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opencc-zeroNov 2020View details →

Data from: Plotting for change: an analytic framework to aid decisions on which lineages are candidate species in phylogenomic species discovery

A recent study argued that coalescent-based models of species delimitation mostly delineate population structure not species, and called for the validation of candidate species using biological information additional to the genetic information, such as phenotypic or ecological data. Here we introduce a framework to interrogate genomic datasets and coalescent-based species trees for the presence of candidate species in situations where additional biological data are unavailable, unobtainable, or uninformative. For de novo genomic studies of species boundaries, we propose six steps: (a) visualize genetic affinities among individuals to identify both discrete and admixed genetic groups from first principles, and to hold aside individuals involved in contemporary admixture for independent consideration; (b) apply phylogenetic techniques to identify lineages; (c) assess diagnosability of those lineages as potential candidate species; (d) interpret the diagnosable lineages in a geographic context (sympatry, parapatry, allopatry); (e) assess significance of difference or trends in the context of sampling intensity; and (f) adopt a holistic approach to available evidence to inform decisions on species status in the difficult cases of allopatry. We use this approach to distinguish candidate species from within-species lineages for a widespread species complex of Australian freshwater fishes (Retropinna spp.). Our framework addresses two cornerstone issues in systematics that are often not explicitly discussed in genomic species discovery: diagnosability and how to determine it, and what criteria should be used to decide whether diagnosable lineages are conspecific or represent different species.

Data from: Discovery and characterization of novel genetic markers for use in the management of Lahontan cutthroat trout (Oncorhynchus clarkii henshawi)

The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) is threatened by habitat destruction, over-harvest and hybridization with nonnative trout. Currently, three Geographic Management Units (GMUs) are recognized within the taxon. Here, we describe a suite of 68 single-nucleotide polymorphism (SNP) genetic markers for use in the study and management of Lahontan cutthroat trout and a closely related subspecies, the Paiute cutthroat trout (O. c. seleneris). These include markers variable within the two subspecies (n = 35), diagnostic for the two subspecies (n = 23) and diagnostic for Yellowstone cutthroat trout (O. c. bouvieri) and other closely related subspecies (n = 10). Sixty-three markers were discovered by Sanger sequencing of 171 EST loci in an ascertainment panel including Lahontan cutthroat trout from four populations representing all GMUs. Five markers were identified in a secondary sequencing effort with a single population of Lahontan cutthroat trout. TaqMan assays were validated on six Lahontan cutthroat trout populations and a diverse panel of other trout. Over 90% of the markers variable in Lahontan cutthroat trout were polymorphic in at least two populations, and 66% were variable within all three GMUs. All Lahontan diagnostic markers were also fixed for the Lahontan allele in Paiute cutthroat trout. Most of the Yellowstone diagnostic markers can also be used for this purpose in other cutthroat trout subspecies. This is the first set of SNP markers to be developed for Lahontan cutthroat trout, and will be an important tool for conservation and management.

opencc-zeroDec 2014View details →

Data from: Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein–DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each siteʼs locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal–Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

Data from: "Polar bear (Ursus maritimus) transcriptome assembly and SNP discovery" in Genomic Resources Notes accepted 1 August 2013-30 September 2013

Polar bears (Ursus maritimus) in the Western Hudson Bay subpopulation have been declining in size and body condition for decades, as climate change causes earlier sea ice breakup, reduced hunting time on the ice, and an increasingly long fasting season. As Western Hudson Bay females have decreased in size, rates of litter production and average litter size have also decreased, while cub mortality and average time to independence have increased. Although these changes have potential evolutionary consequences, little is yet known about the adaptive genetic variation in body size or fat accumulation that would have to underlie any such change. In this study, we used high-throughput Illumina sequencing to develop SNPs from pooled blood and fat transcriptomes, using samples from five adult female polar bears and five (unrelated) dependent cubs. In total, we generated 371,258 transcripts of which 36,755 were deemed to be "full length" (i.e., covered more than 90% of their best BLAST hit), and we identified 63,020 SNPs. Since this study was conducted, we have used a subset of these SNPs to develop an Illumina BeadArray for quantitative genetics research in Western Hudson Bay.

Data from: "White-tailed deer (Odocoileus virginianus) transcriptome assembly and SNP discovery" in Genomic Resources Notes accepted 1 June 2013-31 July 2013

White-tailed deer (Odocoileus virginianus) are among the most abundant and widespread large mammals in the Americas, comprising up to 38 subspecies ranging from Northern Canada to Peru. Although believed to have high genetic diversity, surprisingly few genomic resources are currently available, despite the species' ecological and economic importance. White-tailed deer and other cervids throughout central North America are currently being afflicted by chronic wasting disease (CWD), one of the degenerative prion diseases collectively known as transmissible spongiform encephalopathies. Although CWD is of major importance to white-tailed deer management, little is currently known about innate resistance or susceptibility to CWD outside of polymorphisms in the prion protein gene, Prnp, though a recent study using microsatellites suggests that the disease may have additional underlying genetic components. Further association analysis is hindered by low marker density. In this study, we used high-throughput SOLiD sequencing to create novel sequence data for white-tailed deer and identify single-nucleotide polymorphisms, using the pooled blood transcriptomes of six individuals. In total, we generated 14,010 contigs of length ≥ 200 nt, representing 4,104,760 nt of unique sequence data, and we identified 66,596 SNPs. This data represents one of the largest genetic resources currently available for any cervid. We hope it will facilitate future research for population genomics and assist with the identification of genetic factors that underlie disease resistance and other traits relevant for conservation and management.

opencc-zeroDec 2014View details →

Data from: Discovery of an unknown diversity of Leucinodes species damaging Solanaceae fruits in sub-Saharan Africa and moving in trade (Insecta, Lepidoptera, Pyraloidea)

The larvae of the Old World genera Leucinodes Guenée, 1854 and Sceliodes Guenée, 1854 are internal feeders in the fruits of Solanaceae, causing economic damage to cultivated plants like Solanum melongena and S. aethiopicum. In sub-Saharan Africa five nominal species of Leucinodes and one of Sceliodes occur. One of these species, the eggplant fruit and shoot borer L. orbonalis Guenée, 1854, is regarded as regularly intercepted from Africa and Asia in Europe, North and South America and is therefore a quarantine pest on these continents. We investigate the taxonomy of African Leucinodes and Sceliodes based on morphological characters in wing pattern, genitalia and larvae, as well as mitochondrial DNA, providing these data for identification of all life stages. The results suggest that both genera are congeneric, with Sceliodes syn. n. established as junior subjective synonym of Leucinodes. L. orbonalis is described from Asia and none of the samples investigated from Africa belong to this species. Instead, sub-Saharan Africa harbours a complex of eight endemic Leucinodes species. Among the former nominal species of Leucinodes (and Sceliodes) from Africa, only L. laisalis (Walker, 1859), comb. n. (Sceliodes) is confirmed, with Leucinodes translucidalis Gaede, 1917, syn. n. as a junior subjective synonym. The other African Leucinodes species were unknown to science and are described as new: L. africensis sp. n., L. ethiopica sp. n., L. kenyensis sp. n., L. malawiensis sp. n., L. pseudorbonalis sp. n., L. rimavallis sp. n. and L. ugandensis sp. n. An identification key based on male genitalia is provided for the African Leucinodes species. Most imports of Leucinodes specimens from Africa into Europe refer to Leucinodes africensis, which has been frequently imported with fruits during the last 50 years. In contrast, L. laisalis has been much less frequently recorded, and L. pseudorbonalis as well as L. rimavallis only very recently in fruit imports from Uganda. Accordingly, interceptions of Leucinodes from Africa into other continents will need to be re-investigated for their species identity and will likely require, at least in parts, revisions of the quarantine regulations. The following African taxa are excluded from Leucinodes: Hyperanalyta Strand, 1918, syn. rev. as revised synonym of Analyta Lederer, 1863; Analyta apicalis (Hampson, 1896), comb. n. (Leucinodes); Lygropia aureomarginalis (Gaede, 1916), comb. n. (Leucinodes); Syllepte hemichionalis Mabille, 1900, comb. rev., S. hemichionalis idalis Viette, 1958, comb. rev. and S. vagans (Tutt, 1890), comb. n. (Aphytoceros). Deanolis iriocapna (Meyrick, 1938), comb. n. from Indonesia is originally described and misplaced in Sceliodes, and L. cordalis (Doubleday, 1843), comb. n. (Margaritia) from New Zealand, L. raondry (Viette, 1981), comb. n. (Daraba) from Madagascar as well as L. grisealis (Kenrick, 1912), comb. n. (Sceliodes) from New Guinea are transferred from Sceliodes to Leucinodes. While Leucinodes is now revised from Africa, it still needs further revision in Asia.

opencc-zeroSep 2019View details →

Data from: Females facilitate male food patch discovery in a wild fish population

1. Responding to the information provided by others is an important foraging strategy in many species. Through social foraging, individuals can more efficiently find unpredictable resources and thereby increase their foraging success. 2. When individuals are more socially responsive to particular phenotypes than others, however, the advantage they obtain from foraging socially is likely to depend on the phenotype composition of the social environment. We tested this hypothesis by performing experimental manipulations of guppy, Poecilia reticulata, sex compositions in the wild. 3. Males found fewer novel food patches in the absence of females than in mixed-sex compositions, while female patch discovery did not differ regardless of the presence or absence of males. 4. We argue that these results were driven by sex-dependent mechanisms of social association: Markov chain-based fission-fusion modelling revealed that less social individuals found fewer patches and that males reduced sociality when females were absent. In contrast, females were similarly social with or without males. 5. Our findings highlight the relevance of considering how individual and population-level traits interact in shaping the advantages of social foraging in the wild.

opencc-zeroDec 2014View details →

Data from: Taking the discovery approach in integrative taxonomy: decrypting a complex of narrow-endemic Alpine harvestmen (Opiliones: Phalangiidae: Megabunus)

Species delimitation is fundamental for biological studies, yet precise delimitation is not an easy task, and every involved approach has an inherent failure rate. Integrative taxonomy, a method that merges multiple lines of evidence, can profoundly contribute to reliable alpha taxonomy and shed light on the processes behind speciation. In this study, we explored and validated species limits in a group of closely related Megabunus harvestmen (Eupnoi, Phalangiidae) endemic to the European Alps. Without a priori species hypotheses, we used multiple sources of inference, including mitochondrial and multilocus nuclear DNA, morphometrics, and chemistry. The results of these discovery approaches revealed morphological crypsis and multiple new species within two of the five hitherto known species. Based on our analyses, we discussed the most plausible evolutionary scenarios, invoked the most reasonable species hypotheses, and validated the new species limits. Building upon the achieved rigour, three new species, M. cryptobergomas Muster & Wachter sp. nov., M. coelodonta Muster & Steiner sp. nov., and M. lentipes Muster & Komposch sp. nov., are formally described. In addition, we provide a dichotomous morphological key to the Megabunus species of the Alps. Our work demonstrates the suitability of integrative, discovery-based approaches in combination with validation approaches to precisely characterise species and enabled us to implement nomenclatural consequences for this genus.

Data from: Discovery of a giant chameleon-like lizard (Anolis) on Hispaniola and its significance to understanding replicated adaptive radiations

We report a new chameleon-like Anolis species from Hispaniola that is ecomorphologically similar to congeners found only on Cuba. Lizards from both clades possess short limbs and a short tail and utilize relatively narrow perches, leading us to recognize a novel example of ecomorphological matching among islands in the well-known Greater Antillean anole radiation. This discovery supports the hypothesis that the assembly of island faunas can be substantially deterministic and highlights the continued potential for basic discovery to reveal new insights in well-studied groups. Restricted to a threatened band of midelevation transitional forest near the border of the Dominican Republic and Haiti, this new species appears to be highly endangered.

Data from: Genome-wide SNP discovery in the annual herb, Lasthenia fremontii (Asteraceae): genetic resources for the conservation and restoration of a California vernal pool endemic

California vernal pool (VP) ecosystems support a diverse community of endemic plants that are threatened by multiple anthropogenic pressures, generating a need for molecular tools to quantify the extent and distribution of genetic variation in native populations. Here, we used RADseq to discover single nucleotide polymorphisms (SNPs) for a widespread VP endemic plant species, Lasthenia fremontii. We discovered nuclear-based SNPs using a RAD-tag library of 12 individuals from different VP complexes using SbfI, a restriction enzyme that does not cleave the chloroplast genome in Lasthenia. A total of 316,106 catalog loci were obtained across all twelve individuals in the library. Of these, 713 loci were polymorphic, yielding 3918 candidate SNPs. Next, we genotyped an additional 285 additional plants to validate and characterize 71 of the candidate SNPs. Of these, 44 were polymorphic among VP complexes. A preliminary analysis of the distribution of genetic variation using these loci revealed significant isolation-by-distance across the species' geographic range. Weaker, but in some cases significant, genetic differentiation was detected among subpopulations from different pools within a single VP complex. Thus, in this study, RADseq allowed the discovery of SNP markers that can characterize patterns of genetic variation at multiple spatial scales in L. fremontii, which can be used to inform the conservation and mitigation of VP populations.

opencc-zeroDec 2017View details →

Data from: Insights into VSM diversity and Neoproterozoic biostratigraphy in the light of recent Brazilian discoveries

Vase-shaped microfossils occur in dolostone clasts within conglomerates, breccias and diamictites of the Neoproterozoic Urucum Formation, Jacadigo Group, SW Brazil. Although their taphonomic history is distinct from those of other VSM assemblages, morphometric comparison of Urucum fossils with five others described previously from North America and Europe show that two of the Urucum species --- the long-necked Limeta lageniformis Morais, Fairchild and Lahr, 2017 and the funnel-necked Palaeoamphora urucumense Morais et al., 2017 occur in the Kwagunt and Callison Lake assemblages, as does Pakupaku kabin Riedman, Porter and Calver, 2017 recently described from the Togari Group, Tasmania. Obelix rootsii Cohen, Irvine and Strauss, 2017a (new combination), previously known only from the Callison Lake Formation, is documented here from the Kwagunt Formation. In addition, Trigonocyrillium horodyskii (Bloeser, 1985) and Bonniea dacruchares Porter, Meisterfeld and Knoll 2003, first described from the Kwagunt assemblage, have now been found in the Urucum Formation. In light of this survey, 16 of the 18 validly described VSM species are now known to occur in the Kwagunt Formation and 13 in the Callison Lake Formation, with 12 of them shared by both formations. The fact that the Urucum VSM assemblage exhibits six of seven species in common with the Kwagunt Formation --- L. lageniformis, P. urucumense, Cycliocyrillium simplex Porter et al., 2003, C. torquata Porter et al., 2003, B. dacruchares Porter et al., 2003, and T. horodyskii (Bloeser, 1985) --- and all but the last of these in common with the Callison Lake Formation supports correlation of these three assemblages and indicates that the source of the fossiliferous clasts within the Urucum Formation may well have been a now vanished late Tonian carbonate platform

opencc-zeroDec 2010View details →

Data from: Gene discovery in the Antarctic fur seal (Arctocephalus gazella) skin transcriptome

Next-generation sequencing provides a powerful new approach for developing functional genomic tools for nonmodel species, helping to narrow the gap between studies of model organisms and those of natural populations. Consequently, massively parallel 454 sequencing was used to characterize a normalized cDNA library derived from skin biopsy samples of twelve Antarctic fur seal (Arctocephalus gazella) individuals. Over 412 Mb of sequence data were generated, comprising 1.4 million reads of average length 286 bp. De novo assembly using Newbler 2.3 yielded 156 contigs plus 22 869 isotigs, which in turn clustered into 18 576 isogroups. Almost half of the assembled transcript sequences showed significant similarity to the nr database, revealing a functionally diverse array of genes. Moreover, 97.9% of these mapped to the dog (Canis lupis familiaris) genome, with a strong positive relationship between the number of sequences locating to a given chromosome and the length of that chromosome in the dog indicating a broad genomic distribution. Average depth of coverage was also almost 20-fold, sufficient to detect several thousand putative microsatellite loci and single nucleotide polymorphisms. This study constitutes an important step towards developing genomic resources with which to address consequential questions in pinniped ecology and evolution. It also supports an earlier but smaller study showing that skin tissue can be a rich source of expressed genes, with important implications for studying the genomics not only of marine mammals, but also more generally of species that cannot be destructively sampled.

opencc-zeroDec 2016View details →

Data from: Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.

Data from: Discovery of 20,000 RAD–SNPs and development of a 52-SNP array for monitoring river otters

Many North American river otter (Lontra canadensis) populations are threatened or recovering but are difficult to study because they occur at low densities, it is difficult to visually identify individuals, and they inhabit aquatic environments that accelerate degradation of biological samples. Single nucleotide polymorphisms (SNPs) can improve our ability to monitor demographic and genetic parameters of difficult to study species. We used restriction site associated DNA (RAD) sequencing to discover 20,772 SNPs present in Montana, USA, river otter populations, including 14,512 loci that were also variable in at least one other population range-wide. After applying careful filtering criteria meant to minimize ascertainment bias and identify high quality, highly heterozygous (H o = 0.2–0.50) SNPs, we developed and tested 52 independent SNP qPCR genotyping assays, including 41 that performed well with diluted DNA. The 41 loci provided high power for population assignment tests with only 1 misassignment (1.6 %) between closely neighboring populations. Our SNPs showed high power to differentiate individuals and assign them to population of origin, as well as strong concordance of genotypes from high and diluted concentrations of DNA, and between original RAD and the SNP qPCR array.

opencc-zeroDec 2011View details →

Data from: De novo characterization of the Timema cristinae transcriptome facilitates marker discovery and inference of genetic divergence.

Adaptation to different ecological environments can promote speciation. Although numerous examples of such 'ecological speciation' exist, the genomic basis of the process, and the role of gene flow in it, remains less understood. This is, at least in part, because systems that are well characterized in terms of their ecology often lack genomic resources. In this study we characterize the transcriptome of Timema cristinae stick insects, a system that has been researched intensively in terms of ecological speciation, but for which genomic resources have not been previously developed. Specifically, we obtained >1 million 454 sequencing reads that assembled into 84,937 contigs representing approximately 18,282 unique genes and tens of thousands of potential molecular markers. Second, as an illustration of their utility, we used these genomic resources to assess multi-locus genetic divergence within both an ecotype pair and a species pair of Timema stick insects. The results suggest variable levels of genetic divergence and gene flow among taxon pairs and genes and illustrate a first step towards future genomic work in Timema.

Data from: Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing

Caviar-producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta-samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired-end RAD data focused on the identification of SNPs in the paired-end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population-wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping-by-sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools.

opencc-zeroDec 2017View details →

Data from: Discovery of a multi-species shark aggregation and parturition area in the Ba Estuary, Fiji Islands

Population declines in shark species have been reported on local and global scales, with overfishing, habitat destruction and climate change posing severe threats. The lack of species-specific baseline data on ecology and distribution of many sharks, however, makes conservation measures challenging. Here we present a fisheries-independent shark survey from the Fiji Islands, where scientific knowledge on locally occurring elasmobranchs is largely still lacking despite the location's role as a shark hotspot in the Pacific. Juvenile shark abundance in the fishing grounds of the Ba Estuary (north-western Viti Levu) was assessed with a gillnet- and longline-based survey from December 2015 to April 2016. A total of 103 juvenile sharks identified as blacktip Carcharhinus limbatus (n = 57), scalloped hammerhead Sphyrna lewini (n = 35), and great hammerhead Sphyrna mokarran (n = 11) sharks were captured, tagged, and released. The condition of umbilical scars (68 % open or semi-healed), mean sizes of individuals (± SD) (C. limbatus: 66.5 ± 3.8 cm, S. lewini: 51.8 ± 4.8 cm, S. mokarran 77.4 ± 2.8 cm), and the presence of these species over recent years (based on fishermen interviews), suggest that the Ba Estuary area is a critical habitat for multiple species that are classified as "Near Threatened" or "Endangered". Specifically, the area likely acts as a parturition ground over the studied period, and potentially as a subsequent nursery area. We identified subareas of high abundance and found that temperature, salinity and depth acted as small-scale environmental drivers of shark abundance. The data suggests a tendency for species-specific spatial use, both horizontally (i.e. between sampling areas) and vertically (i.e. across the water column). These results enhance the understanding of shark ecology in Fiji and provide a scientific basis for the implementation of local conservation strategies that contribute to the protection of these threatened species.

Data from: Species discovery and validation in a cryptic radiation of endangered primates: coalescent-based species delimitation in Madagascar's mouse lemurs

Implementation of the coalescent model in a Bayesian framework is an emerging strength in genetically based species delimitation studies. By providing an objective measure of species diagnosis, these methods represent a quantitative enhancement to the analysis of multilocus data, and complement more traditional methods based on phenotypic and ecological characteristics. Recognized as two species 20 years ago, mouse lemurs (genus Microcebus) now comprise more than 20 species, largely diagnosed from mtDNA sequence data. With each new species description, enthusiasm has been tempered with scientific scepticism. Here, we present a statistically justified and unbiased Bayesian approach towards mouse lemur species delimitation. We perform validation tests using multilocus sequence data and two methodologies: (i) reverse-jump Markov chain Monte Carlo sampling to assess the likelihood of different models defined a priori by a guide tree, and (ii) a Bayes factor delimitation test that compares different species-tree models without a guide tree. We assess the sensitivity of these methods using randomized individual assignments, which has been used in bpp studies, but not with Bayes factor delimitation tests. Our results validate previously diagnosed taxa, as well as new species hypotheses, resulting in support for three new mouse lemur species. As the challenge of multiple researchers using differing criteria to describe diversity is not unique to Microcebus, the methods used here have significant potential for clarifying diversity in other taxonomic groups. We echo previous studies in advocating that multiple lines of evidence, including use of the coalescent model, should be trusted to delimit new species.