribed spacer region (ITS) and the partial RNA polymerase I gene (RPB1) for the complete collection; and ITS, RPB1, RNA polymerase II gene (RPB2), and translation elongation factor (EF1-α) for the species-rich Elata Subclade. Analyses of individual and combined data sets revealed that Patagonian morels belong to the Elata Clade and comprised three strongly supported species-level lineages from both Patagonian native forest, and exotic trees introduced from western North America. One lineage was identified as Morchella frustrata phylogenetic species Mel-2, which is known from the USA and Canada. The second lineage, which appeared to be ‘fire-adapted’, was identified as Morchella septimelata phylogenetic species (Mel-7), which is also known from the USA. This species was collected from burned native forests mainly composed of A. chilensis and N. antarctica but also Pseudotsuga menziesii (Mirb.) Blanco, which is native to western North America. The phylogenetic analyses suggested that the third species from Patagonia was nested within the species-rich Elata Subclade and represents a new species-level lineage (informally designated Mel-37) within Elata Clade. The present collections from Patagonia constitute the southernmost latitude from which Morchella has been reported to date. The identification of two Argentine morels as North American taxa is therefore a remarkable biogeographic pattern. In view of the hypothesis that the Elata Clade originated in western North America, we speculate that at least two of the lineages colonized South America from North America via long distance dispersal, migration or, more likely, they were introduced with the exotic tree species that they were collected near.

Integrative taxonomy uncovers high levels of cryptic species diversity in <italic>Hemiphyllodactylus</italic> Bleeker, 1860 (Squamata: Gekkonidae) and the description of a new species from Peninsular Malaysia

Conserving the planet's biodiversity is greatly handicapped, in that only a small fraction of it (∼14–75%) has been described so far. Integrative taxonomy is making significant inroads in light of this challenge by incorporating multiple data sets across a wide range of disciplines that simultaneously elucidate phylogenetic structure and delimit species-level lineages within a unified species concept. An integrative taxonomic approach to the classification of the gekkonid genus Hemiphyllodactylus Bleeker, 1860 reveals that it is far more diverse than posited by a recent taxonomic revision based solely on morphology, and that it is composed of at least 19 species, most of which are montane upland or insular endemics. Three new species (Hemiphyllodactylus dushanensis sp. nov., Hemiphyllodactylus jinpingensis sp. nov., and Hemiphyllodactylus longlingensis sp. nov.) from southern China previously considered to be subspecies of Hemiphyllodactylus yunnanensis (Boulenger, 1903) are elevated to full species status, and 10 new species-level lineages ranging from Vietnam, Laos, Thailand, Myanmar, Peninsular Malaysia, the Philippines, and Indonesia are identified. One new species, Hemiphyllodactylus tehtarik sp. nov. from Gunung Tebu, Terengganu, Peninsular Malaysia, is described herein, and is differentiated from all other species of Hemiphyllodactylus on the basis of morphology, colour pattern, and an 18.1–31.5% sequence divergence from all other congeners. Hemiphyllodactylus larutensis (Boulenger, 1900) is removed from the synonymy of H. harterti (Werner, 1900). Using an integrative taxonomic approach imbues the revised classification of Hemiphyllodactylus with more objectivity, stability, and phylogenetic history, while identifying undescribed species-level lineages in potential need of conservation. © 2013 The Linnean Society of London

Integrative taxonomy uncovers high levels of cryptic species diversity inHemiphyllodactylus Bleeker, 1860 (Squamata: Gekkonidae) and the description of a new species from Peninsular Malaysia

Conserving the planet's biodiversity is greatly handicapped, in that only a small fraction of it (∼14–75%) has been described so far. Integrative taxonomy is making significant inroads in light of this challenge by incorporating multiple data sets across a wide range of disciplines that simultaneously elucidate phylogenetic structure and delimit species-level lineages within a unified species concept. An integrative taxonomic approach to the classification of the gekkonid genus Hemiphyllodactylus Bleeker, 1860 reveals that it is far more diverse than posited by a recent taxonomic revision based solely on morphology, and that it is composed of at least 19 species, most of which are montane upland or insular endemics. Three new species (Hemiphyllodactylus dushanensis sp. nov., Hemiphyllodactylus jinpingensis sp. nov., and Hemiphyllodactylus longlingensis sp. nov.) from southern China previously considered to be subspecies of Hemiphyllodactylus yunnanensis (Boulenger, 1903) are elevated to full species status, and 10 new species-level lineages ranging from Vietnam, Laos, Thailand, Myanmar, Peninsular Malaysia, the Philippines, and Indonesia are identified. One new species, Hemiphyllodactylus tehtarik sp. nov. from Gunung Tebu, Terengganu, Peninsular Malaysia, is described herein, and is differentiated from all other species of Hemiphyllodactylus on the basis of morphology, colour pattern, and an 18.1–31.5% sequence divergence from all other congeners. Hemiphyllodactylus larutensis (Boulenger, 1900) is removed from the synonymy of H. harterti (Werner, 1900). Using an integrative taxonomic approach imbues the revised classification of Hemiphyllodactylus with more objectivity, stability, and phylogenetic history, while identifying undescribed species-level lineages in potential need of conservation. © 2013 The Linnean Society of London

Starting from scratch: Evolution of the lichen thallus in the basidiolichen Dictyonema (Agaricales: Hygrophoraceae)

Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought to represent only a single genus with few species, includes several well-supported genus-level clades, all of which form associations with a unique lineage of obligately lichenized cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nuclear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny obtained via maximum likelihood and Bayesian approaches indicates the presence of five genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species, a paraphyletic grade representing Dictyonema s.str., and three clades representing the genera Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the lichenized thallus in the group, ancestral character state reconstruction was done using six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal sheath and cyphelloid–stereoid basidiocarps partially incorporated into the lichen thallus in Dictyonema, to squamulose–foliose thalli with corticioid basidiocarps entirely supported by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that, at least in this case, lichenized thalli may have evolved from reproductive structures in their nonlichenized ancestors.

Novel Rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas

The taxonomic affiliations of nineteen root-nodule bacteria isolated from the common bean (Phaseolus vulgaris L.) in Mexico, Ecuador and Brazil were investigated by analyses of 16S rRNA and of four protein-coding housekeeping genes. One strain from Mexico could be assigned to Rhizobium etli and two from Brazil to Rhizobium leucaenae, whereas another from Mexico corresponded to a recently described bean-nodulating species-level lineage related to R. etli and Rhizobium phaseoli. Ten strains isolated in Ecuador and Mexico corresponded to three novel Rhizobium lineages that fall into the R. phaseoli/R. etli/Rhizobium leguminosarum clade. One of those lineages, with representatives isolated mostly from Ecuador, seems to be dominant in beans from that Andean region. Only one of the Mexican strains clustered within the Rhizobium tropici clade, but as an independent lineage. Interestingly, four strains were affiliated with species within the Rhizobium radiobacter clade. The existence of yet non-described native Rhizobium lineages in both the Andean and Mesoamerican areas is discussed in relation to common-bean diversity and environmental conditions.

DNA barcode identification of lichen-forming fungal species in the Rhizoplaca melanophthalma species-complex (Lecanorales, Lecanoraceae), including five new species

Recent studies using sequence data from multiple loci and coalescent-based species delimitation have revealed several species-level lineages within the phenotypically circumscribed taxon Rhizoplaca melanophthalma sensu lato. Here, we formally describe five new species within this group, R. occulta, R. parilis, R. polymorpha, R. porterii, and R. shushanii, using support from the coalescent-based species delimitation method implemented in the program Bayesian Phylogenetics and Phylogeography (BPP) as the diagnostic feature distinguishing new species. We provide a reference DNA sequence database using the ITS marker as a DNA barcode for identifying species within this complex. We also assessed intraspecific genetic distances within the six R. melanophthalma sensu lato species. While intraspecific genetic distances within the five new species were less than or equal to the lowest interspecific pairwise comparison values, an overlap in genetic distances within the R. melanophthalma sensu stricto clade suggests the potential for additional phenotypically cryptic lineages within this broadly distributed lineage. Overall, our results demonstrate the potential for accurately identifying species within the R. melanophthalma group by using molecular-based identification methods.

Molecular phylogeny of the genus Sticta (lichenized Ascomycota: Lobariaceae) in Colombia

We present a molecular phylogenetic study of the lichen genus Sticta focusing on Colombia, using the ITS fungal barcoding gene for a total of 370 ingroup OTUs, with 322 newly generated sequences. The topology resulting from a maximum likelihood approach does not support current species concepts in Sticta, which use a morphological concept, but in contrast shows that similar morphodemes evolved multiple times independently within the genus. As a consequence, currently applied names such as S. fuliginosa and S. weigelii comprise numerous (up to more than 20) unrelated species-level lineages, which can be distinguished also phenotypically using previously unrecognized characters such as lobe configuration, lobe surface structure, tomentum type, and anatomy of the basal membrane of the cyphellae. We conclude that the genus Sticta contains about four to five times the number of species currently recognized. In Colombia alone, approximately 150 species of Sticta are present.

Leptochlorella corticola gen. et sp. nov. and Kalinella apyrenoidosa sp. nov.: two novel Chlorella-like green microalgae (Trebouxiophyceae, Chlorophyta) from subaerial habitats

The diversity of green microalgae in subaerial habitats remains largely unexplored and a number of new genus- and species-level lineages have been discovered recently. The traditional green algal genus, Chlorella, which accommodated coccoid unicellular green algal species with globular to oval cells, reproducing entirely by autospores, has been found to be polyphyletic. In this study, we provide a detailed characterization of two strains of microalgae isolated from tree bark in the Mediterranean. These algae share the general Chlorella-like morphology and their 18S rRNA and rbcL gene sequences place them in the Trebouxiophyceae. Strain CAUP H8401 forms an independent trebouxiophycean lineage, together with three previously published 18S rRNA gene environmental sequences of undescribed microalgae, which were retrieved from profoundly different habitats. In contrast, strain CAUP H7902 is related to Kalinella bambusicola in the Watanabea clade of the Trebouxiophyceae on the basis of its 18S rRNA gene sequence. This relationship is also supported by the rbcL gene sequence, acquired from the type strain of K. bambusicola. The investigated strains are described as representatives of a novel species in a new genus, Leptochlorella corticola gen. et sp. nov., and a novel species, Kalinella apyrenoidosa sp. nov., according to the International Code of Nomenclature for Algae, Fungi and Plants.

Miocene divergence, phenotypically cryptic lineages, and contrasting distribution patterns in common lichen-forming fungi (Ascomycota: Parmeliaceae)

Despite the recent advancements in recognizing diversity in lichen-forming fungi, assessing the timing of diversification remains largely unexplored in these important fungal symbionts. To better understand the evolutionary processes driving diversification in common lichen-forming fungi, we investigated the phylogeny and biogeography of the broadly distributed Melanelixia fuliginosa/M. glabratula group, using molecular data from six nuclear markers. Phylogenetic analyses of individual gene alignments and combined data provide strong evidence for five species-level lineages within this species complex. Three of these lineages correspond to the previously described species M. fuliginosa, M. glabratula, and M. subaurifera. The remaining two lineages, ‘M. sp. 1’ and ‘M. sp. 2’, merit species recognition based on genealogical concordance. Both M. glabratula and M. subaurifera had broad intercontinental distributions, sharing identical haplotypes among intercontinental populations. Based on the current sampling, M. fuliginosa s.s. was represented exclusively by European material and was not collected in North America. ‘M. sp. 1’ was represented by collections from Scotland and Spain; and ‘M. sp. 2’ was represented by collections in California, USA. Environmental factors driving the contrasting distribution patterns in this group remain unknown. Divergence times estimated using a coalescence-based multilocus species-tree approach suggest that diversification within the M. fuliginosa/M. glabratula group occurred exclusively during the Miocene. The results of the present study indicate that phenotypically cryptic lichen-forming fungal species-level lineages may be relatively ancient and do not necessarily reflect recent divergence events. Furthermore, diagnosable phenotypic differences may be absent even millions of years after the initial divergence. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●●, ●●–●●.

A phylogeny and taxonomy of the Thai-Malay Peninsula Bent-toed Geckos of the Cyrtodactylus pulchellus complex (Squamata: Gekkonidae): combined morphological and molecular analyses with descriptions of seven new species

An integrative taxonomic analysis using color pattern, morphology and 1497 base pairs of the ND2 mitochondrial geneand its five flanking tRNAs demonstrated that nine monophyletic species-level lineages occur within the Cyrtodactyluspulchellus complex (Cyrtodactylus pulchellus sensu strictu and C. macrotuberculatus) of the Thai-Malay Peninsula thathave a sequence divergence between them ranging from 5.9–16.8%. Additionally, each lineage is discretely diagnosablefrom one another based on morphology and color pattern and most occur in specific geographic regions (upland areas orislands) that prevent or greatly restrict interpopulation gene flow. Six of these lineages were masquerading under thenomen C. pulchellus and are described as the following: Cyrtodactylus astrum sp. nov. from northwestern PeninsularMalaysia and southwestern Thailand; C. langkawiensis sp. nov., at this point endemic to Langkawi Island, Malaysia; C.bintangrendah sp. nov., a lowland species surrounding the Banjaran (=mountain range) Bintang of northwesternPeninsular Malaysia; C. bintangtinggi sp. nov., endemic to the upland regions of the Banjaran Bintang of northwesternPeninsular Malaysia; C. trilatofasciatus sp. nov., endemic to upland regions of Cameron Highlands in the central portionof the Banjaran Titiwangsa in Peninsular Malaysia; and C. australotitiwangsaensis sp. nov. from the more southerlyupland regions of the Banjaran Titiwangsa. An additional species, Cyrtodactylus lekaguli sp. nov. from Satun, Trang, SuratThani, and Phang-nga provinces in southern Thailand, was identified on the basis of morphology and color pattern and is hypothesized to be part of a clade containing C. astrum sp. nov. and C. langkawiensis sp. nov.

Genetic Diversity of Neotropical Myotis (Chiroptera: Vespertilionidae) with an Emphasis on South American Species

BackgroundCryptic morphological variation in the Chiropteran genus Myotis limits the understanding of species boundaries and species richness within the genus. Several authors have suggested that it is likely there are unrecognized species-level lineages of Myotis in the Neotropics. This study provides an assessment of the diversity in New World Myotis by analyzing cytochrome-b gene variation from an expansive sample ranging throughout North, Central, and South America. We provide baseline genetic data for researchers investigating phylogeographic and phylogenetic patterns of Myotis in these regions, with an emphasis on South America.Methodology and Principal FindingsCytochrome-b sequences were generated and phylogenetically analyzed from 215 specimens, providing DNA sequence data for the most species of New World Myotis to date. Based on genetic data in our sample, and on comparisons with available DNA sequence data from GenBank, we estimate the number of species-level genetic lineages in South America alone to be at least 18, rather than the 15 species currently recognized.ConclusionsOur findings provide evidence that the perception of lower species richness in South American Myotis is largely due to a combination of cryptic morphological variation and insufficient sampling coverage in genetic-based systematic studies. A more accurate assessment of the level of diversity and species richness in New World Myotis is not only helpful for delimiting species boundaries, but also for understanding evolutionary processes within this globally distributed bat genus.

Multilocus phylogeny of the lichen-forming fungal genus Melanohalea (Parmeliaceae, Ascomycota): Insights on diversity, distributions, and a comparison of species tree and concatenated topologies

Accurate species circumscriptions are central for many biological disciplines and have critical implications for ecological and conservation studies. An increasing body of evidence suggests that in some cases traditional morphology-based taxonomy have underestimated diversity in lichen-forming fungi. Therefore, genetic data play an increasing role for recognizing distinct lineages of lichenized fungi that it would otherwise be improbable to recognize using classical phenotypic characters. Melanohalea (Parmeliaceae, Ascomycota) is one of the most widespread and common lichen-forming genera in the northern Hemisphere. In this study, we assess traditional phenotype-based species boundaries, identify previously unrecognized species-level lineages and discuss biogeographic patterns in Melanohalea. We sampled 487 individuals worldwide, representing 18 of the 22 described Melanohalea species, and generated DNA sequence data from mitochondrial, nuclear ribosomal, and protein-coding markers. Diversity previously hidden within traditional species was identified using a genealogical concordance approach. We inferred relationships among sampled species-level lineages within Melanohalea using both concatenated phylogenetic methods and a coalescent-based multilocus species tree approach. Although lineages identified from genetic data are largely congruent with traditional taxonomy, we found strong evidence supporting the presence of previously unrecognized species in six of the 18 sampled taxa. Strong nodal support and overall congruence among independent loci suggest long-term reproductive isolation among most species-level lineages. While some Melanohalea taxa are truly widespread, a limited number of clades appear to have much more restricted distributional ranges. In most instances the concatenated gene tree and multilocus species tree approaches provided similar estimates of relationships. However, nodal support was generally higher in the phylogeny estimated from concatenated data, and relationships among taxa within one major clade were largely unresolved in the species tree. This study contributes to our understanding of diversity and evolution in common lichen-forming fungi by incorporating multiple locus sequence data to circumscribe morphologicallly cryptic lineages and infer relationships within a coalescent-based species tree approach.

Diversity and systematics of the sequestrate genus Octaviania in Japan: two new subgenera and eleven new species

The sequestrate fungi of Japan, including truffle and truffle-like fungi, have not been well characterized but are potentially diverse. We investigated the diversity and phylogeny of Japanese Octaviania specimens using a multifaceted approach including scanning and transmission electron microscopy as well as analysis of nuclear ribosomal DNA (ITS and LSU) and EF-1α (tef1) sequences. Phylogenetic analyses indicate that the genus Octaviania is divided into three major clades, and that there are at least 12 species-level lineages in Japan. Accordingly, we describe two new subgenera, Parcaea and Fulvoglobus, and eleven new species. Subgenus Parcaea accommodates four highly divergent, but macromorphologically almost indiscernible cryptic species. We discuss not only the diversity and species delimitation within the genus Octaviania but also the phylogeography of the Japanese taxa and their relatives.

A gleam in the dark: Phylogenetic species delimitation in the confusing spring-snail genus Bythinella Moquin-Tandon, 1856 (Gastropoda: Rissooidea: Amnicolidae)

We re-assess the value of morphological specific descriptors within the spring-snail genus Bythinella by sequencing mitochondrial COI and nuclear ITS1 gene fragments. Taxonomic coverage represents 16 nominal species sampled among 35 populations from France. Application of monophyly and cohesive haplotype networks as criteria to delineate species allow us to identify 10 mitochondrial species-level lineages, all but one of which are recovered by ITS1. COI species thresholds that are estimated from newly delimited species (ca. 1.5%) agree with values found among other hydrobioids. Our results strongly suggest that classical morphological descriptors may not constitute valid specific criteria within Bythinella. Our analyses support a complex scenario of invasions of subterranean habitats, as illustrated by the syntopy of several mitochondrial lineages or the conflicting evolutionary histories between COI and ITS1 in caves. In addition, morphological convergence related to subterranean ecological constraints that affect shell shape and size among the hypogean springsnails studied is suspected.