How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

Slow and steady wins the race: Diversification rate is independent from body size and lifestyle in Malagasy skinks (Squamata: Scincidae: Scincinae).

Most of the unique and diverse vertebrate fauna that inhabits Madagascar derives from in situ diversification from colonisers that reached this continental island through overseas dispersal. The endemic Malagasy Scincinae lizards are amongst the most species-rich squamate groups on the island. They colonised all bioclimatic zones and display many ecomorphological adaptations to a fossorial (burrowing) lifestyle. Here we propose a new phylogenetic hypothesis for their diversification based on the largest taxon sampling so far compiled for this group. We estimated divergence times and investigated several aspects of their diversification (diversification rate, body size and fossorial lifestyle evolution, and biogeography). We found that diversification rate was constant throughout most of the evolutionary history of the group, but decreased over the last 6-4 million years and independently from body size and fossorial lifestyle evolution. Fossoriality has evolved from fully quadrupedal ancestors at least five times independently, which demonstrates that even complex morphological syndromes - in this case involving traits such as limb regression, body elongation, modification of cephalic scalation, depigmentation, and eyes and ear-opening regression - can evolve repeatedly and independently given enough time and eco-evolutionary advantages. Initial diversification of the group likely occurred in forests, and the divergence of sand-swimmer genera around 20 Ma appears linked to a period of aridification. Our results show that the large phenotypic variability of Malagasy Scincinae has not influenced diversification rate and that their rich species diversity results from a constant accumulation of lineages through time. By compiling large geographic and trait-related datasets together with the computation of a new time tree for the group, our study contributes important insights on the diversification of Malagasy vertebrates.

Computational molecular species delimitation and taxonomic revision of the gecko genus Ebenavia Boettger, 1878.

Cryptic species have been detected in many groups of organisms and must be assumed to make up a significant portion of global biodiversity. We study geckos of the Ebenavia inunguis complex from Madagascar and surrounding islands and use species delimitation algorithms (GMYC, BOLD, BPP), COI barcode divergence, diagnostic codon indels in the nuclear marker PRLR, diagnostic categorical morphological characters, and significant differences in continuous morphological characters for its taxonomic revision. BPP yielded ≥ 10 operational taxonomic units, whereas GMYC (≥ 27) and BOLD (26) suggested substantial oversplitting. In consequnce, we resurrect Ebenavia boettgeri Boulenger 1885 and describe Ebenavia tuelinae sp. nov., Ebenavia safari sp. nov., and Ebenavia robusta sp. nov., increasing the number of recognised species in Ebenavia from two to six. Further lineages of Ebenavia retrieved by BPP may warrant species or subspecies status, but further taxonomic conclusions are postponed until more data become available. Finally, we present an identification key to the genus Ebenavia, provide an updated distribution map, and discuss the diagnostic values of computational species delimitation as well as morphological and molecular diagnostic characters.

Reconciling molecular phylogeny, morphological divergence and classification of Madagascan narrow-mouthed frogs (Amphibia: Microhylidae).

A recent study clarified several aspects of microhylid phylogeny by combining DNA sequences from Sanger sequencing and anchored phylogenomics, although numerous aspects of tree topology proved highly susceptible to data partition and chosen model. Although the phylogenetic results of the study were in conflict with previous publications, the authors made several changes to the taxonomy of Madagascar's cophyline microhylids. We re-analyzed part of their data together with our own molecular and morphological data. Based on a supermatrix of 11 loci, we propose a new phylogeny of the Cophylinae, and discuss it in the context of a newly generated osteological dataset. We found several sample misidentifications, partially explaining their deviant results, and propose to resurrect the genera Platypelis and Stumpffia from the synonymy of Cophyla and Rhombophryne, respectively. We provide support for the previous genus-level taxonomy of this subfamily, and erect a new genus, Anilany gen. nov., in order to eliminate paraphyly of Stumpffia and to account for the osteological differences observed among these groups. Deep nodes in our phylogeny remain poorly supported, and future works will certainly refine our classification, but we are confident that these will not produce large-scale rearrangements.

Vertebrate time-tree elucidates the biogeographic pattern of a major biotic change around the K-T boundary in Madagascar.

The geographic and temporal origins of Madagascar's biota have long been in the center of debate. We reconstructed a time-tree including nearly all native nonflying and nonmarine vertebrate clades present on the island, from DNA sequences of two single-copy protein-coding nuclear genes (BDNF and RAG1) and a set of congruent time constraints. Reconstructions calculated with autocorrelated or independent substitution rates over clades agreed in placing the origins of the 31 included clades in Cretaceous to Cenozoic times. The two clades with sister groups in South America were the oldest, followed by those of a putative Asian ancestry that were significantly older than the prevalent clades of African ancestry. No colonizations from Asia occurred after the Eocene, suggesting that dispersal and vicariance of Asian/Indian groups were favored over a comparatively short period during, and shortly after, the separation of India and Madagascar. Species richness of clades correlates with their age but those clades that have a large proportion of species diversity in rainforests are significantly more species-rich. This finding suggests an underlying pattern of continuous speciation through time in Madagascar's vertebrates, with accelerated episodes of adaptive diversification in those clades that succeeded radiating into the rainforests.

Alike but different: the evolution of the Tubifex tubifex species complex (Annelida, Clitellata) through polyploidization.

BACKGROUND: Tubifex tubifex is a widespread annelid characterized by considerable variability in its taxonomic characteristics and by a mixed reproductive strategy, with both parthenogenesis and biparental reproduction. In a molecular phylogenetic analysis, we detected substantial genetic variability among sympatric Tubifex spp. from the Lambro River (Milano, Italy), which we suggested comprise several cryptic species. To gain insights into the evolutionary events that generated this differentiation, we performed a cytogenetic analysis in parallel with a molecular assay. Approximately 80 cocoons of T. tubifex and T. blanchardi were collected and dissected. For each cocoon, we sequenced a fragment of the 16S rRNA from half of the sibling embryos and karyotyped the other half. To generate a robust phylogeny enabling the reconstruction of the evolutionary processes shaping the diversity of these sympatric lineages, we complemented our original 16S rRNA gene sequences with additional COI sequences. RESULTS: The chromosome number distribution was consistent with the presence of at least six sympatric euploid chromosome complements (one diploid, one triploid, three tetraploids and one hexaploid), as confirmed by a FISH assay performed with an homologous 18S rDNA probe. All the worms with 2n = 50 chromosomes belonged to an already identified sibling species of T. tubifex, T. blanchardi. The six euploid sets were coherently arranged in the phylogeny, with each lineage grouping specimens with the same chromosome complement. CONCLUSIONS: These results are compatible with the hypothesis that multiple polyploidization events, possibly enhanced by parthenogenesis, may have driven the evolution of the T. tubifex species complex.

PARSID: a Python script for automatic analysis of local BLAST results for a rapid molecular taxonomic identification.

OBJECTIVE: A reliable taxonomic identification of species from molecular samples is the first step for many studies. For researchers unfamiliar with programming, running a BLAST analysis, filtering, and organizing results for hundreds of sequences through the BLAST web interface can be difficult. Additionally, sequences deposited in GenBank can have outdated taxonomic identification. The use of reliable Reference Sequences Library (RSL) containing accurate taxonomically-identified sequences facilitates this task. Pending the availability of a RSL with the user, we developed a tool that automates the molecular taxonomic identification of sequences. RESULTS: We developed PARSID, a Python script running through the command-line that automates the routine workflow of blasting an input sequence file against the user's RSL, and retrieves the matches with the highest percentage of identity in five steps. PARSID accepts cut-off parameters and supplementary information in a.csv file for filtering the results. The final output is visualized in a spreadsheet. We tested its functioning using 10 input sequences simulating different situations of the molecular taxonomic identification of sequences against an example RSL containing 25 sequences. Step-by-step instructions and test files are publicly available at https://github.com/kokinide/PARSID.git .

Genomic Tools in Biological Invasions: Current State and Future Frontiers.

Human activities are accelerating rates of biological invasions and climate-driven range expansions globally, yet we understand little of how genomic processes facilitate the invasion process. Although most of the literature has focused on underlying phenotypic correlates of invasiveness, advances in genomic technologies are showing a strong link between genomic variation and invasion success. Here, we consider the ability of genomic tools and technologies to (i) inform mechanistic understanding of biological invasions and (ii) solve real-world issues in predicting and managing biological invasions. For both, we examine the current state of the field and discuss how genomics can be leveraged in the future. In addition, we make recommendations pertinent to broader research issues, such as data sovereignty, metadata standards, collaboration, and science communication best practices that will require concerted efforts from the global invasion genomics community.

An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula.

Four species of legless anguid lizard genus Anguis have been currently recognized: A. fragilis from western and central Europe, A. colchica from eastern Europe and western Asia, A. graeca from southern Balkans, and A. cephallonica from the Peloponnese. Slow worms from the Italian Peninsula have been considered conspecific with A. fragilis, despite the fact that the region served as an important speciation center for European flora and fauna, and included some Pleistocene glacial refugia. We used mitochondrial and nuclear DNA sequences to investigate the systematic and phylogenetic position of the Italian slow-worm populations and morphological analyses to test for phenotypic differentiation from A. fragilis from other parts of Europe. Our phylogenetic analyses revealed that Italian slow worms form a distinct deeply differentiated mtDNA clade, which presumably diverged during or shortly after the basal radiation within the genus Anguis. In addition, the specimens assigned to this clade bear distinct haplotypes in nuclear PRLR gene and show morphological differentiation from A. fragilis. Based on the differentiation in all three independent markers, we propose to assign the Italian clade species level under the name Anguis veronensisPollini, 1818. The newly recognized species is distributed throughout the Italian Peninsula to the Southern Alps and south-eastern France. We hypothesize that the Tertiary Alpine orogeny with subsequent vicariance might have played a role in differentiation of this species. The current genetic variability was later presumably shaped in multiple glacial refugia within the Italian Peninsula, with the first splitting event separating populations from the region of the Dolomite Mountains.

Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana.

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

Food load manipulation ability shapes flight morphology in females of central-place foraging Hymenoptera.

BACKGROUND: Ecological constraints related to foraging are expected to affect the evolution of morphological traits relevant to food capture, manipulation and transport. Females of central-place foraging Hymenoptera vary in their food load manipulation ability. Bees and social wasps modulate the amount of food taken per foraging trip (in terms of e.g. number of pollen grains or parts of prey), while solitary wasps carry exclusively entire prey items. We hypothesized that the foraging constraints acting on females of the latter species, imposed by the upper limit to the load size they are able to transport in flight, should promote the evolution of a greater load-lifting capacity and manoeuvrability, specifically in terms of greater flight muscle to body mass ratio and lower wing loading. RESULTS: Our comparative study of 28 species confirms that, accounting for shared ancestry, female flight muscle ratio was significantly higher and wing loading lower in species taking entire prey compared to those that are able to modulate load size. Body mass had no effect on flight muscle ratio, though it strongly and negatively co-varied with wing loading. Across species, flight muscle ratio and wing loading were negatively correlated, suggesting coevolution of these traits. CONCLUSIONS: Natural selection has led to the coevolution of resource load manipulation ability and morphological traits affecting flying ability with additional loads in females of central-place foraging Hymenoptera. Release from load-carrying constraints related to foraging, which took place with the evolution of food load manipulation ability, has selected against the maintenance of a powerful flight apparatus. This could be the case since investment in flight muscles may have to be traded against other life-history traits, such as reproductive investment.

Skin and gut microbiomes of tadpoles vary differently with host and water environment: a short-term experiment using 16S metabarcoding.

The host-microbiome community is influenced by several host and environmental factors. In order to disentangle the individual effects of host and environment, we performed a laboratory experiment to assess the effects of the exposure to different water sources on the skin and gut microbiome of two amphibian species (Pelophylax perezi and Bufo spinosus). We observed that the bacterial communities greatly varied with water environment and host identity. Tadpoles of B. spinosus collected from a waterbody with poorer bacterial diversity exhibited a more diverse skin and gut microbiome after exposed to a richer water source. Tadpoles of P. perezi, originally collected from a richer water environment, exhibited less marked alterations in diversity patterns independently of the water source but showed alterations in gut composition. These results highlight that environment alterations, such as the water source, combined with the host effect, impact the microbiome of amphibian species in different ways; the population history (e.g., previous water environment and habitat) of the host species may also influence future alterations on tadpole microbiome.

Spatial ecology of the invasive Asian common toad in Madagascar and its implications for invasion dynamics.

Invasion dynamics are determined, among other aspects, by the spatial behaviour of invasive populations. The invasive toad Duttaphrynus melanostictus is spreading inland from the eastern coast of Madagascar, causing considerable ecological impacts. Understanding the basic factors determining the spread dynamics can inform management strategies and provide insights into spatial evolutionary processes. We radio-tracked 91 adult toads in three localities along the invasion gradient to determine whether spatial sorting of dispersive phenotypes is occurring, and investigate intrinsic and extrinsic determinants of spatial behaviour. Overall, toads in our study appeared as habitat generalists, and their sheltering behaviour was tied to water proximity, with toads changing shelter more frequently closer to waterbodies. Toads showed low displacement rates (mean = 4.12 m/day) and quite a philopatric behaviour but were able to perform daily movements of over 50 m. We did not detect any spatial sorting of dispersal-relevant traits nor sex- or size-biased dispersal. Our results suggest that toads are more likely to expand their range during the wet season, and that the range expansion is probably dominated by short-distance dispersal at this stage of the invasion, although a future increase in invasion speed is expected, due to the capacity for long-distance movements of this species.

Integrative revision of the Blommersia wittei complex, with description of a new species of frog from western and north-western Madagascar.

Frogs of the Blommersia wittei complex are widespread in western and northern Madagascar, and are one of two clades of the family Mantellidae that have colonized the Comoran island of Mayotte. Based on a comprehensive set of DNA sequences of the mitochondrial 16S rRNA gene and the nuclear-encoded RAG1 and SACS genes, integrated with morphological and bioacoustic data, we here analyze the genetic differentiation of populations of this complex across Madagascar. We confirm that a candidate species named B. sp. Ca5 in previous studies represents a genetically well-defined evolutionary lineage distributed over much of western Madagascar, which we describe herein as Blommeria bara sp. nov. based on its molecular and bioacoustic differentiation. Blommersia wittei occurs across northern Madagascar but its type locality Ambanja, at the lower Sambirano river, is very close to the range of another, newly discovered microendemic lineage that was only found at two sites along the upper Sambirano river (here named as candidate species B. sp. Ca12). The B. wittei complex thus provides an example of a clade of closely related Malagasy frogs that contains species widespread over hundreds of kilometers, as well as extreme microendemics. For a full resolution of this species complex, more data need to be collected on the geographical contact among these two lineages, on the morphology and bioacoustics of B. sp. Ca12, and on the north-eastern populations of B. wittei at Sambava, which are weakly differentiated in mitochondrial genes but differ in bioacoustics and possibly in the extent of foot webbing.

Another step through the crux: a new microendemic rock-dwelling Paroedura (Squamata, Gekkonidae) from south-central Madagascar.

Using an integrative taxonomic approach including genetic and morphological data, we formally describe a new microendemic gecko species belonging to the Paroedurabastardi clade, previously referred to as P.bastardi D. We name this taxon currently known from Anja Reserve and Tsaranoro Valley Forest (south-central Madagascar), as P.manongavatosp. nov. The new species differs from other species of the P.bastardi clade by ≥ 12.4% uncorrected p-distance at the mitochondrial 16S rRNA gene and it forms a monophyletic group in the COI mtDNA phylogenetic tree. It lacks haplotype sharing at the nuclear KIAA1239 and CMOS genes with the other species of the same complex, including the syntopic P.rennerae. Given its limited extent of occurrence and high levels of habitat fragmentation linked to forest clearances and fires, we propose the IUCN Red List Category of Critically Endangered, based on the B1ab(iii) criterion. The conservation value of Anja Reserve and Tsaranoro Valley Forest is remarkable. Preserving the remaining deciduous forest habitat is of paramount importance to protect these narrow-range reptile species.

Life History Traits and Longevity of the Invasive Asian Common Toad Duttaphrynus melanostictus (Schneider, 1799) in Madagascar.

We analyzed the body length, age structure, and age at sexual maturity of the invasive Asian common toad Duttaphrynus melanostictus from different sites in Toamasina, east Madagascar. We used skeletochronology as a proxy for age estimation, while gonads were histologically analyzed to determine the age of sexual maturity. The analysis of pooled age data from three sites investigated in 2016 showed that both sexes were larger, although not older, than those of native populations. For the individuals from Madagascar, the males were significantly smaller and younger (mean ± SD, SVL: 71.4 ± 1.6 mm; age: 1.8 ± 0.7 years) than the females (SVL: 78.42 ± 1.9 mm; age: 2.7 ± 1.3 years), when the data were pooled, but when the data were analyzed separately for each of the three sites, similar results were obtained only for one site. The oldest recorded male and female were 3 and 6 years old, respectively. Gonadal histology showed that the males and females reach sexual maturity after the first and second years of age, respectively. Further studies are needed to understand if the larger size and faster growth rates observed in the invasive population of D. melanostictus in Madagascar are a consequence of more favorable environmental conditions with respect to the native range (e.g., the availability of larger trophic niches, a lack of competitors, and lower predatory pressure), and we suggest to extend the monitoring of these life history traits to understand how they might influence the invasion.

Gray versus yellow ventral coloration: Identity, distribution, color polymorphism and molecular relationships of the microhylid frog Platypelis mavomavo Andreone, Fenolio & Walvoord, 2003.

The Malagasy frog Platypelis mavomavo from Ambolokopatrika in the North East of Madagascar was originally diagnosed based on its bright yellow venter, but only limited information on this species has become available after its initial description in 2003. Several Platypelis specimens with yellow ventral color have been erroneously assigned to this species due to a lack of DNA sequences from the P. mavomavo type series. On the other hand, the candidate species Platypelis sp. Ca10 from Andranomapanga in the Northern Central East of Madagascar with gray ventral color has been defined based on its genetic differentiation from other nominal Platypelis species. Here we study the genetic variation of P. mavomavo and P. sp. Ca10 based on mitochondrial (16S rRNA) and nuclear-encoded (RAG-1) genes, including a newly determined sequence from the P. mavomavo holotype, which was studied using a museomics approach. We find only limited genetic variation among the samples studied, and this variation is unlinked to ventral coloration but instead reflects geographic distribution. We, therefore, conclude that P. sp. Ca10 is a gray-colored variant of P. mavomavo, and that P. mavomavo is rather widespread in the North East and Northern Central East of Madagascar, with populations in areas bordering the North West (Ambohitantely) and Sambirano (Ampotsidy) geographic regions, and the yellow-bellied morph restricted to the North East (Makira, Ambolokopatrika). Due to the range extension of P. mavomavo, the conservation status of the species requires re-assessment.

A multigene species tree for Western Mediterranean painted frogs (Discoglossus).

Painted frogs (Discoglossus) are an anuran clade that originated in the Upper Miocene. Extant species are morphologically similar and have a circum-Mediterranean distribution. We assembled a multilocus dataset from seven nuclear and four mitochondrial genes for several individuals of all but one of the extant species and reconstructed a robust phylogeny by applying a coalescent-based species-tree method and a concatenation approach, both of which gave congruent results. The earliest phylogenetic split within Discoglossus separates D. montalentii from a clade comprising all other species. Discoglossus montalentii is monophyletic for haplotype variation at all loci and has distinct morphological, bioacoustic and karyotypic characters. We find moderate support for a sister-group relationship between the Iberian taxa and the Moroccan D. scovazzi, and high support for a D. pictus -D. sardus clade distributed around the Tyrrhenian basin. Topological discordance among gene trees during the speciation of D. galganoi, D. scovazzi, D. pictus and D. sardus is interpreted as the consequence of nearly simultaneous, vicariant diversification. The timing of these events is unclear, but possibly coincided with the final geotectonic rearrangement of the Western Mediterranean in the Middle Miocene or later during the Messinian salinity crisis. The Iberian taxa D. galganoi galganoi and D. g. jeanneae are reciprocally monophyletic in mitochondrial DNA but not in nuclear gene trees, and are therefore treated as subspecies of D. galganoi.

Comparative cytogenetics on eight Malagasy Mantellinae (Anura, Mantellidae) and a synthesis of the karyological data on the subfamily.

We performed a molecular and cytogenetic analysis on different Mantellinae species and revised the available chromosomal data on this group to provide an updated assessment of its karyological diversity and evolution. Using a fragment of the mitochondrial 16S rRNA, we performed a molecular taxonomic identification of the samples that were used for cytogenetic analyses. A comparative cytogenetic analysis, with Giemsa's staining, Ag-NOR staining and sequential C-banding + Giemsa + CMA + DAPI was performed on eight species: Gephyromantis sp. Ca19, G.striatus (Vences, Glaw, Andreone, Jesu et Schimmenti, 2002), Mantidactylus (Chonomantis) sp. Ca11, M. (Brygoomantis) alutus (Peracca, 1893), M. (Hylobatrachus) cowanii (Boulenger, 1882), Spinomantispropeaglavei "North" (Methuen et Hewitt, 1913), S.phantasticus (Glaw et Vences, 1997) and S. sp. Ca3. Gephyromantisstriatus, M. (Brygoomantis) alutus and Spinomantispropeaglavei "North" have a karyotype of 2n = 24 chromosomes while the other species show 2n = 26 chromosomes. Among the analysed species we detected differences in the number and position of telocentric elements, location of NOR loci (alternatively on the 6th, 7th or 10th pair) and in the distribution of heterochromatin, which shows species-specific patterns. Merging our data with those previously available, we propose a karyotype of 2n = 26 with all biarmed elements and loci of NORs on the 6th chromosome pair as the ancestral state in the whole family Mantellidae. From this putative ancestral condition, a reduction of chromosome number through similar tandem fusions (from 2n = 26 to 2n = 24) occurred independently in Mantidactylus Boulenger, 1895 (subgenus Brygoomantis Dubois, 1992), Spinomantis Dubois, 1992 and Gephyromantis Methuen, 1920. Similarly, a relocation of NORs, from the putative primitive configuration on the 6th chromosome, occurred independently in Gephyromantis, Blommersia Dubois, 1992, Guibemantis Dubois, 1992, Mantella Boulenger, 1882 and Spinomantis. Chromosome inversions of primitive biarmed elements likely generated a variable number of telocentric elements in Mantellanigricans Guibé, 1978 and a different number of taxa of Gephyromantis (subgenera Duboimantis Glaw et Vences, 2006 and Laurentomantis Dubois, 1980) and Mantidactylus (subgenera Brygoomantis, Chonomantis Glaw et Vences, 1994, Hylobatrachus Laurent, 1943 and Ochthomantis Glaw et Vences, 1994).

Integrative revision of the Lygodactylus madagascariensis group reveals an unexpected diversity of little brown geckos in Madagascars rainforest.

The Lygodactylus madagascariensis species group, constituting the subgenus Domerguella, currently contains five valid species of inconspicuous dwarf geckos from Madagascars humid forests, but at least 18 deep genetic lineages have been revealed by recent molecular studies. Given the high morphological similarity of these lineages, taxonomic resolution of this astonishing diversity requires efforts to correctly delimit species, as well as assigning the available nomina to the species-level lineages identified. We here combine DNA sequences of one mitochondrial and two nuclear-encoded gene fragments with morphometric measurements and scale counts, and report evidence for a species status of most of the previously identified lineages. In particular, we rely on sympatric and often even syntopic occurrence of several of these lineages without evidence for genetic admixture, and consistent with subtle morphological differences. Furthermore, the very high divergences of 7.423.8% pairwise distances in the relatively conserved mitochondrial 16S rRNA gene, combined with a lack of haplotype sharing in the nuclear-encoded genes and differences in scale counts convinced us that most of the other, allopatrically distributed lineages also represent distinct species. We elevate L. madagascariensis petteri to species level and formally name eight new species: L. salvi sp. nov., a species from the Sambirano region in northern Madagascar, previously called L. sp. 8; L. tantsaha sp. nov. (L. sp. 10), a species occurring sympatrically with L. madagascariensis and L. petteri on Montagne dAmbre in far northern Madagascar; L. roellae sp. nov. (L. sp. 17), a species characterized by a striped coloration in all known specimens, from northern Madagascar; L. winki sp. nov. (L. sp. 18), an unstriped species from northern Madagascar but belonging to a subclade mostly distributed in the eastern rainforests of the island; L. ulli sp. nov. (L. sp. 21), a species from the same subclade as L. winki but known only from the Marojejy Massif in the North East; L. fritzi sp. nov. (L. sp. 11), a further species of this subclade from coastal lowlands in the Northern Central East; L. hodikazo sp. nov. (L. sp. 23) known from a single specimen collected at the Tsingy de Bemaraha and therefore the only Domerguella species known from the West region of Madagascar; and L. hapei sp. nov. (L. sp. 26), an enigmatic species from the Sambirano region characterized by a striped pattern on the throat that is otherwise unknown in the subgenus. Three additional deep mitochondrial lineages of Domerguella were identified in our analysis, but could not be further analyzed due to the lack or scarcity of voucher specimens. More field work and collection of voucher specimens is needed to understand their status. Furthermore, the taxonomy of the Domerguella subclade occurring in eastern Madagascar, with three described species (L. guibei, L. miops, L. fritzi), two synonyms (L. septemtuberculatus, Microscalabotes spinulifer) and at least two further deep genetic lineages co-occurring in a relatively small area, requires further revisionary work, possibly aided by target-enrichment sequencing of the respective name-bearing types.