On and off the rocks: persistence and ecological diversification in a tropical Australian lizard radiation.

BACKGROUND: Congruent patterns in the distribution of biodiversity between regions or habitats suggest that key factors such as climatic and topographic variation may predictably shape evolutionary processes. In a number of tropical and arid biomes, genetic analyses are revealing deeper and more localised lineage diversity in rocky ranges than surrounding habitats. Two potential drivers of localised endemism in rocky areas are refugial persistence through climatic change, or ecological diversification and specialisation. Here we examine how patterns of lineage and phenotypic diversity differ across two broad habitat types (rocky ranges and open woodlands) in a small radiation of gecko lizards in the genus Gehyra (the australis group) from the Australian Monsoonal Tropics biome. RESULTS: Using a suite of approaches for delineating evolutionarily independent lineages, we find between 26 and 41 putative evolutionary units in the australis group (versus eight species currently recognised). Rocky ranges are home to a greater number of lineages that are also relatively more restricted in distribution, while lineages in open woodland habitats are fewer, more widely distributed, and, in one case, show evidence of range expansion. We infer at least two shifts out of rocky ranges and into surrounding woodlands. Phenotypic divergence between rocky ranges specialist and more generalist taxa is detected, but no convergent evolutionary regimes linked to ecology are inferred. CONCLUSIONS: In climatically unstable biomes such as savannahs, rocky ranges have functioned as zones of persistence, generators of diversity and a source of colonists for surrounding areas. Phenotypic divergence can also be linked to the use of differing habitat types, however, the extent to which ecological specialisation is a primary driver or secondary outcome of localised diversification remains uncertain.

Phylogenomic analysis of Apoidea sheds new light on the sister group of bees.

BACKGROUND: Apoid wasps and bees (Apoidea) are an ecologically and morphologically diverse group of Hymenoptera, with some species of bees having evolved eusocial societies. Major problems for our understanding of the evolutionary history of Apoidea have been the difficulty to trace the phylogenetic origin and to reliably estimate the geological age of bees. To address these issues, we compiled a comprehensive phylogenomic dataset by simultaneously analyzing target DNA enrichment and transcriptomic sequence data, comprising 195 single-copy protein-coding genes and covering all major lineages of apoid wasps and bee families. RESULTS: Our compiled data matrix comprised 284,607 nucleotide sites that we phylogenetically analyzed by applying a combination of domain- and codon-based partitioning schemes. The inferred results confirm the polyphyletic status of the former family "Crabronidae", which comprises nine major monophyletic lineages. We found the former subfamily Pemphredoninae to be polyphyletic, comprising three distantly related clades. One of them, Ammoplanina, constituted the sister group of bees in all our analyses. We estimate the origin of bees to be in the Early Cretaceous (ca. 128 million years ago), a time period during which angiosperms rapidly radiated. Finally, our phylogenetic analyses revealed that within the Apoidea, (eu)social societies evolved exclusively in a single clade that comprises pemphredonine and philanthine wasps as well as bees. CONCLUSION: By combining transcriptomic sequences with those obtained via target DNA enrichment, we were able to include an unprecedented large number of apoid wasps in a phylogenetic study for tracing the phylogenetic origin of bees. Our results confirm the polyphyletic nature of the former wasp family Crabonidae, which we here suggest splitting into eight families. Of these, the family Ammoplanidae possibly represents the extant sister lineage of bees. Species of Ammoplanidae are known to hunt thrips, of which some aggregate on flowers and feed on pollen. The specific biology of Ammoplanidae as predators indicates how the transition from a predatory to pollen-collecting life style could have taken place in the evolution of bees. This insight plus the finding that (eu)social societies evolved exclusively in a single subordinated lineage of apoid wasps provides new perspectives for future comparative studies.

Analysis of the genome of the New Zealand giant collembolan (Holacanthella duospinosa) sheds light on hexapod evolution.

BACKGROUND: The New Zealand collembolan genus Holacanthella contains the largest species of springtails (Collembola) in the world. Using Illumina technology we have sequenced and assembled a draft genome and transcriptome from Holacanthella duospinosa (Salmon). We have used this annotated assembly to investigate the genetic basis of a range of traits critical to the evolution of the Hexapoda, the phylogenetic position of H. duospinosa and potential horizontal gene transfer events. RESULTS: Our genome assembly was ~375 Mbp in size with a scaffold N50 of ~230 Kbp and sequencing coverage of ~180×. DNA elements, LTRs and simple repeats and LINEs formed the largest components and SINEs were very rare. Phylogenomics (370,877 amino acids) placed H. duospinosa within the Neanuridae. We recovered orthologs of the conserved sex determination genes thought to play a role in sex determination. Analysis of CpG content suggested the absence of DNA methylation, and consistent with this we were unable to detect orthologs of the DNA methyltransferase enzymes. The small subunit rRNA gene contained a possible retrotransposon. The Hox gene complex was broken over two scaffolds. For chemosensory ability, at least 15 and 18 ionotropic glutamate and gustatory receptors were identified, respectively. However, we were unable to identify any odorant receptors or their obligate co-receptor Orco. Twenty-three chitinase-like genes were identified from the assembly. Members of this multigene family may play roles in the digestion of fungal cell walls, a common food source for these saproxylic organisms. We also detected 59 and 96 genes that blasted to bacteria and fungi, respectively, but were located on scaffolds that otherwise contained arthropod genes. CONCLUSIONS: The genome of H. duospinosa contains some unusual features including a Hox complex broken over two scaffolds, in a different manner to other arthropod species, a lack of odorant receptor genes and an apparent lack of environmentally responsive DNA methylation, unlike many other arthropods. Our detection of candidate horizontal gene transfer candidates confirms that this phenomenon is occurring across Collembola. These findings allow us to narrow down the regions of the arthropod phylogeny where key innovations have occurred that have facilitated the evolutionary success of Hexapoda.

Transcriptome and target DNA enrichment sequence data provide new insights into the phylogeny of vespid wasps (Hymenoptera: Aculeata: Vespidae).

The wasp family Vespidae comprises more than 5000 described species which represent life history strategies ranging from solitary and presocial to eusocial and socially parasitic. The phylogenetic relationships of the major vespid wasp lineages (i.e., subfamilies and tribes) have been investigated repeatedly by analyzing behavioral and morphological traits as well as nucleotide sequences of few selected genes with largely incongruent results. Here we reconstruct their phylogenetic relationships using a phylogenomic approach. We sequenced the transcriptomes of 24 vespid wasp and eight outgroup species and exploited the transcript sequences for design of probes for enriching 913 single-copy protein-coding genes to complement the transcriptome data with nucleotide sequence data from additional 25 ethanol-preserved vespid species. Results from phylogenetic analyses of the combined sequence data revealed the eusocial subfamily Stenogastrinae to be the sister group of all remaining Vespidae, while the subfamily Eumeninae turned out to be paraphyletic. Of the three currently recognized eumenine tribes, Odynerini is paraphyletic with respect to Eumenini, and Zethini is paraphyletic with respect to Polistinae and Vespinae. Our results are in conflict with the current tribal subdivision of Eumeninae and thus, we suggest granting subfamily rank to the two major clades of "Zethini": Raphiglossinae and Zethinae. Overall, our findings corroborate the hypothesis of two independent origins of eusociality in vespid wasps and suggest a single origin of using masticated and salivated plant material for building nests by Raphiglossinae, Zethinae, Polistinae, and Vespinae. The inferred phylogenetic relationships and the open access vespid wasp target DNA enrichment probes will provide a valuable tool for future comparative studies on species of the family Vespidae, including their genomes, life styles, evolution of sociality, and co-evolution with other organisms.

Leaves that walk and eggs that stick: comparative functional morphology and evolution of the adhesive system of leaf insect eggs (Phasmatodea: Phylliidae).

Phylliidae are herbivorous insects exhibiting impressive cryptic masquerade and are colloquially called "walking leaves". They imitate angiosperm leaves and their eggs often resemble plant seeds structurally and in some cases functionally. Despite overall morphological similarity of adult Phylliidae, their eggs reveal a significant diversity in overall shape and exochorionic surface features. Previous studies have shown that the eggs of most Phylliidae possess a specialised attachment mechanism with hierarchical exochorionic fan-like structures (pinnae), which are mantled by a film of an adhesive secretion (glue). The folded pinnae and glue respond to water contact, with the fibrous pinnae expanding and the glue being capable of reversible liquefaction. In general, the eggs of phylliids appear to exhibit varying structures that were suggested to represent specific adaptations to the different environments the eggs are deposited in. Here, we investigated the diversity of phylliid eggs and the functional morphology of their exochorionic structure. Based on the examination of all phylliid taxa for which the eggs are known, we were able to characterise eleven different morphological types. We explored the adhesiveness of these different egg morphotypes and experimentally compared the attachment performance on a broad range of substrates with different surface roughness, surface chemistry and tested whether the adhesion is replicable after detachment in multiple cycles. Furthermore, we used molecular phylogenetic methods to reconstruct the evolutionary history of different egg types and their adhesive systems within this lineage, based on 53 phylliid taxa. Our results suggest that the egg morphology is congruent with the phylogenetic relationships within Phylliidae. The morphological differences are likely caused by adaptations to the specific environmental requirements for the particular clades, as the egg morphology has an influence on the performance regarding the surface roughness. Furthermore, we show that different pinnae and the adhesive glue evolved convergently in different species. While the evolution of the Phylliidae in general appears to be non-adaptive judging on the strong similarity of the adults and nymphs of most species, the eggs represent a stage with complex and rather diverse functional adaptations including mechanisms for both fixation and dispersal of the eggs.

On seven undescribed leaf insect species revealed within the recent "Tree of Leaves" (Phasmatodea, Phylliidae).

With the recent advance in molecular phylogenetics focused on the leaf insects (Phasmatodea, Phylliidae), gaps in knowledge are beginning to be filled. Yet, shortcomings are also being highlighted, for instance, the unveiling of numerous undescribed phylliid species. Here, some of these taxa are described, including Phylliumiyadaonsp. nov. from Mindoro Island, Philippines; Phylliumsamarensesp. nov. from Samar Island, Philippines; Phylliumortizisp. nov. from Mindanao Island, Philippines; Pulchriphylliumheraclessp. nov. from Vietnam; Pulchriphylliumdelisleisp. nov. from South Kalimantan, Indonesia; and Pulchriphylliumbhaskaraisp. nov. from Java, Indonesia. Several additional specimens of these species together with a seventh species described herein, Pulchriphylliumanangusp. nov. from southwestern India, were incorporated into a newly constructed phylogenetic tree. Additionally, two taxa that were originally described as species, but in recent decades have been treated as subspecies, are elevated back to species status to reflect their unique morphology and geographic isolation, creating the following new combinations: Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. from Bangladesh and northeastern India, and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from the Seychelles islands. Lectotype specimens are also designated for Pulchriphylliumscythe (Gray, 1843) stat. rev., comb. nov. and Pulchriphylliumcrurifolium (Audinet-Serville, 1838) stat. rev., comb. nov. from original type material.

A second view on the evolution of flight in stick and leaf insects (Phasmatodea).

BACKGROUND: The re-evolution of complex characters is generally considered impossible, yet, studies of recent years have provided several examples of phenotypic reversals shown to violate Dollo's law. Along these lines, the regain of wings in stick and leaf insects (Phasmatodea) was hypothesised to have occurred several times independently after an ancestral loss, a scenario controversially discussed among evolutionary biologists due to overestimation of the potential for trait reacquisition as well as to the lack of taxonomic data. RESULTS: We revisited the recovery of wings by reconstructing a phylogeny based on a comprehensive taxon sample of over 500 representative phasmatodean species to infer the evolutionary history of wings. We additionally explored the presence of ocelli, the photoreceptive organs used for flight stabilisation in winged insects, which might provide further information for interpreting flight evolution. Our findings support an ancestral loss of wings and that the ancestors of most major lineages were wingless. While the evolution of ocelli was estimated to be dependent on the presence of (fully-developed) wings, ocelli are nevertheless absent in the majority of all examined winged species and only appear in the members of few subordinate clades, albeit winged and volant taxa are found in every euphasmatodean lineage. CONCLUSION: In this study, we explored the evolutionary history of wings in Phasmatodea and demonstrate that the disjunct distribution of ocelli substantiates the hypothesis on their regain and thus on trait reacquisition in general. Evidence from the fossil record as well as future studies focussing on the underlying genetic mechanisms are needed to validate our findings and to further assess the evolutionary process of phenotypic reversals.

A tree of leaves: Phylogeny and historical biogeography of the leaf insects (Phasmatodea: Phylliidae).

The insect order Phasmatodea is known for large slender insects masquerading as twigs or bark. In contrast to these so-called stick insects, the subordinated clade of leaf insects (Phylliidae) are dorso-ventrally flattened and therefore resemble leaves in a unique way. Here we show that the origin of extant leaf insects lies in the Australasian/Pacific region with subsequent dispersal westwards to mainland Asia and colonisation of most Southeast Asian landmasses. We further hypothesise that the clade originated in the Early Eocene after the emergence of angiosperm-dominated rainforests. The genus Phyllium to which most of the ~100 described species pertain is recovered as paraphyletic and its three non-nominate subgenera are recovered as distinct, monophyletic groups and are consequently elevated to genus rank. This first phylogeny covering all major phylliid groups provides the basis for future studies on their taxonomy and a framework to unveil more of their cryptic and underestimated diversity.

Cryptophyllium, the hidden leaf insects - descriptions of a new leaf insect genus and thirteen species from the former celebicum species group (Phasmatodea, Phylliidae).

While the leaf insects (Phylliidae) are a well-supported group within Phasmatodea, the genus Phyllium Illiger, 1798 has repeatedly been recovered as paraphyletic. Here, the Phyllium (Phyllium) celebicum species group is reviewed and its distinctiveness from the remaining Phylliini genera and subgenera in a phylogenetic context based on morphological review and a phylogenetic analysis of three genes (nuclear gene 28S and mitochondrial genes COI and 16S) from most known and multiple undescribed species is shown. A new genus, Cryptophyllium gen. nov., is erected to partially accommodate the former members of the celebicum species group. Two species, Phyllium ericoriaiHennemann et al., 2009 and Phyllium bonifacioi Lit & Eusebio, 2014 morphologically and molecularly do not fall within this clade and are therefore left within Phyllium (Phyllium). The transfer of the remaining celebicum group members from Phyllium Illiger, 1798 to this new genus creates the following new combinations; Cryptophyllium athanysus (Westwood, 1859), comb. nov.; Cryptophyllium celebicum (de Haan, 1842), comb. nov.; Cryptophyllium chrisangi (Seow-Choen, 2017), comb. nov.; Cryptophyllium drunganum (Yang, 1995), comb. nov.; Cryptophyllium oyae (Cumming & Le Tirant, 2020), comb. nov.; Cryptophyllium parum (Liu, 1993), comb. nov.; Cryptophyllium rarum (Liu, 1993), comb. nov.; Cryptophyllium tibetense (Liu, 1993), comb. nov.; Cryptophyllium westwoodii (Wood-Mason, 1875), comb. nov.; Cryptophyllium yapicum (Cumming & Teemsma, 2018), comb. nov.; and Cryptophyllium yunnanense (Liu, 1993), comb. nov. The review of specimens belonging to this clade also revealed 13 undescribed species, which are described within as: Cryptophyllium animatum gen. et sp. nov. from Vietnam: Quang Nam Province; Cryptophyllium bankoi gen. et sp. nov. from Vietnam: Quang Ngai, Thua Thien Hue, Da Nang, Gia Lai, Quang Nam, and Dak Nong Provinces; Cryptophyllium bollensi gen. et sp. nov. from Vietnam: Ninh Thuan Province; Cryptophyllium daparo gen. et sp. nov. from China: Yunnan Province; Cryptophyllium echidna gen. et sp. nov. from Indonesia: Wangi-wangi Island; Cryptophyllium faulkneri gen. et sp. nov. from Vietnam: Quang Ngai and Lam Dong Provinces; Cryptophyllium icarus gen. et sp. nov. from Vietnam: Lam Dong and Dak Lak Provinces; Cryptophyllium khmer gen. et sp. nov. from Cambodia: Koh Kong and Siem Reap Provinces; Cryptophyllium limogesi gen. et sp. nov. from Vietnam: Lam Dong, Dak Lak, and Dak Nong Provinces; Cryptophyllium liyananae gen. et sp. nov. from China: Guangxi Province; Cryptophyllium nuichuaense gen. et sp. nov. from Vietnam: Ninh Thuan Province; Cryptophyllium phami gen. et sp. nov. from Vietnam: Dong Nai and Ninh Thuan Provinces; and Cryptophyllium wennae gen. et sp. nov. from China: Yunnan Province. All newly described species are morphologically described, illustrated, and molecularly compared to congenerics. With the molecular results revealing cryptic taxa, it was found necessary for Cryptophyllium westwoodii (Wood-Mason, 1875), comb. nov. to have a neotype specimen designated to allow accurate differentiation from congenerics. To conclude, male and female dichotomous keys to species for the Cryptophyllium gen. nov. are presented.

Notes on the leaf insects of the genus Phyllium of Sumatra and Java, Indonesia, including the description of two new species with purple coxae (Phasmatodea, Phylliidae).

Within the last two years, the leaf insects of the genus Phyllium of both the islands of Java and Sumatra have been reviewed extensively based on morphological observations. However, cryptic species which cannot be differentiated morphologically may be present among the various populations. Since it has frequently been demonstrated that analyses based on molecular data can bring clarity in such cases, we conducted a phylogenetic analysis based on three genes (nuclear gene 28S and mitochondrial genes COI and 16S) from the Phyllium species of these islands. The results show distinct molecular divergence for several populations and suggest the presence of two new cryptic species, morphologically inseparable from Phyllium hausleithneri Brock, 1999. From Sumatra, the population originally thought to be a range expansion for Phyllium hausleithneri, is now here described as Phyllium nisus sp. nov., with the only consistent morphological difference being the color of the eggs between the two populations (dark brown in P. hausleithneri and tan in P. nisus sp. nov.). Further, an additional population with purple coxae from Java was morphologically examined and found to have no consistent features to separate it morphologically from the other purple coxae species. This cryptic species from Java was however shown to be molecularly distinct from the other purple coxae populations from Sumatra and Peninsular Malaysia and is here described as Phyllium gardabagusi sp. nov. In addition, Phyllium giganteum is here officially reported from Java for the first time based on both historic and modern records of male specimens.

The Orphan Cytokine Receptor CRLF3 Emerged With the Origin of the Nervous System and Is a Neuroprotective Erythropoietin Receptor in Locusts.

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.

Cryptic lineage diversity, body size divergence, and sympatry in a species complex of Australian lizards (Gehyra).

Understanding the joint evolutionary and ecological underpinnings of sympatry among close relatives remains a key challenge in biology. This problem can be addressed through joint phylogenomic and phenotypic analysis of complexes of closely related lineages within, and across, species and hence representing the speciation continuum. For a complex of tropical geckos from northern Australia-Gehyra nana and close relatives-we combine mtDNA phylogeography, exon-capture sequencing, and morphological data to resolve independently evolving lineages and infer their divergence history and patterns of morphological evolution. Gehyra nana is found to include nine divergent lineages and is paraphyletic with four other species from the Kimberley region of north-west Australia. Across these 13 taxa, 12 of which are restricted to rocky habitats, several lineages overlap geographically, including on the diverse Kimberley islands. Morphological evolution is dominated by body size shifts, and both body size and shape have evolved gradually across the group. However, larger body size shifts are observed among overlapping taxa than among closely related parapatric lineages of G. nana, and sympatric lineages are more divergent than expected at random. Whether elevated body size differences among sympatric lineages are due to ecological sorting or character displacement remains to be determined.