Familiarity with, perceptions of and attitudes toward butterflies of urban park users in megacities across East and Southeast Asia.

Perceptions of, and attitudes toward, wildlife are influenced by exposure to, and direct experiences with, nature. Butterflies are a conspicuous and ubiquitous component of urban nature across megacities that are highly urbanized with little opportunity for human-nature interactions. We evaluated public familiarity with, perceptions of and attitudes toward butterflies across nine megacities in East and Southeast Asia through face-to-face interviews with 1774 urban park users. A total of 79% of respondents had seen butterflies in their cities mostly in urban parks, indicating widespread familiarity with butterflies. Those who had seen butterflies also had higher perceptions of butterflies, whereas greater than 50% of respondents had positive attitudes toward butterflies. Frequent visits to natural places in urban neighbourhoods was associated with (i) sightings of caterpillars, indicating increased familiarity with urban wildlife, and (ii) increased connectedness to nature. We found two significant positive relationships: (i) between connectedness to nature and attitudes toward butterflies and (ii) between connectedness to nature and perceptions of butterflies, firmly linking parks users' thoughts and feelings about butterflies with their view of nature. This suggests that butterflies in urban parks can play a key role in building connectedness to nature and consequently pro-environmental behaviours and support for wildlife conservation among urban residents.

Butterflies in urban parks in the Bangkok Metropolitan Region, Thailand.

BACKGROUND: For residents of East-Southeast Asia's megacities, interactions with "nature" may be largely limited to interactions taking place in urban parks. Urban parks provide refuges for ecologically-important biodiversity, such as insect pollinators. While residents may be unlikely to notice small insects, butterflies are more likely to be noticed and to provide positive human-"nature" interactions. Engaging residents and city planners in promoting habitat for butterflies is valid conservation practice and has well-understood educational and well-being benefits. Surveying and monitoring is an essential activity to corroborate, improve and communicate the outcomes of conservation practices amongst city governments, scientists and other stakeholders. Here we present the data from a survey of butterflies in urban parks in the megacity of the Bangkok Metropolitan Region as part of the "Urban biodiversity and human well-being in East-Southeast Asia's megacities" project organised by the "Urban Butterflies in Asia Research Network". NEW INFORMATION: We recorded 51 species of butterflies from ten urban parks in the Bangkok Metropolitan Region. This was more than double the 25 species reported in Bangkok's City Biodiversity Index application. However, this was lower than that recorded in other megacities in Southeast Asia, such as Kuala Lumpur at 60 species. Most of the butterflies recorded were common and widespread species. DNA barcodes are provided for most of the butterflies sampled.

Ring roads and urban biodiversity: distribution of butterflies in urban parks in Beijing city and correlations with other indicator species.

The capital of China, Beijing, has a history of more than 800 years of urbanization, representing a unique site for studies of urban ecology. Urbanization can severely impact butterfly communities, yet there have been no reports of the species richness and distribution of butterflies in urban parks in Beijing. Here, we conducted the first butterfly survey in ten urban parks in Beijing and estimated butterfly species richness. Subsequently, we examined the distribution pattern of butterfly species and analyzed correlations between butterfly species richness with park variables (age, area and distance to city center), and richness of other bioindicator groups (birds and plants). We collected 587 individual butterflies belonging to 31 species from five families; 74% of the species were considered cosmopolitan. The highest butterfly species richness and abundance was recorded at parks located at the edge of city and species richness was significantly positively correlated with distance from city center (p < 0.05). No significant correlations were detected between the species richness and park age, park area and other bioindicator groups (p > 0.05). Our study provides the first data of butterfly species in urban Beijing, and serves as a baseline for further surveys and conservation efforts.

High-throughput terrestrial biodiversity assessments: mitochondrial metabarcoding, metagenomics or metatranscriptomics?

Consensus on the optimal high-throughput sequencing (HTS) approach to examine biodiversity in mixed terrestrial arthropod samples has not been reached. Metatranscriptomics could increase the proportion of taxonomically informative mitochondrial reads in HTS outputs but has not been investigated for terrestrial arthropod samples. We compared the efficiency of 16S rRNA metabarcoding, metagenomics and metatranscriptomics for detecting species in a mixed terrestrial arthropod sample (pooled DNA/RNA from 38 taxa). 16S rRNA metabarcoding and nuclear rRNA-depleted metatranscriptomics had the highest detection rate with 97% of input species detected. Based on cytochrome c oxidase I, metagenomics had the highest detection rate with 82% of input species detected, but metatranscriptomics produced a larger proportion of reads matching (Sanger) reference sequences. Metatranscriptomics with nuclear rRNA depletion may offer advantages over metabarcoding through reducing the number of spurious operational taxonomic units while retaining high detection rates, and offers natural enrichment of mitochondrial sequences which may enable increased species detection rates compared with metagenomics.

DNA barcodes for dragonflies and damselflies (Odonata) of Mindanao, Philippines.

Reliable species identification provides a sounder basis for use of species in the order Odonata as biological indicators and for their conservation, an urgent concern as many species are threatened with imminent extinction. We generated 134 COI barcodes from 36 morphologically identified species of Odonata collected from Mindanao Island, representing 10 families and 19 genera. Intraspecific sequence divergences ranged from 0 to 6.7% with four species showing more than 2%, while interspecific sequence divergences ranged from 0.5 to 23.3% with seven species showing less than 2%. Consequently, no distinct gap was observed between intraspecific and interspecific DNA barcode divergences. The numerous islands of the Philippine archipelago may have facilitated rapid speciation in the Odonata and resulted in low interspecific sequence divergences among closely related groups of species. This study contributes DNA barcodes for 36 morphologically identified species of Odonata reported from Mindanao including 31 species with no previous DNA barcode records.

Tracking the southern river terrapin (Batagur affinis) through environmental DNA: prospects and challenges.

Environmental DNA detection has emerged as a powerful tool to monitor aquatic species without the need for capture or visual identification and is particularly useful for rare or elusive species. Our objective was to develop an eDNA approach for detecting the southern river terrapin (Batagur affinis) in Malaysia. We designed species-specific primers for a fragment of B. affinis mtDNA and evaluated their effectiveness in silico, in vitro and in situ. The primers amplified 110 bp of the cytochrome b mtDNA sequence of B. affinis from aquarium water samples housing nine juvenile B. affinis. We also successfully detected B. affinis eDNA from river samples taken from a site where turtles were known to be in the vicinity. Prospects and challenges of using an eDNA approach to help determine the distribution of B. affinis, essential information for an effective conservation plan, are discussed.

Dynamic microbiome evolution in social bees.

The highly social (eusocial) corbiculate bees, comprising the honey bees, bumble bees, and stingless bees, are ubiquitous insect pollinators that fulfill critical roles in ecosystem services and human agriculture. Here, we conduct wide sampling across the phylogeny of these corbiculate bees and reveal a dynamic evolutionary history behind their microbiota, marked by multiple gains and losses of gut associates, the presence of generalist as well as host-specific strains, and patterns of diversification driven, in part, by host ecology (for example, colony size). Across four continents, we found that different host species have distinct gut communities, largely independent of geography or sympatry. Nonetheless, their microbiota has a shared heritage: The emergence of the eusocial corbiculate bees from solitary ancestors appears to coincide with the acquisition of five core gut bacterial lineages, supporting the hypothesis that host sociality facilitates the development and maintenance of specialized microbiomes.

Unexpected diversity of sandflies (Diptera: Psychodidae) in tourist caves in Northern Thailand.

Certain species of Phlebotomine sandflies (Diptera: Psychodidae) are vectors of the protozoa which causes leishmaniasis. Sandflies are found breeding in enclosed places like caves. Thailand is a popular tourist destination, including for ecotourism activities like caving, which increases the risk of contact between tourists and sandflies. Surveillance of sandflies is important for monitoring this risk but identification of species based on morphology is challenged by phenotypic plasticity and cryptic diversity. DNA barcodes have been used for the identification of sandflies in Thailand. We collected sandflies using CDC light trap from four tourist caves in Northern Thailand. Female sandflies were provisionally sorted into 13 morphospecies and 19 unidentified specimens. DNA was extracted from the thorax and legs of sandflies and the DNA barcode region of cytochrome c oxidase I mtDNA amplified and sequenced. The specimens were sorted into 22 molecular operational taxonomic units (MOTU) based on the 145 DNA barcodes, which is significantly more than the morphospecies. Several of the taxa thought to be present in multiple caves, based on morphospecies sorting, split into cave-specific MOTU which likely represent cryptic species. Several MOTU reported in an earlier study from Wihan Cave, Thailand, were also found in these caves. This supports the use of DNA barcodes to investigate species diversity of sandflies and their useful role in surveillance of sandflies in Thailand.

Application of DNA barcodes in wildlife conservation in Tropical East Asia.

Over the past 50 years, Tropical East Asia has lost more biodiversity than any tropical region. Tropical East Asia is a megadiverse region with an acute taxonomic impediment. DNA barcodes are short standardized DNA sequences used for taxonomic purposes and have the potential to lessen the challenges of biodiversity inventory and assessments in regions where they are most needed. We reviewed DNA barcoding efforts in Tropical East Asia relative to other tropical regions. We suggest DNA barcodes (or metabarcodes from next-generation sequencers) may be especially useful for characterizing and connecting species-level biodiversity units in inventories encompassing taxa lacking formal description (particularly arthropods) and in large-scale, minimal-impact approaches to vertebrate monitoring and population assessments through secondary sources of DNA (invertebrate derived DNA and environmental DNA). We suggest interest and capacity for DNA barcoding are slowly growing in Tropical East Asia, particularly among the younger generation of researchers who can connect with the barcoding analogy and understand the need for new approaches to the conservation challenges being faced.

DNA barcodes and citizen science provoke a diversity reappraisal for the "ring" butterflies of Peninsular Malaysia (Ypthima: Satyrinae: Nymphalidae: Lepidoptera).

The "rings" belonging to the genus Ypthima are amongst the most common butterflies in Peninsular Malaysia. However, the species can be difficult to tell apart, with keys relying on minor and often non-discrete ring characters found on the hindwing. Seven species have been reported from Peninsular Malaysia, but this is thought to be an underestimate of diversity. DNA barcodes of 165 individuals, and wing and genital morphology, were examined to reappraise species diversity of this genus in Peninsular Malaysia. DNA barcodes collected during citizen science projects-School Butterfly Project and Peninsular Malaysia Butterfly Count-recently conducted in Peninsular Malaysia were included. The new DNA barcodes formed six groups with different Barcode Index Numbers (BINs) representing four species reported in Peninsular Malaysia. When combined with public DNA barcodes from the Barcode Of Life Datasystems, several taxonomic issues arose. We consider the taxon Y. newboldi, formerly treated as a subspecies of Y. baldus, as a distinct species. DNA barcodes also supported an earlier suggestion that Y. nebulosa is a synonym under Y. horsfieldii humei. Two BINs of the genus Ypthima comprising DNA barcodes collected during citizen science projects did not correspond to any species previously reported in Peninsular Malaysia.

Can butterflies cope with city life? Butterfly diversity in a young megacity in southern China.

During 30 years of unprecedented urbanization, plant diversity in Shenzhen, a young megacity in southern China, has increased dramatically. Although strongly associated with plant diversity, butterfly diversity generally declines with urbanization, but this has not been investigated in Shenzhen. Considering the speed of urbanization in Shenzhen and the large number of city parks, we investigated butterfly diversity in Shenzhen parks. We measured butterfly species richness in four microhabitats (groves, hedges, flowerbeds, and unmanaged areas) across 10 parks and examined the relationship with three park variables: park age, park size, and distance from the central business district. Butterflies were identified based on wing morphology and DNA barcoding. We collected 1933 butterflies belonging to 74 species from six families; 20% of the species were considered rare. Butterfly species richness showed weak negative correlations with park age and distance from the central business district, but the positive correlation with park size was statistically significant (p = 0.001). Among microhabitat types, highest species richness was recorded in unmanaged areas. Our findings are consistent with others in suggesting that to promote urban butterfly diversity it is necessary to make parks as large as possible and to set aside areas for limited management. In comparison to neighbouring cities, Shenzhen parks have high butterfly diversity.

Diversity and human perceptions of bees (Hymenoptera: Apoidea) in Southeast Asian megacities.

Urbanization requires the conversion of natural land cover to cover with human-constructed elements and is considered a major threat to biodiversity. Bee populations, globally, are under threat; however, the effect of rapid urban expansion in Southeast Asia on bee diversity has not been investigated. Given the pressing issues of bee conservation and urbanization in Southeast Asia, coupled with complex factors surrounding human-bee coexistence, we investigated bee diversity and human perceptions of bees in four megacities. We sampled bees and conducted questionnaires at three different site types in each megacity: a botanical garden, central business district, and peripheral suburban areas. Overall, the mean species richness and abundance of bees were significantly higher in peripheral suburban areas than central business districts; however, there were no significant differences in the mean species richness and abundance between botanical gardens and peripheral suburban areas or botanical gardens and central business districts. Urban residents were unlikely to have seen bees but agreed that bees have a right to exist in their natural environment. Residents who did notice and interact with bees, even though being stung, were more likely to have positive opinions towards the presence of bees in cities.

Towards monitoring the sandflies (Diptera: Psychodidae) of Thailand: DNA barcoding the sandflies of Wihan Cave, Uttaradit.

Sandflies vary in their distributions and role in pathogen transmission. Attempts to record distributions of sandflies in Thailand have faced difficulties due to their high abundance and diversity. We aim to provide an insight into the diversity of sandflies in Thailand by (i) conducting a literature review, and (ii) DNA barcoding sandflies collected from Wihan Cave where eight morphologically characterized species were recorded. DNA barcodes generated for 193 sandflies fell into 13 distinct species clusters under four genera (Chinius, Idiophlebotomus, Phlebotomus and Sergentomyia). Five of these species could be assigned Linnaean species names unambiguously and two others corresponded to characterized morphospecies. Two species represented a complex under the name Sergentomyia barraudi while the remaining four had not been recognized before in any form. The resulting species checklist and DNA barcode library contribute to a growing set of records for sandflies which is useful for monitoring and vector control.

Reading Mammal Diversity from Flies: The Persistence Period of Amplifiable Mammal mtDNA in Blowfly Guts (Chrysomya megacephala) and a New DNA Mini-Barcode Target.

Most tropical mammal species are threatened or data-deficient. Data collection is impeded by the traditional monitoring approaches which can be laborious, expensive and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates are emerging as cost- and time-effective alternatives. As a step towards development of blowfly-derived DNA as an effective method for mammal monitoring in the biodiversity hotspot of Peninsular Malaysia, our objectives were (i) to determine the persistence period of amplifiable mammal mtDNA in blowfly guts through a laboratory feeding experiment (ii) to design and test primers that can selectively amplify mammal COI DNA mini-barcodes in the presence of high concentrations of blowfly DNA. The persistence period of amplifiable mammal mtDNA in blowfly guts was 24 h to 96 h post-feeding indicating the need for collecting flies within 24 h of capture to detect mammal mtDNA of sufficient quantity and quality. We designed a new primer combination for a COI DNA mini-barcode that did not amplify blowfly DNA and showed 89% amplification success for a dataset of mammals from Peninsular Malaysia. The short (205 bp) DNA mini-barcode could distinguish most mammal species (including separating dark taxa) and is of suitable length for high-throughput sequencing. Our new DNA mini-barcode target and a standardized trapping protocol with retrieval of blowflies every 24 h could point the way forward in the development of blowfly-derived DNA as an effective method for mammal monitoring.

Citizen Science: The First Peninsular Malaysia Butterfly Count.

BACKGROUND: Over the past 50 years, Southeast Asia has suffered the greatest losses of biodiversity of any tropical region in the world. Malaysia is a biodiversity hotspot in the heart of Southeast Asia with roughly the same number of mammal species, three times the number of butterfly species, but only 4% of the land area of Australia. Consequently, in Malaysia, there is an urgent need for biodiversity monitoring and also public engagement with wildlife to raise awareness of biodiversity loss. Citizen science is "on the rise" globally and can make valuable contributions to long-term biodiversity monitoring, but perhaps more importantly, involving the general public in science projects can raise public awareness and promote engagement. Butterflies are often the focus of citizen science projects due to their charisma and familiarity and are particularly valuable "ambassadors" of biodiversity conservation for public outreach. NEW INFORMATION: Here we present the data from our citizen science project, the first "Peninsular Malaysia Butterfly Count". Participants were asked to go outdoors on June 6, 2015, and (non-lethally) sample butterfly legs for species identification through DNA barcoding. Fifty-seven citizens responded to our adverts and registered to take part in the butterfly count with many registering on behalf of groups. Collectively the participants sampled 220 butterfly legs from 26 mostly urban and suburban sampling localities. These included our university campus, a highschool, several public parks and private residences. On the basis of 192 usable DNA barcodes, 43 species were sampled by the participants. The most sampled species was Appias olferna, followed by Junonia orithya and Zizina otis. Twenty-two species were only sampled once, five were only sampled twice, and four were only sampled three times. Three DNA barcodes could not be assigned species names. The sampled butterflies revealed that widely distributed, cosmopolitan species, often those recently arrived to the peninsula or with documented "invasive" potential, dominated the habitat types sampled by the participants. Data from this first Butterfly Count helps establish a baseline from which we can monitor the patterns and changes in butterfly communities in Peninsular Malaysia.

Mercury accumulation in bats near hydroelectric reservoirs in Peninsular Malaysia.

In large man-made reservoirs such as those resulting from hydroelectric dam construction, bacteria transform the relatively harmless inorganic mercury naturally present in soil and the submerged plant matter into toxic methylmercury. Methylmercury then enters food webs and can accumulate in organisms at higher trophic levels. Bats feeding on insects emerging from aquatic systems can show accumulation of mercury consumed through their insect prey. In this study, we investigated whether the concentration of mercury in the fur of insectivorous bat species was significantly higher than that in the fur of frugivorous bat species, sampled near hydroelectric reservoirs in Peninsular Malaysia. Bats were sampled at Temenggor Lake and Kenyir Lake and fur samples from the most abundant genera of the two feeding guilds-insectivorous (Hipposideros and Rhinolophus) and frugivorous (Cynopterus and Megaerops) were collected for mercury analysis. We found significantly higher concentrations of total mercury in the fur of insectivorous bats. Mercury concentrations also differed significantly between insectivorous bats sampled at the two sites, with bats from Kenyir Lake, the younger reservoir, showing higher mercury concentrations, and between the insectivorous genera, with Hipposideros bats showing higher mercury concentrations. Ten bats (H. cf. larvatus) sampled at Kenyir Lake had mercury concentrations approaching or exceeding 10 mg/kg, which is the threshold at which detrimental effects occur in humans, bats and mice.

Building a DNA barcode reference library for the true butterflies (Lepidoptera) of Peninsula Malaysia: what about the subspecies?

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.