Novel wing display and divergent agonistic behaviors of two incipient Psolodesmus damselflies.

Sexual selection via male competition is a strong evolutionary force that can drive rapid changes in competitive traits and subsequently lead to population divergence and speciation. Territorial males of many odonates are known to use their colorful wings as visual signals and to perform agonistic displays toward intruders. Psolodesmus mandarinus dorothea and Psolodesmus mandarinus mandarinus are two parapatrically distributed sister damselflies that share similar ecological characteristics but differ markedly in wing coloration. The wings of P. m. dorothea are mostly clear, whereas those of P. m. mandarinus have a large area of black pigmentation and a central white patch. We investigated whether territorial males of the two damselflies at breeding sites display distinct agonistic behaviors associated with their respective wing colors. Behavioral interactions between territorial and intruder males and their wing kinematics were filmed and analyzed for P. m. dorothea in Lienhuachih of central Taiwan, and P. m. mandarinus in Tianxiyuan and Fusan of northern Taiwan. We observed that the P. m. mandarinus males exhibited a novel set of perched wing displays, which was not only absent in its sister P. m. dorothea but also previously unknown in Odonata. At breeding sites, perched rival males of P. m. mandarinus with pigmented wings exhibited escalating agonistic wing-flapping and wing-hitting displays toward each other. In contrast, territorial males of P. m. dorothea with clear wings engaged only in aerial chase or face-to-face hovering when intruder males approached from the air. These results indicate that the two sister P. mandarinus damselflies diverged behaviorally in territorial contests and support the hypothesis of coadaptation on the basis of wing colors and types of wing movement in Odonata. Our findings further suggest that divergent agonistic wing displays may play a pivotal role in the speciation mechanism of P. mandarinus damselflies. The sequential analyses of behavioral characteristics and progression suggest that P. m. mandarinus damselflies likely use mutual assessment of rivals in territorial contests.

Comparative phylogeography of two hemipteran species (Geisha distinctissima and Megacopta cribraria) in the Zhoushan Archipelago of China reveals contrasting genetic structures despite concordant historical demographies.

Continental islands are useful models to explore the roles of shared historical factors in the evolution of sympatric species. However, China's largest continental group of islands, the Zhoushan Archipelago, was neglected by most studies focusing on biodiversity hotspots. Here we investigated the phylogeographic patterns and the historical demography of two sympatric hemipteran insects (Geisha distinctissima and Megacopta cribraria), which shared historical factors in the Zhoushan Archipelago. The results based on mtDNA (COX1, COX2-COX3, and CYTB) and nDNA (28S and ITS2) showed that G. distinctissima diverged into three genetic lineages (L1-L3) ~8.9-13.7 thousand years ago (kya), which coincided with the period of island isolation. However, the three lineages exhibit no clear phylogeographic patterns for frequent asymmetrical gene flow (starting around 5 kya) from the mainland and adjacent islands to other distant islands due to subsequent human activities. In contrast, only one genetic lineage exists for M. cribraria, without any phylogeographic structures. The ancestral range in the mainland as well as in neighboring islands, together with the frequent asymmetrical gene flow of M. cribraria (from the mainland and neighboring islands to more distant islands) within the last 5000 years suggests that human activities may have lead to the colonization of this species in the Zhoushan Archipelago. The contrasting genetic structures indicate shared historical factors but independent evolutionary histories for the two sympatric species in the Zhoushan Archipelago. Our demographic analysis clearly showed that both species underwent population expansion before 5 kya during the post-LGM (Last Glacial Maximum), which indicates that the two species shared concordant historical demographies. This result suggests that the population size of the two species was affected similarly by the climatic oscillations of post-LGM in Eastern China. Together, our findings reveal that the two insect species in the Zhoushan Archipelago exhibit contrasting genetic structures despite concordant historical demographies, which provides an important framework for the exploration of the evolution patterns of sympatric species in the continental island.

Endocuticle sclerotisation increases the mechanical stability of cuticle.

The cuticle plays an important role in the evolutionary success of insects. Many studies on insect cuticles have reported a soft, resilin-rich endocuticle. However, a recent study indicated the presence of a sclerotised endocuticle in the weevil Pachyrhynchus sarcitis kotoensis, which contradicts former knowledge. To understand the degree of sclerotisation in the endocuticle of the weevil and its potential function, we first examined the endocuticle by microscopic and staining techniques. We next performed mechanical tests to measure the material properties of the endocuticle, and numerical simulations to predict the structural effect of the sclerotisation. Our results provide the first evidence of the existence of a sclerotised endocuticle and its remarkable function in improving the mechanical stability of the cuticle. This study highlights the finding of a high degree of sclerotisation in the stiff endocuticle of the weevil, especially the matrix surrounding the fibres. This novel case brings new understanding of cuticle properties and gives promising insights into biomaterial design.

Too hard to swallow: a secret secondary defence of an aposematic insect.

Anti-predator strategies are significant components of adaptation in prey species. Aposematic prey are expected to possess effective defences that have evolved simultaneously with their warning colours. This study tested the hypothesis of the defensive function and ecological significance of the hard body in aposematic Pachyrhynchus weevils pioneered by Alfred Russel Wallace nearly 150 years ago. We used predation trials with Japalura tree lizards to assess the survivorship of 'hard' (mature) versus 'soft' (teneral) and 'clawed' (intact) versus 'clawless' (surgically removed) weevils. The ecological significance of the weevil's hard body was evaluated by assessing the hardness of the weevils, the local prey insects, and the bite forces of the lizard populations. The existence of toxins or deterrents in the weevil was examined by gas chromatography-mass spectrometry (GC-MS). All 'hard' weevils were instantly spat out after being bitten once and survived attacks by the lizards. In contrast, the 'soft' weevils were chewed and subsequently swallowed. The results were the same regardless of the presence or absence of the weevil's tarsal claws. The hardness of 'hard' Pachyrhynchus weevils was significantly higher than the average hardness of other prey insects in the same habitat and the mean bite forces of the local lizards. The four candidate compounds of the weevil identified by GC-MS had no known toxic or repellent functions against vertebrates. These results reveal that the hardness of aposematic prey functions as an effective secondary defence, and they provide a framework for understanding the spatio-temporal interactions between vertebrate predators and aposematic insect prey.

Rafting on floating fruit is effective for oceanic dispersal of flightless weevils.

Terrestrial species, especially non-vagile ones (those unable to fly or swim), cannot cross oceans without exploiting other animals or floating objects. However, the colonisation history of flightless Pachyrhynchus weevils, inferred from genetic data, reveals their ability to travel long distances to colonise remote islands. Here, we used captive-bred Pachyrhynchus jitanasaius to analyse (i) the physiological tolerance of weevils (egg, larva and adult stages) to different levels of salinity; (ii) the survival rate of larvae in a simulated ocean environment in the laboratory; and (iii) the survival rate of larvae in a field experiment in the ocean using fruit of the fish poison tree floating on the Kuroshio current in the Pacific Ocean. We found that the survival rate of larvae in seawater was lower than in fresh water, although if the larvae survived 7 days of immersion in seawater, some emerged as adults in the subsequent rearing process. No adults survived for more than 2 days, regardless of salinity level. After floating separately for 6 days in salt water in the laboratory and in the Kuroshio current, two of 18 larvae survived in the fruit. This study provides the first empirical evidence that P. jitanasaius larvae can survive 'rafting' on ocean currents and that the eggs and larvae of these weevils have the highest probability of crossing the oceanic barrier. This ability may facilitate over-the-sea dispersal of these flightless insects and further shape their distribution and speciation pattern in the Western Pacific islands.

Phylogenetic Relationships of Japanese Auritibicen Species (Hemiptera: Cicadidae: Cryptotympanini) Inferred from Mitochondrial and Nuclear Gene Sequences.

We investigated the phylogenetic relationships and divergence times within the genus Auritibicen(Cicadidae: Cicadinae: Cryptotympanini), analyzing five Japanese species (A. japonicus, A. bihamatus,A. kyushyuensis, A. esakii and A. flammatus) and three species from East Asian mainland and Taiwan (A. atrofasciatus, A. intermedius and A. chujoi) using mitochondrial cytochrome oxidase subunit I (COI) and nuclear elongation factor 1-alpha (EF-1a) gene sequences. Although the EF-1a gene tree did not resolve the relationships among these Auritibicen species, the trees based on COI gene and the combined data set showed that Japanese taxa comprised three distinct lineages: the individual species A. flammatus and A. bihamatus, and the A. japonicus group, comprising A. japonicus, A. esakii and A. kyushyuensis from Japan and A. intermedius from Korea. In A. kyushyuensis, which comprises three populations in Kyushu, western Honshu and Shikoku, the specimens from western Honshu and Shikoku were closely related to each other, but not to the specimen from Kyushu; instead, they were sister to the Korean A. intermedius. The incongruence between the gene tree and species tree necessitates further population genetic and morphological studies to confirm the classification and species status of the western Honshu and Shikoku populations of A. kyushyuensis, which were originally described as two independent species. Divergence time estimation suggested that the most recent common ancestor of Auritibicen species studied dated back to the late Pliocene and that the species of the A. japonicus group diverged during the mid Pleistocene. Thus, the Pleistocene climatic fluctuation may have promoted the divergence of the Auritibicen species.

Phylogeography of the Rice Spittle Bug (Callitettix versicolor) Implies Two Long-Term Mountain Barriers in South China.

South China is a region of remarkable topographic complexity. However, the impact of climate fluctuations in the Pleistocene on the local fauna and especially insects has not been extensively studied. We integrated mitochondrial DNA (mtDNA) and microsatellite data of the rice spittle bug, Callitettix versicolor, to determine the genetic structure, potential biogeographic barriers, and historical demography of this species. The mtDNA data revealed two distinct lineages (Western and Eastern) congruent with the geographically separated western and eastern sub-regions of the Hengduan Mountains. The Eastern lineage was subdivided into two sub-lineages, E1 and E2, congruent with the geographically separated northern and southern sub-regions of the Dabie Mountains. E2 was further subdivided into two sub-groups, E2-1 and E2-2, with a hybrid zone (Guizhou and Hunan Provinces) in which their areas were contiguous. The genetic structures constructed using mtDNA were corroborated by four clusters (G1-G4) of microsatellite data. The populations of each cluster were nearly consistent with a sub-lineage of the mtDNA gene tree (G1-G4 corresponded to the Western, E1, E2-2 and E2-1 lineages, respectively). The divergence time estimated between the Western and Eastern lineages was 1.17 (0.50-2.37) to 0.89 (0.39-1.78) Mya, indicating that the lineages diversified on both geographic and temporal scales. The historical demography of the Eastern lineage showed continuous population growth after the Last Interglacial (LIG) and a stable population during the Last Glacial Maximum (LGM) period. However, the Western lineage remained largely unchanged during the LIG and LGM periods. This suggests that the historical demography of C. versicolor is probably related not only to the paleoclimate of South China, but also to the geological restriction and specific habitat preferences of species.

Dietary Niche Partitioning of Euphaea formosa and Matrona cyanoptera (Odonata: Zygoptera) on the Basis of DNA Barcoding of Larval Feces.

Odonate larvae are commonly considered opportunistic general predators in freshwater ecosystems. However, the dietary breadth of most odonate larvae in forest streams is still poorly documented. We characterized the prey species and estimated the level of dietary niche overlap of two damselflies, Euphaea formosa Hagen 1869 and Matrona cyanoptera Hämäläinen and Yeh, 2000 in a forest stream of central Taiwan on the basis of DNA barcoding of larval feces. A collection of 23 successfully identified cytochrome c oxidase 1 (CO1) barcoding sequences suggested that the mayflies (Ephemeroptera), caddisflies (Trichoptera), and midges (Diptera) comprise the majority (43%, 6/14) of prey species consumed by E. formosa larvae, whereas the identified prey for M. cyanoptera were mainly zooplankton (56%, 5/9). Statistical analysis of dietary overlap indicated that these two species occupy different dietary niches (Pianka's index = 0.219). DNA barcoding analysis of damselfly larval feces was effective in detecting less sclerotized prey such as vertebrates (fish and frog) and small zooplankton. However, a moderately successful rate (<70%) of PCR amplification by universal CO1 primers and a low percentage (<60%) of identifiable sequences in public databases indicate the limitations of naive DNA barcoding in fecal analysis.

Ultrastructure of dragonfly wing veins: composite structure of fibrous material supplemented by resilin.

Dragonflies count among the most skilful of the flying insects. Their exceptional aerodynamic performance has been the subject of various studies. Morphological and kinematic investigations have showed that dragonfly wings, though being rather stiff, are able to undergo passive deformation during flight, thereby improving the aerodynamic performance. Resilin, a rubber-like protein, has been suggested to be a key component in insect wing flexibility and deformation in response to aerodynamic loads, and has been reported in various arthropod locomotor systems. It has already been found in wing vein joints, connecting longitudinal veins to cross veins, and was shown to endow the dragonfly wing with chordwise flexibility, thereby most likely influencing the dragonfly's flight performance. The present study revealed that resilin is not only present in wing vein joints, but also in the internal cuticle layers of veins in wings of Sympetrum vulgatum (SV) and Matrona basilaris basilaris (MBB). Combined with other structural features of wing veins, such as number and thickness of cuticle layers, material composition, and cross-sectional shape, resilin most probably has an effect on the vein's material properties and the degree of elastic deformations. In order to elucidate the wing vein ultrastructure and the exact localisation of resilin in the internal layers of the vein cuticle, the approaches of bright-field light microscopy, wide-field fluorescence microscopy, confocal laser-scanning microscopy, scanning electron microscopy and transmission electron microscopy were combined. Wing veins were shown to consist of up to six different cuticle layers and a single row of underlying epidermal cells. In wing veins of MBB, the latter are densely packed with light-scattering spheres, previously shown to produce structural colours in the form of quasiordered arrays. Longitudinal and cross veins differ significantly in relative thickness of exo- and endocuticle, with cross veins showing a much thicker exocuticle. The presence of resilin in the unsclerotised endocuticle suggests its contribution to an increased energy storage and material flexibility, thus to the prevention of vein damage. This is especially important in the highly stressed longitudinal veins, which have much lower possibility to yield to applied loads with the aid of vein joints, as the cross veins do. These results may be relevant not only for biologists, but may also contribute to optimise the design of micro-air vehicles.

Integrative species delimitation and five new species of lynx spiders (Araneae, Oxyopidae) in Taiwan.

An accurate assessment of species diversity is a cornerstone of biology and conservation. The lynx spiders (Araneae: Oxyopidae) represent one of the most diverse and widespread cursorial spider groups, however their species richness in Asia is highly underestimated. In this study, we revised species diversity with extensive taxon sampling in Taiwan and explored species boundaries based on morphological traits and genetic data using a two-step approach of molecular species delimitation. Firstly, we employed a single COI dataset and applied two genetic distance-based methods: ABGD and ASAP, and two topology-based methods: GMYC and bPTP. Secondly, we further analyzed the lineages that were not consistently delimited, and incorporated H3 to the dataset for a coalescent-based analysis using BPP. A total of eight morphological species were recognized, including five new species, Hamataliwa cordivulva sp. nov., Hamat. leporauris sp. nov., Tapponia auriola sp. nov., T. parva sp. nov. and T. rarobulbus sp. nov., and three newly recorded species, Hamadruas hieroglyphica (Thorell, 1887), Hamat. foveata Tang & Li, 2012 and Peucetia latikae Tikader, 1970. All eight morphological species exhibited reciprocally monophyletic lineages. The results of molecular-based delimitation analyses suggested a variety of species hypotheses that did not fully correspond to the eight morphological species. We found that Hamat. cordivulva sp. nov. and Hamat. foveata showed shallow genetic differentiation in the COI, but they were unequivocally distinguishable according to their genitalia. In contrast, T. parva sp. nov. represented a deep divergent lineage, while differences of genitalia were not detected. This study highlights the need to comprehensively employ multiple evidence and methods to delineate species boundaries and the values of diagnostic morphological characters for taxonomic studies in lynx spiders.

Strong attachment as an adaptation of flightless weevils on windy oceanic islands.

Enhanced attachment ability is common in plants on islands to avoid potential fatal passive dispersal. However, whether island insects also have increased attachment ability remains unclear. Here we measured the attachment of a flightless weevil, Pachyrhynchus sarcitis kotoensis, from tropical islands, and compared it with documented arthropods from the mainland. We examined the morphology and material gradient of its attachment devices to identify the specific adaptive modifications for attachment. We find that the weevil has much stronger attachment force and higher safety factor than previously studied arthropods, regardless of body size and substrate roughness. This probably results from the specific flexible bases of the adhesive setae on the third footpad of the legs. This softer material on the setal base has not been reported hitherto and we suggest that it acts as a flexible hinge to form intimate contact to substrate more effectively. By contrast, no morphological difference in tarsomeres and setae between the weevil and other beetles is observed. Our results show the remarkably strong attachment of an island insect and highlights the potential adaptive benefits of strong attachment in windy island environment. The unique soft bases of the adhesive hairs may inspire the development of strong biomimetic adhesives.

Evolution of horn length and lifting strength in the Japanese rhinoceros beetle Trypoxylus dichotomus.

What limits the size of nature's most extreme structures? For weapons like beetle horns, one possibility is a tradeoff associated with mechanical levers: as the output arm of the lever system-the beetle horn-gets longer, it also gets weaker. This "paradox of the weakening combatant" could offset reproductive advantages of additional increases in weapon size. However, in contemporary populations of most heavily weaponed species, males with the longest weapons also tend to be the strongest, presumably because selection drove the evolution of compensatory changes to these lever systems that ameliorated the force reductions of increased weapon size. Therefore, we test for biomechanical limits by reconstructing the stages of weapon evolution, exploring whether initial increases in weapon length first led to reductions in weapon force generation that were later ameliorated through the evolution of mechanisms of mechanical compensation. We describe phylogeographic relationships among populations of a rhinoceros beetle and show that the "pitchfork" shaped head horn likely increased in length independently in the northern and southern radiations of beetles. Both increases in horn length were associated with dramatic reductions to horn lifting strength-compelling evidence for the paradox of the weakening combatant-and these initial reductions to horn strength were later ameliorated in some populations through reductions to horn length or through increases in head height (the input arm for the horn lever system). Our results reveal an exciting geographic mosaic of weapon size, weapon force, and mechanical compensation, shedding light on larger questions pertaining to the evolution of extreme structures.

Pleistocene speciation with and without gene flow in Euphaea damselflies of subtropical and tropical East Asian islands.

Climatic oscillations during the Pleistocene period could have had a profound impact on the origin of tropical species by the alternation of allopatric isolation and interpopulation gene flow cycles. However, whether tropical speciation involves strictly allopatric isolation, or proceeds in the face of homogenizing gene flow, is relatively unclear. Here, we investigated geographical modes of speciation in four closely related Euphaea damselfly species endemic to the subtropical and tropical East Asian islands using coalescent analyses of a multilocus data set. The reconstructed phylogenies demonstrated distinct species status for each of the four species and the existence of two sister species pairs, Euphaea formosa/E. yayeyamana and E. decorata/E. ornata. The species divergence time of the sibling Euphaea damselflies dates back to within the last one Mya of the Middle to Lower Pleistocene. The speciation between the populous E. formosa of Taiwan and the less numerous E. yayeyamana of the Yaeyama islands occurred despite significant bidirectional, asymmetric gene flow, which is strongly inconsistent with a strictly allopatric model. In contrast, speciation of the approximately equal-sized populations of E. decorata of the southeast Asian mainland and E. ornata of Hainan is inferred to have involved allopatric divergence without gene flow. Our findings suggest that differential selection of natural or sexual environments is a prominent driver of species divergence in subtropical E. formosa and E. yayeyamana; whereas for tropical E. decorata and E. ornata at lower latitudes, allopatric isolation may well be a pivotal promoter of species formation.

Morphometric and genetic differentiation of two sibling gossamer-wing damselflies, Euphaea formosa and E. yayeyamana, and adaptive trait divergence in subtropical East Asian islands.

Insular species frequently demonstrate different tendencies to become smaller or larger than their continental relatives. Two sibling gossamer-wing damselflies, Euphaea formosa (Odonata: Euphaeidae) from Taiwan and E. yayeyamana from the Yaeyama Islands of Japan, have no clear structural differentiation, and can only be recognized by their geographical distribution, sizes, and subtle differences in wing shape and coloration. This study combined morphometric and genetic techniques to investigate the adaptive significance of trait divergence and species status in these two Euphaea damselflies. Phylogenetic analyses of the mitochondrial cox2 sequences demonstrated that the two damselflies are monophyletic lineages and constitute valid phylogenetic species. The landmark-based geometric morphometrics indicated that the two damselflies are different morphological species characterized by distinctive wing shapes. The larger E. formosa exhibited broader hind wings, whereas E. yayeyamana had narrower and elongated forewings. The body size and wing shape variations among populations of the two species do not follow the expected pattern of neutral evolution, suggesting that the evolutionary divergence of these two traits is likely to be subjected to natural or sexual selection. The decreased body size, elongated forewings, and narrower hind wings of E. yayeyamana may represent insular adaptation to limited resources and reduced territorial competition on smaller islands.

Stag beetle Cyclommatus mniszechi employs both mutual- and self-assessment strategies in male-male combat.

Animals may base contest decisions on their fighting ability alone (self-assessment) or also their opponents' (mutual assessment). Many male stag beetles develop disproportionately enlarged mandibles and use them as weapons. Information on their assessment strategy is limited. To investigate their assessment strategy and whether they adopt the same strategy at different stages of contests, we used food to encourage male Cyclommatus mniszechi of different (random pairings) or similar (ML-matched pairings) mandible length (ML) to interact. For the random pairings, losers had shorter mandibles than winners and were faster to feed. Overall contest duration and the tendency to escalate to tussles associated positively with winners' ML and average ML in the random and the ML-matched pairings, respectively, consistent with self-assessment. Non-tussle phase duration associated positively with average ML in the ML-matched pairings, consistent with self-assessment. Tussle phase duration, however, positively associated with losers' ML in the random pairings and had no association with average ML in the ML-matched pairings, consistent with mutual assessment. These results show that (1) the males employ both assessment strategies, (2) winners have more control over contest intensity than losers, and (3) males with shorter mandibles are quicker to feed and also more likely to lose fights.

doublesex Controls Both Hindwing and Abdominal Mimicry Traits in the Female-Limited Batesian Mimicry of Papilio memnon.

Papilio butterflies are known to possess female-limited Batesian mimicry polymorphisms. In Papilio memnon, females have mimetic and non-mimetic forms, whereas males are monomorphic and non-mimetic. Mimetic females are characterized by color patterns and tails in the hindwing and yellow abdomens. Recently, an analysis of whole-genome sequences has shown that an approximately 160 kb region of chromosome 25 is responsible for mimicry and has high diversity between mimetic (A) and non-mimetic (a) alleles (highly diversified region: HDR). The HDR includes three genes, UXT, doublesex (dsx), and Nach-like, but the functions of these genes are unknown. Here, we investigated the function of dsx, a gene involved in sexual differentiation, which is expected to be functionally important for hindwing and abdominal mimetic traits in P. memnon. Expression analysis by reverse transcription quantitative PCR (RT-qPCR) and RNA sequencing showed that mimetic dsx (dsx-A) was highly expressed in the hindwings in the early pupal stage. In the abdomen, both dsx-A and dsx-a were highly expressed during the early pupal stage. When dsx was knocked down using small interfering RNAs (siRNAs) designed in the common region of dsx-A and dsx-a, a male-like pattern appeared on the hindwings of mimetic and non-mimetic females. Similarly, when dsx was knocked down in the abdomen, the yellow scales characteristic of mimetic females changed to black. Furthermore, when dsx-a was specifically knocked down, the color pattern of the hindwings changed, as in the case of dsx knockdown in non-mimetic females but not mimetic females. These results suggest that dsx-a is involved in color pattern formation on the hindwings of non-mimetic females, whereas dsx-A is involved in hindwing and abdominal mimetic traits. dsx was involved in abdominal and hindwing mimetic traits, but dsx expression patterns in the hindwing and abdomen were different, suggesting that different regulatory mechanisms may exist. Our study is the first to show that the same gene (dsx) regulates both the hindwing and abdominal mimetic traits. This is the first functional analysis of abdominal mimicry in butterflies.

Lineage-specific late pleistocene expansion of an endemic subtropical gossamer-wing damselfly, Euphaea formosa, in Taiwan.

BACKGROUND: Pleistocene glacial oscillations have significantly affected the historical population dynamics of temperate taxa. However, the general effects of recent climatic changes on the evolutionary history and genetic structure of extant subtropical species remain poorly understood. In the present study, phylogeographic and historical demographic analyses based on mitochondrial and nuclear DNA sequences were used. The aim was to investigate whether Pleistocene climatic cycles, paleo-drainages or mountain vicariance of Taiwan shaped the evolutionary diversification of a subtropical gossamer-wing damselfly, Euphaea formosa. RESULTS: E. formosa populations originated in the middle Pleistocene period (0.3 Mya) and consisted of two evolutionarily independent lineages. It is likely that they derived from the Pleistocene paleo-drainages of northern and southern Minjiang, or alternatively by divergence within Taiwan. The ancestral North-central lineage colonized northwestern Taiwan first and maintained a slowly growing population throughout much of the early to middle Pleistocene period. The ancestral widespread lineage reached central-southern Taiwan and experienced a spatial and demographic expansion into eastern Taiwan. This expansion began approximately 30,000 years ago in the Holocene interglacial period. The ancestral southern expansion into eastern Taiwan indicates that the central mountain range (CMR) formed a barrier to east-west expansion. However, E. formosa populations in the three major biogeographic regions (East, South, and North-Central) exhibit no significant genetic partitions, suggesting that river drainages and mountains did not form strong geographical barriers against gene flow among extant populations. CONCLUSIONS: The present study implies that the antiquity of E. formosa's colonization is associated with its high dispersal ability and larval tolerance to the late Pleistocene dry grasslands. The effect of late Pleistocene climatic changes on the subtropical damselfly's historical demography is lineage-specific, depending predominantly on its colonization history and geography. It is proposed that the Riss and Würm glaciations in the late Pleistocene period had a greater impact on the evolutionary diversification of subtropical insular species than the last glacial maximum (LGM).

Species delimitation and taxonomic revision of Oxyopes (Araneae: Oxyopidae) of Taiwan, with description of two new species.

This study revised the spider genus Oxyopes Latreille, 1804 in Taiwan and delineated the species boundaries based on morphological and molecular characters. A total of seven Oxyopes spiders were recognized, including two newly described species, O. hasta sp. nov. and O. taiwanensis sp. nov. Oxyopes fujianicus Song Zhu 1993 from Yilan County, Nantou County, and Kaohsuing City, and O. striagatus Song 1999 from New Taipei City, Taichung City, Nantou County, and Kaohsiung City were recorded for the first time in Taiwan. An identification key and a distributional map of Taiwanese Oxyopes species were provided. Partial COI sequences were obtained for molecular phylogenetic and species delimitation analyses. Maximum likelihood and Bayesian phylogenies, and DNA barcoding gap analysis supported morphologically defined species. However, molecular species delimitation based on Automatic Barcode Gap Discovery (ABGD), PID (Liberal), and generalized mixed Yule coalescent (GMYC) were incongruent in species assignment. The results showed that the interspecific genetic divergence between O. sertatus and O. taiwanensis was relatively low (1.28 ± 0.43%), and the intraspecific genetic divergence of O. striagatus was relatively high (1.69 ± 0.35%). Ecological data, additional samples and genetic loci are required to further examine the level of reproductive isolation and patterns of population genetic structure in Taiwanese Oxyopes.

Diversification in subtropical mountains: phylogeography, Pleistocene demographic expansion, and evolution of polyphenic mandibles in Taiwanese stag beetle, Lucanus formosanus.

Pleistocene glacial oscillations have had profound impacts on the historical population dynamics of extant species. However, the genetic consequences of past climatic changes depend largely on the latitude and topography of the regions in question. This study investigates the effect of Pleistocene glacial periods and the Central Mountain Range on the phylogeography, historical demography, and phenotypic differentiation of a montane forest-dwelling stag beetle, Lucanus formosanus (Coleoptera: Lucanidae), which exhibits extensive mandible variations across mountain ranges in subtropical Taiwan. Analyses of mitochondrial (cox1) and nuclear (wg) loci reveal that L. formosanus originated nearly 1.6 million years ago (Mya) in the early Pleistocene period and consisted of geographically overlapping Alishan and Widespread clades. A drastic population expansion starting approximately 0.2 Mya in the Widespread clade likely resulted from altitudinal range shift of the temperate forests, which was closely tied to the arrival of the Riss glacial period in the late Middle Pleistocene. A ring-like pattern of historical gene flow among neighboring populations in the vicinity of the Central Mountain Range indicates that the mountains constitute a strong vicariant barrier to the east-west gene flow of L. formosanus populations. A geographic cline of decreasing mandible size from central to north and south, and onto southeast of Taiwan is inconsistent with the low overall phylogeographic structures. The degree of mandible variation does not correlate with the expected pattern of neutral evolution, indicating that the evolutionary diversification of this morphological weapon is most likely subject to sexual or natural selection. We hypothesize that the adaptive evolution of mandibles in L. formosanus is shaped largely by the habitat heterogeneity.

Allometry and Fighting Behaviour of a Dimorphic Stag Beetle Cyclommatus mniszechi (Coleoptera: Lucanidae).

Male stag beetles (Coleoptera: Lucanidae) use their mandibles as weapons to compete for resources and reproduction. Mandible size in stag beetles can be associated with different behaviours and the outcome of male contests. We investigated the allometric relationship between mandible and body size in males of the stag beetle Cyclommatus mniszechi to uncover distinct morphs. The results divided male C. mniszechi into majors and minors with the switch point of mandible length at 14.01 mm. The allometric slope of mandibles was positive for both morphs but was steeper for the minors. We also characterised the fighting behaviour of the different morphs in size-matched contests using sequential analyses. Males matched each other's behaviour in contests with many physical contacts, no injury and a progression from low towards high aggression and rare de-escalation. Major and minor males employed the same behavioural elements in contests, but major males were more likely to escalate directly into more aggressive phases and minor males tended to stay within phases. This finding suggests that major males may compete more aggressively than minor males in contests.