Phylogenetic insights into Central European Chorthippus and Pseudochorthippus (Orthoptera: Acrididae) species using ddRADseq data.

The evolution of several orthopteran groups, especially within the grasshopper family Acrididae, remains poorly understood. This is particularly true for the subfamily Gomphocerinae, which comprises cryptic sympatric and syntopic species. Previous mitochondrial studies have highlighted major discrepancies between taxonomic and phylogenetic hypotheses, thereby emphasizing the necessity of genome-wide approaches. In this study, we employ double-digest restriction site-associated DNA sequencing (ddRADseq) to reconstruct the evolution of Central European Chorthippus and Pseudochorthippus species, especially C.smardai, P.tatrae and the C.biguttulus group. Our phylogenomic analyses recovered deep discordance with mitochondrial DNA barcoding, emphasizing its unreliability in Gomphocerinae grasshoppers. Specifically, our data robustly distinguished the C.biguttulus group and confirmed the distinctiveness of C.eisentrauti, also shedding light on its presence in the Berchtesgaden Alps. Moreover, our results support the reclassification of C.smardai to the genus Pseudochorthippus and of P.tatrae to the genus Chorthippus. Our study demonstrates the efficiency of high-throughput genomic methods such as RADseq without prior optimization to elucidate the complex evolution of grasshopper radiations with direct taxonomic implications. While RADseq has predominantly been utilized for population genomics and within-genus phylogenomics, its application extends to resolve relationships between deeply-diverged clades representative of distinct genera.

Evolution of mitogenomic gene order in Orthoptera.

Mitochondrial gene order has contributed to the elucidation of evolutionary relationships in several animal groups. It generally has found its application as a phylogenetic marker for deep nodes. Yet, in Orthoptera limited research has been performed on the gene order, although the group represents one of the oldest insect orders. We performed a comprehensive study on mitochondrial genome rearrangements (MTRs) within Orthoptera in the context of mitogenomic sequence-based phylogeny. We used 280 published mitogenome sequences from 256 species, including three outgroup species, to reconstruct a molecular phylogeny. Using a heuristic approach, we assigned MTR scenarios to the edges of the phylogenetic tree and reconstructed ancestral gene orders to identify possible synapomorphies in Orthoptera. We found all types of MTRs in our dataset: inversions, transpositions, inverse transpositions, and tandem-duplication/random loss events (TDRL). Most of the suggested MTRs were in single and unrelated species. Out of five MTRs which were unique in subgroups of Orthoptera, we suggest four of them to be synapomorphies; those were in the infraorder Acrididea, in the tribe Holochlorini, in the subfamily Pseudophyllinae, and in the two families Phalangopsidae and Gryllidae or their common ancestor (leading to the relationship ((Phalangopsidae + Gryllidae) + Trigonidiidae)). However, similar MTRs have been found in distant insect lineages. Our findings suggest convergent evolution of specific mitochondrial gene orders in several species, deviant from the evolution of the mitogenome DNA sequence. As most MTRs were detected at terminal nodes, a phylogenetic inference of deeper nodes based on MTRs is not supported. Hence, the marker does not seem to aid resolving the phylogeny of Orthoptera, but adds further evidence for the complex evolution of the whole group, especially at the genetic and genomic levels. The results indicate a high demand for more research on patterns and underlying mechanisms of MTR events in Orthoptera.

Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones.

The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions.

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Repositories for Taxonomic Data: Where We Are and What is Missing.

Natural history collections are leading successful large-scale projects of specimen digitization (images, metadata, DNA barcodes), thereby transforming taxonomy into a big data science. Yet, little effort has been directed towards safeguarding and subsequently mobilizing the considerable amount of original data generated during the process of naming 15,000-20,000 species every year. From the perspective of alpha-taxonomists, we provide a review of the properties and diversity of taxonomic data, assess their volume and use, and establish criteria for optimizing data repositories. We surveyed 4113 alpha-taxonomic studies in representative journals for 2002, 2010, and 2018, and found an increasing yet comparatively limited use of molecular data in species diagnosis and description. In 2018, of the 2661 papers published in specialized taxonomic journals, molecular data were widely used in mycology (94%), regularly in vertebrates (53%), but rarely in botany (15%) and entomology (10%). Images play an important role in taxonomic research on all taxa, with photographs used in >80% and drawings in 58% of the surveyed papers. The use of omics (high-throughput) approaches or 3D documentation is still rare. Improved archiving strategies for metabarcoding consensus reads, genome and transcriptome assemblies, and chemical and metabolomic data could help to mobilize the wealth of high-throughput data for alpha-taxonomy. Because long-term-ideally perpetual-data storage is of particular importance for taxonomy, energy footprint reduction via less storage-demanding formats is a priority if their information content suffices for the purpose of taxonomic studies. Whereas taxonomic assignments are quasifacts for most biological disciplines, they remain hypotheses pertaining to evolutionary relatedness of individuals for alpha-taxonomy. For this reason, an improved reuse of taxonomic data, including machine-learning-based species identification and delimitation pipelines, requires a cyberspecimen approach-linking data via unique specimen identifiers, and thereby making them findable, accessible, interoperable, and reusable for taxonomic research. This poses both qualitative challenges to adapt the existing infrastructure of data centers to a specimen-centered concept and quantitative challenges to host and connect an estimated $ \le $2 million images produced per year by alpha-taxonomic studies, plus many millions of images from digitization campaigns. Of the 30,000-40,000 taxonomists globally, many are thought to be nonprofessionals, and capturing the data for online storage and reuse therefore requires low-complexity submission workflows and cost-free repository use. Expert taxonomists are the main stakeholders able to identify and formalize the needs of the discipline; their expertise is needed to implement the envisioned virtual collections of cyberspecimens. [Big data; cyberspecimen; new species; omics; repositories; specimen identifier; taxonomy; taxonomic data.].

Integrative evidence confirms new endemic island frogs and transmarine dispersal of amphibians between Madagascar and Mayotte (Comoros archipelago).

Previous genetic studies of frogs from Mayotte Island (a French Overseas Department in the Comoros Archipelago) in the Western Indian Ocean have provided evidence for oceanic dispersal in amphibians, which is a rare phenomenon due to the osmotic intolerance of amphibians to saline water. Using an integrative approach including morphological, bioacoustic, and genetic evidence, we here confirm that these frogs correspond to two new species and are the only representatives of the family Mantellidae not endemic to Madagascar. Blommersia transmarina sp. nov. differs from its sister taxon, B. wittei, by several morphological differences including larger body size (snout-vent length up to 34.5 mm) and by slight differences in advertisement calls. Boophis nauticus sp. nov. differs from its closest relatives, B. tephraeomystax and B. doulioti, by slight morphological differences (including larger body size), a reddish (vs. silvery or golden) iris coloration in life, and slightly different advertisement calls. The two new species differ from their closest relatives by a substantial genetic differentiation, with pairwise genetic distances > 5% in the mitochondrial 16S rRNA gene, and based on the limited available data, also by distinct differences in nuclear DNA. They also are both larger than their closest relatives from Madagascar and B. transmarina sp. nov. is the largest Blommersia species, suggesting a moderate form of island gigantism. The Madagascan sister species B. wittei and B. doulioti are among the relatively few amphibian species occurring in the arid western biomes of the island, are adapted to open landscape, and reproduce in stagnant water bodies, which we hypothesize may represent important preadaptations for successful overseas colonization.

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.

Unveiling the diversification dynamics of Australasian predaceous diving beetles in the Cenozoic.

During the Cenozoic, Australia experienced major climatic shifts that have had dramatic ecological consequences for the modern biota. Mesic tropical ecosystems were progressively restricted to the coasts and replaced by arid-adapted floral and faunal communities. Whilst the role of aridification has been investigated in a wide range of terrestrial lineages, the response of freshwater clades remains poorly investigated. To gain insights into the diversification processes underlying a freshwater radiation, we studied the evolutionary history of the Australasian predaceous diving beetles of the tribe Hydroporini (147 described species). We used an integrative approach including the latest methods in phylogenetics, divergence time estimation, ancestral character state reconstruction, and likelihood-based methods of diversification rate estimation. Phylogenies and dating analyses were reconstructed with molecular data from seven genes (mitochondrial and nuclear) for 117 species (plus 12 outgroups). Robust and well-resolved phylogenies indicate a late Oligocene origin of Australasian Hydroporini. Biogeographic analyses suggest an origin in the East Coast region of Australia, and a dynamic biogeographic scenario implying dispersal events. The group successfully colonized the tropical coastal regions carved by a rampant desertification, and also colonized groundwater ecosystems in Central Australia. Diversification rate analyses suggest that the ongoing aridification of Australia initiated in the Miocene contributed to a major wave of extinctions since the late Pliocene probably attributable to an increasing aridity, range contractions and seasonally disruptions resulting from Quaternary climatic changes. When comparing subterranean and epigean genera, our results show that contrasting mechanisms drove their diversification and therefore current diversity pattern. The Australasian Hydroporini radiation reflects a combination of processes that promoted both diversification, resulting from new ecological opportunities driven by initial aridification, and a subsequent loss of mesic adapted diversity due to increasing aridity.

Phylogeny of dwarf geckos of the genus Lygodactylus (Gekkonidae) in the Western Indian Ocean.

Diurnal dwarf geckos of the genus Lygodactylus are distributed in tropical and subtropical regions and live in highly diverse habitats. The genus currently comprises 79 species and several candidates for new species or subspecies. Most of these taxa occur in Sub-Saharan Africa and Madagascar, with only two described species in South America. Although the main center of diversity of Lygodactylus currently is Africa, the genus probably has a Malagasy origin, followed by two or three independent transoceanic dispersal events between Madagascar and Africa and one trans-Atlantic dispersal from Africa to South America. A few species colonised islands in the Western Indian Ocean belonging to the Zanzibar Archipelago and to the Îles Éparses. Here we examined L. grotei pakenhami from Pemba Island, L. insularis from Juan de Nova, and L. verticillatus from Europa Island to clarify their taxonomic status and their origin. Concerning L. grotei pakenhami and L. insularis, preceding studies pointed to a relation to species of the African L. capensis group. In contrast, L. verticillatus on Europa Island is considered to be conspecific with Malagasy populations. Therefore, we conducted a phylogenetic study of the African L. capensis group and the Malagasy L. verticillatus group, and examined color pattern, selected morphological characters and two mitochondrial markers (ND2 for African and 16S rRNA for Malagasy Lygodactylus). Lygodactylus grotei pakenhami from Pemba and L. grotei from mainland Africa cannot be distinguished by their scalation, but their reciprocal monophyly suggested by mitochondrial DNA, conspicuously different coloration (both in adults and hatchlings) and their high genetic distances (16.3% in ND2) support the hypothesis that these taxa represent two distinct species. Consequently, we elevate L. grotei pakenhami to species level, as Lygodactylus pakenhami Loveridge, 1941. Lygodactylus pakenhami is endemic to Pemba Island which was possibly separated from the African mainland during the late Miocene or Early Pliocene (6 million years ago). The simplest explanation for the existence of L. pakenhami on Pemba is vicariance. A recent, human-mediated transportation is excluded, as the molecular data clearly indicate a longer period of isolation. Lygodactylus insularis has been supposed to be related to the taxa 'capensis' or 'grotei'. However, it is impossible to discern the relationship of L. insularis, L. capensis and L. grotei by means of scalation or coloration alone. Our molecular phylogenetic analyses reveal that L. insularis is embedded within the L. capensis group, clearly indicating its African origin. The single gene (ND2) as well as the multigene analyses fully support a closer common origin of L. insularis and L. capensis than of L. insularis and L. grotei. However, the position of L. insularis within the clade formed by L. insularis, L. nyaneka, L. capensis sensu stricto and six L. aff. capensis groups is not clearly resolved. Lygodactylus insularis is endemic on Juan de Nova Island, an old low elevation atoll. That all L. insularis mitochondrial sequences are very similar to each other and together form a monophyletic lineage is in agreement with the hypothesis of a single dispersal event to the island. For the L. verticillatus population from Europa Island our mitochondrial data suggest close relationships to conspecific samples from the coastal regions of south-western Madagascar. As we found no relevant morphological or genetic differences between the insular and the Malagasy populations of L. verticillatus, and no remarkable genetic variation within the monophyletic lineage on Europa, we suggest a single, very recent dispersal event, perhaps human-mediated. Although the genus Lygodactylus colonised Africa, islands in the Gulf of Guinea, South America and some islands in the Western Indian Ocean, it seems-compared to other lizard genera-to be only moderately successful in transoceanic long-distance dispersal.

New estimates of genome size in Orthoptera and their evolutionary implications.

Animal genomes vary widely in size, and much of their architecture and content remains poorly understood. Even among related groups, such as orders of insects, genomes may vary in size by orders of magnitude-for reasons unknown. The largest known insect genomes were repeatedly found in Orthoptera, e.g., Podisma pedestris (1C = 16.93 pg), Stethophyma grossum (1C = 18.48 pg) and Bryodemella holdereri (1C = 18.64 pg). While all these species belong to the suborder of Caelifera, the ensiferan Deracantha onos (1C = 19.60 pg) was recently found to have the largest genome. Here, we present new genome size estimates of 50 further species of Ensifera (superfamilies Gryllidea, Tettigoniidea) and Caelifera (Acrididae, Tetrigidae) based on flow cytometric measurements. We found that Bryodemella tuberculata (Caelifera: Acrididae) has the so far largest measured genome of all insects with 1C = 21.96 pg (21.48 gBp). Species of Orthoptera with 2n = 16 and 2n = 22 chromosomes have significantly larger genomes than species with other chromosome counts. Gryllidea genomes vary between 1C = 0.95 and 2.88 pg, and Tetrigidae between 1C = 2.18 and 2.41, while the genomes of all other studied Orthoptera range in size from 1C = 1.37 to 21.96 pg. Reconstructing ancestral genome sizes based on a phylogenetic tree of mitochondrial genomic data, we found genome size values of >15.84 pg only for the nodes of Bryodemella holdereri / B. tuberculata and Chrysochraon dispar / Euthystira brachyptera. The predicted values of ancestral genome sizes are 6.19 pg for Orthoptera, 5.37 pg for Ensifera, and 7.28 pg for Caelifera. The reasons for the large genomes in Orthoptera remain largely unknown, but a duplication or polyploidization seems unlikely as chromosome numbers do not differ much. Sequence-based genomic studies may shed light on the underlying evolutionary mechanisms.

The Genomics of Isolated Populations of Gampsocleis glabra (Orthoptera: Tettigoniidae) in Central and Western Europe.

Habitat destruction and fragmentation are among the major current threats to global biodiversity. Fragmentation may also affect species with good dispersal abilities. We study the heath bushcricket Gampsocleis glabra, a specialist of steppe-like habitats across Europe that are highly fragmented, investigating if these isolated populations can be distinguished using population genomics and if there are any traces of admixture or dispersal among them. We try to answer these questions using genome-wide SNP data generated with ddRAD sequencing. We calculated F-statistics and visualized differentiation using STRUCTURE plots. While limited by the difficulty of sampling this threatened species, our results show that all populations except one that was represented by a singleton were clearly distinct, with pairwise FST values between 0.010 and 0.181. STRUCTURE indicated limited but visible admixture across most populations and probably also an exchange of individuals between populations of Germany and The Netherlands. We conclude that in G. glabra, a certain amount of gene flow has persisted, at least in the past, also among populations that are isolated today. We also detect a possibly more recent dispersal event between a population in The Netherlands and one in Germany, which may be human aided. We suggest that the conservation of larger populations should be maintained, that efforts should be taken to restore abandoned habitat, that the preservation even of small habitat fragments may be beneficial for the conservation of this species, and that these habitats should be regularly monitored for possible (re-)colonization.

Pleistocene climate change promoted rapid diversification of aquatic invertebrates in Southeast Australia.

BACKGROUND: The Pleistocene Ice Ages were the most recent geohistorical event of major global impact, but their consequences for most parts of the Southern hemisphere remain poorly known. We investigate a radiation of ten species of Sternopriscus, the most species-rich genus of epigean Australian diving beetles. These species are distinct based on genital morphology but cannot be distinguished readily by mtDNA and nDNA because of genotype sharing caused by incomplete lineage sorting. Their genetic similarity suggests a Pleistocene origin. RESULTS: We use a dataset of 3858 bp of mitochondrial and nuclear DNA to reconstruct a phylogeny of Sternopriscus using gene and species trees. Diversification analyses support the finding of a recent rapid speciation event with estimated speciation rates of up to 2.40 species per MY, which is considerably higher than the proposed average rate of 0.16 species per MY for insects. Additionally, we use ecological niche modeling and analyze data on habitat preferences to test for niche divergence between species of the recent Sternopriscus radiation. These analyses show that the species can be characterized by a set of ecological variables referring to habitat, climate and altitude. CONCLUSIONS: Our results suggest that the repeated isolation of populations in glacial refugia might have led to divergent ecological adaptations and the fixation of morphological traits supporting reproductive isolation and therefore may have promoted speciation. The recent Sternopriscus radiation fulfills many characteristics of a species flock and would be the first described example of an aquatic insect species flock. We argue that the species of this group may represent a stage in speciation past the species flock condition because of their mostly broad and often non-overlapping ranges and preferences for different habitat types.

Molecular phylogeny of the squeak beetles, a family with disjunct Palearctic-Australian range.

Many higher groups of plants and animals show distributional patterns which have been shown or have at some point in time been suggested to be correlated with plate tectonics and the ancient supercontinents Laurasia and Gondwana. Here, we study the family of squeak beetles (Coleoptera: Adephaga: Hygrobiidae) and its enigmatic distribution pattern, with one species in the Western Palearctic, one in China and four in Australia. We present a molecular phylogeny including five of the six extant species, showing the monophyly of the Australian radiation. We use a molecular clock approach, which indicates that Hygrobiidae is an ancient group dating back to the breakup of Pangea and discuss the possibility of vicariance as explanation for its current distribution.

Methodological challenges in the genomic analysis of an endangered mammal population with low genetic diversity.

Recently, populations of various species with very low genetic diversity have been discovered. Some of these persist in the long term, but others could face extinction due to accelerated loss of fitness. In this work, we characterize 45 individuals of one of these populations, belonging to the Iberian desman (Galemys pyrenaicus). For this, we used the ddRADseq technique, which generated 1421 SNPs. The heterozygosity values of the analyzed individuals were among the lowest recorded for mammals, ranging from 26 to 91 SNPs/Mb. Furthermore, the individuals from one of the localities, highly isolated due to strong barriers, presented extremely high inbreeding coefficients, with values above 0.7. Under this scenario of low genetic diversity and elevated inbreeding levels, some individuals appeared to be almost genetically identical. We used different methods and simulations to determine if genetic identification and parentage analysis were possible in this population. Only one of the methods, which does not assume population homogeneity, was able to identify all individuals correctly. Therefore, genetically impoverished populations pose a great methodological challenge for their genetic study. However, these populations are of primary scientific and conservation interest, so it is essential to characterize them genetically and improve genomic methodologies for their research.

Morphological, osteological, and genetic data support a new species of Madatyphlops (Serpentes: Typhlopidae) endemic to Mayotte Island, Comoros Archipelago.

Blind snakes (Typhlopidae) are an enigmatic group of small burrowing snakes whose anatomy, phylogenetics, and biodiversity remain poorly known. Madatyphlops comorensis (Boulenger, 1889), endemic to the Comoros Archipelago in the Western Indian Ocean, is one of many species whose phylogenetic placement and generic assignment is unclear. We used DNA barcoding, external morphological examination, and osteological data from 3D reconstruction with micro-CT to study specimens of Madatyphlops from the Comoros Archipelago. Our results support the placement of M. comorensis in Madatyphlops and the recognition of the specimens from Mayotte Island as a closely related but distinct species, which we describe as Madatyphlops eudelini sp. nov. In this context, we present the first detailed osteological descriptions of any species of Madatyphlops, which we hope will serve as groundwork for further osteological studies in this genus and contribute to our limited but growing understanding of the osteology of typhlopid snakes.

Extreme miniaturization of a new amniote vertebrate and insights into the evolution of genital size in chameleons.

Evolutionary reduction of adult body size (miniaturization) has profound consequences for organismal biology and is an important subject of evolutionary research. Based on two individuals we describe a new, extremely miniaturized chameleon, which may be the world's smallest reptile species. The male holotype of Brookesia nana sp. nov. has a snout-vent length of 13.5 mm (total length 21.6 mm) and has large, apparently fully developed hemipenes, making it apparently the smallest mature male amniote ever recorded. The female paratype measures 19.2 mm snout-vent length (total length 28.9 mm) and a micro-CT scan revealed developing eggs in the body cavity, likewise indicating sexual maturity. The new chameleon is only known from a degraded montane rainforest in northern Madagascar and might be threatened by extinction. Molecular phylogenetic analyses place it as sister to B. karchei, the largest species in the clade of miniaturized Brookesia species, for which we resurrect Evoluticauda Angel, 1942 as subgenus name. The genetic divergence of B. nana sp. nov. is rather strong (9.9‒14.9% to all other Evoluticauda species in the 16S rRNA gene). A comparative study of genital length in Malagasy chameleons revealed a tendency for the smallest chameleons to have the relatively largest hemipenes, which might be a consequence of a reversed sexual size dimorphism with males substantially smaller than females in the smallest species. The miniaturized males may need larger hemipenes to enable a better mechanical fit with female genitals during copulation. Comprehensive studies of female genitalia are needed to test this hypothesis and to better understand the evolution of genitalia in reptiles.

Towards completion of the species inventory of small-sized leaf-tailed geckos: two new species of Uroplatus from northern Madagascar.

The northern part of Madagascar is well known for its high species diversity and endemism. Exceptional species richness is related to the existence of large forest blocks and mountain complexes. These areas shelter a diverse variety of habitats occupied by a wide diversity of species, including leaf-tailed geckos of the genus Uroplatus. Based on morphological and molecular evidence, we here formally name two evolutionary lineages as new species that previously had been considered as candidate species (Uroplatus spp. Ca3 and Ca4), both small-sized species of the Uroplatus ebenaui group. Genetically, both new species are related to U. finiavana with a genetic divergence (uncorrected pairwise distance) of 10.3-12.8% in a fragment of the mitochondrial 16S rRNA gene, and separated from each other by 10.3-10.7%. Uroplatus fangorn sp. nov. is described from the Marojejy massif and is also known from Andrevorevo, Lohanandroranga and Sorata; it is similar to U. ebenaui and U. fetsy but is distinguishable by its rather short tail and only partially black pigmented oral mucosa. Uroplatus fivehy sp. nov. is described from the Sorata Massif and is wider-ranging, occurring in an area from Marotandrano and Makira to Sorata, comprising Marojejy, Anjanaharibe-Sud, Ankitsika and Betaolana; it is morphologically similar to U. finiavana but distinguishable by tail size and shape. Both newly described species are found in rainforest from mid to high elevation and range respectively from 840-1417 m for U. fivehy sp nov. and 1300-1800 m for U. fangorn sp. nov. According to their respective geographical distributions, we propose to classify the two new species as Vulnerable under the IUCN Red List criteria, due to their occurrence in some forests outside of the protected area network plus the continuing decline of forest patches in the north of Madagascar.

A pipeline for metabarcoding and diet analysis from fecal samples developed for a small semi-aquatic mammal.

Metabarcoding allows the genetic analysis of pooled samples of various sources. It is becoming popular in the study of animal diet, especially because it allows the analysis of the composition of feces without the need of handling animals. In this work, we studied the diet of the Pyrenean desman (Galemys pyrenaicus), a small semi-aquatic mammal endemic to the Iberian Peninsula and the Pyrenees, by sequencing COI minibarcodes from feces using next-generation sequencing techniques. For the identification of assembled sequences, we employed a tree-based identification method that used a reference tree of sequences of freshwater organisms. The comparison of freshly collected fecal samples and older samples showed that fresh samples produced significantly more sequencing reads. They also rendered more operational taxonomical units (OTUs), but not significantly. Our analyses of 41 samples identified 224 OTUs corresponding to species of the reference tree. Ephemeroptera, Diptera excl. Chironomidae, and Chironomidae were the most highly represented groups in terms of reads as well as samples. Other groups of freshwater organisms detected were Plecoptera, Trichoptera, Neuropteroida, Coleoptera, Crustacea, and Annelida. Our results are largely in line with previous morphological and genetic studies on the diet of the Pyrenean desman, but allowed the identification of a higher diversity of OTUs in each sample. Additionally, the bioinformatic pipeline we developed for deep sequencing of fecal samples will enable the quantitative analysis of the diet of this and other species, which can be highly useful to determine their ecological requirements.

A review of the taxonomy and osteology of the Rhombophryne serratopalpebrosa species group (Anura: Microhylidae) from Madagascar, with comments on the value of volume rendering of micro-CT data to taxonomists.

Over the last three years, three new species of saw-browed diamond frogs (Rhombophryne serratopalpebrosa species group)-a clade of cophyline microhylid frogs native to northern and eastern Madagascar-have been described. We here review the taxonomy of these frogs based on a new multi-gene phylogeny of the group, which confirms its monophyly but is insufficiently resolved to clarify most intra-group relationships. We confirm Rhombophryne guentherpetersi (Guibé, 1974) to be a member of this group, and we re-describe it based on its type series and newly collected material; the species is characterised by small superciliary spines (overlooked in its original description), as well as large tibial glands and an unusually laterally compressed pectoral girdle. We go on to describe two new species of this group from northern Madagascar: both R. diadema sp. nov. from the Sorata Massif and R. regalis sp. nov. from several sites in the northeast of the island possess three superciliary spines, but they are characterised by several subtle morphological and osteological differences. The new species are separated from all known congeners by an uncorrected pairwise distance of at least 5.1% in a ca. 550 bp fragment of the 16S rRNA gene. In order to highlight the significance of the skeleton in the taxonomy of this group, we provide a detailed description of its generalized osteology based on volume-rendered micro-CT scans of all described members, revisiting already-described skeletons of some species, and describing the skeletons of R. guentherpetersi, R. coronata, and the new taxa for the first time. Use of volume rendering, instead of surface rendering of micro-CT data, resulted in some discrepancies due to the properties of each method. We discuss these inconsistencies and their bearing on the relative value of surface and volume rendering in the taxonomist's toolkit. We argue that, while surface models are more practical for the reader, volumes are generally a more objective representation of the data. Thus, taxonomic description work should be based on volume rendering when possible, with surface models presented as an aid to the reader.

Phylogenetic niche conservatism explains an inverse latitudinal diversity gradient in freshwater arthropods.

The underlying mechanisms responsible for the general increase in species richness from temperate regions to the tropics remain equivocal. Many hypotheses have been proposed to explain this astonishing pattern but additional empirical studies are needed to shed light on the drivers at work. Here we reconstruct the evolutionary history of the cosmopolitan diving beetle subfamily Colymbetinae, the majority of which are found in the Northern hemisphere, hence exhibiting an inversed latitudinal diversity gradient. We reconstructed a dated phylogeny using 12 genes, to investigate the biogeographical history and diversification dynamics in the Colymbetinae. We aimed to identify the role that phylogenetic niche conservatism plays in the inversed diversification pattern seen in this group. Our results suggest that Colymbetinae originated in temperate climates, which supports the hypothesis that their distribution is the result of an ancestral adaptation to temperate environmental conditions rather than tropical origins, and that temperate niche conservatism can generate and/or maintain inverse latitudinal diversity gradients.