Discovering a resilient and hyperdiverse midge fly fauna in a Singaporean swamp forest

Discovering a resilient and hyperdiverse midge fly fauna in a Singaporean swamp forest

Discovering a resilient and hyperdiverse midge fly fauna in a Singaporean swamp forest

Researchers uncover a highly unique and diverse chironomid community in a Singaporean swamp forest highlighting the importance of these ecosystems and the power of Next-Generation Sequencing for biomonitoring efforts.

Nee Soon Swamp Forest, Singapore

PHOTO CREDIT: Wang Luan Keng

Benthic macroinvertebrates – those animals that live at the bottoms of water bodies – are abundant, diverse, relatively immobile, and responsive to environmental stresses, and these traits make them ideal indicators of the quality of aquatic ecosystems. Our study demonstrates the utility of Next-Generation Sequencing (NGS) platforms as an efficient and rapid tool for monitoring efforts.

In freshwater ecosystems, non-biting midges (Diptera: Chironomidae) often constitute the majority of diversity and biomass with different chironomid species varying in their sensitivity to environmental changes. But, when monitoring these habitats, chironomids are either ignored entirely or not studied at a species-level because morphological assessments are expensive and laborious, and the identification literature is based on adults while larvae are most often collected.

Chironomid adults collected from Nee Soon Swamp Forest. Different chironomid species vary in their sensitivity to environmental parameters.

PHOTO CREDIT: Bilgenur Baloglu

The solution? NGS platforms. They allow for fast and effective species-level assessments of large-scale samples at low cost (less than $0.40 USD/specimen). Moreover, there is a high congruence between molecular and morphological identification, enabling a detailed examination of the composition of taxonomically complex communities1,2.

Freshwater swamp forests – the forested wetlands occurring along rivers and lakes – are home to various endemic and endangered species with 33% of birds and 45% of mammals either threatened or endangered on the IUCN Red List3, and with most of the insect fauna unknown. These ecosystems are under threat worldwide from habitat destruction, pollution, and climate crisis. Most of the world’s tropical swamp forests are found in Southeast Asia’s Indo-Malayan region collectively occupying more than 13 million ha4 among many geographically separated peninsulas and islands. Nee Soon swamp forest is the largest remnant (90 ha) of its kind in Singapore and thus of high national conservation value.

Bilge Baloglu sampling water DNA from Singapore’s largest swamp forest remnant.
PHOTO CREDIT: Dickson Ng

We generated DNA barcodes using NGS to study chironomids among the natural swamp forest Nee Soon and three adjacent man-made reservoirs. We wanted to understand the effects of urbanization and to know whether the chironomid fauna of Nee Soon is resistant to, that is, minimally impacted by, the adjacent reservoirs. We sampled >14,000 chironomid specimens (both adults and larvae) as part of a freshwater quality monitoring program, and quantified species richness and compositional changes using NGS and DNA barcoding.

Our study showed that Singapore’s biggest swamp forest remnant maintains a rich and largely unique fauna of about 350 species. The minimal species overlap between sites indicated that the Nee Soon swamp forest is resistant against the invasion of species from surrounding artificial reservoirs. 

 These findings suggest that even small or fragmented swamp forests can be suitable habitats for chironomids, shedding light on many other swamp forests in Southeast Asia that collectively occupy a much larger area and that are threatened by destruction for oil palm plantations and paper pulp production. Overall, our study exposes the enormous power of NGS and DNA barcoding in ecological research to study ecosystem health, biological diversity, and habitat conservation.

For full details on the study, see Baloğlu et al. 2018.

References:

1. Brodin Y, Ejdung G, Strandberg J, Lyrholm T (2013) Improving environmental and biodiversity monitoring in the Baltic Sea using DNA barcoding of Chironomidae (Diptera). Molecular Ecology Resources 13:996–1004.

2. Montagna M, Mereghetti V, Lencioni V, Rossaro B (2016) Integrated taxonomy and DNA barcoding of alpine midges (Diptera: Chironomidae). PLoS One 11:e0149673

3. Posa MR (2011) Peat swamp forest avifauna of Central Kalimantan, Indonesia: Effects of habitat loss and degradation. Biological Conservation 144(10):2548-2556.

4. Hooijer A, Page S, Canadell JG, Silvius M, Kwadijk J, Wösten H, Jauhiainen J (2010) Current and future CO2 emissions from drained peatlands in Southeast Asia. Biogeosciences 7:1505–1514

Written by

Bilgenur Baloğlu

Bilgenur Baloğlu

Centre for Biodiversity Genomics, Guelph, ON, Canada

September 6, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5525

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The Diversification and Evolution of Marine Invertebrates in Oceanic Islands

The Diversification and Evolution of Marine Invertebrates in Oceanic Islands

The Diversification and Evolution of Marine Invertebrates in Oceanic Islands

Unravelling long-established divergence patterns and remarkable geographic segregation that has endured over millions of years in marine invertebrates.

Macaronesian rocky shores.
PHOTO CREDIT: Pedro Vieira, Mafalda Tavares, Henrique Queiroga

Oceanic islands constitute prime evolutionary grounds for terrestrial organisms, promoting extensive isolation and harboring exceptional levels of diversity and endemism. Marine organisms, on the contrary, are not expected to experience evolutionary forces with the same intensity and, therefore, their diversification and evolution in oceanic landscapes has been somewhat disregarded.

Shore-dwelling marine benthic invertebrates are unique relative to both terrestrial organisms and other marine taxa. Most shore species have planktonic larvae that facilitate dispersal over open water. However, some small invertebrates, such as free-living peracarids (Peracarida: Crustacea), are more prone to isolation due to life histories characterized by direct development and putatively reduced vagility.

As a result of a DNA barcode-based screening of the diversity of littoral peracarids along Macaronesia and nearby continental shores, we have found an exceptional amount of cryptic diversity, together with a very structured geographic assortment within multiple morphospecies. We investigated, with particular detail, two common and distinct peracarid taxa present in the Macaronesian archipelagos of Azores, Madeira, and Canaries, namely 3 morphospecies of the isopod genus Dynamene1 and 7 morphospecies of the amphipod family Hyalidae2. We unraveled an additional 34 suspected new species (75% endemic of Macaronesia) with no apparent discriminative morphological features, including the noteworthy cases of the isopod Dynamene edwardsi and the amphipod Apohyale stebbingi comprising 9 and 13 putative species, respectively.

Adding to this surprising cryptic diversity, lineages within each morphospecies were also frequently exclusive to one island (50%), despite the general geographic proximity of the islands within each archipelago (e.g., from as little as 50 km between Porto Santo and Madeira). Globally, the genetic divergences among lineages within a morphospecies were also comparatively high (e.g., D. edwardsi up to 22% and A. stebbingi up to 21%), indicating a very deep evolutionary history in the region which pre-dates the Pleistocene glacial cycles (D. edwardsi lineages probably started diverging between 20 and 30 MYA).

 

Representative study species; not to scale.
PHOTO CREDIT: Pedro Vieira

There were several noteworthy findings in these studies. First, the unexpectedly high amount of diversity and endemism in these Macaronesian marine invertebrates, even within the same archipelago. Although peracarids can be assumed to have comparatively low vagility, since they lack a planktonic larval stage, there is extensive evidence for their dispersal capability and population connectivity. Namely, both our and other authors’ studies report peracarid morphospecies displaying little genetic structure over wide geographic ranges, for example, along the European continental Atlantic coasts.

Second, we were surprised to find marked geographic segregation among the newly found species. They were frequently restricted to a single island where they constituted the only representative of the cryptic complex. To a certain extent, the geographical segregation of these peracarids more closely resemble what would be expected for terrestrial organisms than marine invertebrates.

 

A. Sampling locations for each Dynamene species. B. Reduced median network of COI data from the genus Dynamene. Size of the circles are proportional to the number of similar haplotypes. Number of mutations separating each haplotype and inferred ancestors (median vectors) are displayed in black. Links displaying a single mutation do not display the number.

PHOTO CREDIT: Pedro Vieira

Finally, the combined evidence on diversity, geographic segregation, and divergence times, unraveled long-established divergence patterns and a remarkable geographic segregation that endured over millions of years till present. Therefore, the current distribution patterns of many of these peracarids cannot be elucidated through common accounts of marine invertebrates in the north-eastern Atlantic, namely processes involving dispersal, geographic proximity or Pleistocene glacial cycles. We propose alternative mechanisms for the speciation of these invertebrates in Macaronesia, such as those involving priority effects and pre-emptive exclusion, which have been seldom evoked to explain the deep segregation in the open ocean.

In the near future, we intend to investigate the genetic variability of taxa with planktonic larvae from these oceanic islands to verify if the long-term segregation patterns are exclusive of peracarid crustaceans or, instead, are more widespread patterns in marine invertebrates in Macaronesia.

References

  1. Vieira PE, Desiderato A, Holdich DM, Soares P, Creer S, Carvalho GR, Costa FO, Queiroga H (2019) Deep segregation in the open ocean: Macaronesia as an evolutionary hotspot for low dispersal marine invertebrates. Molecular Ecology. https://doi.org/10.1111/mec.15052
  2. Desiderato A, Costa FO, Serejo CS, Abbiati M, Queiroga H, Vieira PE (2019) Macaronesian islands as promoters of diversification in amphipods: The remarkable case of the family Hyalidae (Crustacea, Amphipoda). Zoologica Scripta. https://doi.org/10.1111/zsc.12339

Written by

Pedro Vieira

Pedro Vieira

University of Minho, Braga, Portugal

Filipe Costa

Filipe Costa

University of Minho, Braga, Portugal

April 7, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5477

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Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Researchers provide new insights into biodiversity using DNA barcoding in Fiji's topographically complex archipelago.

Homalictus hadrander, one of the four described species previously known from Fiji.
PHOTO CREDIT: James Dorey

Fiji’s entomological diversity has historically been considered depauperate. Recent widespread DNA barcoding efforts, however, from the South Australian Museum, Flinders University, and University of South Australia, along with a flurry of undergraduate, honours, and PhD students, have helped to uncover some of the hidden secrets of biodiversity within this topographically complex archipelago.

Since 2010, funding from the Australian & Pacific Science Foundation and Australian Commonwealth New Colombo Plan, along with support from students, has enabled fieldwork focused on collecting bees, wasps, and butterflies across all the major Fijian islands. Trekking up the tallest mountains, four-wheel driving across challenging terrain, and following the meandering rivers of inland Fiji has revealed that initial estimations of Fiji’s entomological fauna have been severely underestimated.

DNA barcoding over 1,000 bee specimens has increased species richness estimates from 4 species (known since 1979) up to 26 endemic species in the genus Homalictus. Interestingly, 60% of these new species are only found above 800 m elevation which comprise a mere 2% of land area of Fiji, and they are often restricted to single mountain tops (Figure 1). From extensive DNA barcoding, mitochondrial haplotype diversity was used to explore the level of intraspecific gene flow in the widespread species of the genus (Figure 2).

Figure 1: (a) The number of species (species richness) plotted against land area available at each elevational gradient. (b) Map of Fiji showing the land area available. Colours correspond to those used in (a).

CREATED BY: Cale Matthews

These results also indicate that gene flow is being restricted within highland localities of the most widespread Homalictus species. Dispersal from a species home range does not appear to be occurring in Fiji, which may be presenting a contemporary model of speciation that is predominantly influenced by past climatic fluctuations. There is an estimated crown age of 400 ka for the initial Fijian Homalictus colonisation, which would result in the genus being present for several glacial cycles. During glacial maxima, cooler climates would be ubiquitous throughout Fiji, however during glacial minima and interglacial periods there is a distinction between cool highland and warm lowland climate. DNA barcoding results indicate that the largest diversification of this genus is concordant with the most recent glacial minima, as species that were freely dispersing during glacial maxima are forced to retreat into highland refugia. Combined with the inferred haplotype networks, these results indicate that restriction due to low thermal tolerance of lowland climate is driving the extraordinary highland species richness in Fiji.

 

Figure 2: (a) Haplotype network of all sequenced Homalictus fijiensis (N=358) coloured by sampling locality. Hash marks represent nucleotide changes between each haplotype. Shared haplotypes represented by circles with multiple colours. Circle outline representing highland or lowland sampling. (b) Sampling map of H. fijiensis coloured by geographic sampling locality.

CREATED BY: Cale Matthews

Further to the work on bees, we have also started barcoding Fiji’s butterfly fauna, along with the first ever species of Gasteruption, a parasitoid wasp genus, found in Fiji. The species,Gasteruption tomanivi(Published in Zootaxa by PhD student Ben Parslow), was found at the peak of Fiji’s highest mountain. These discoveries have highlighted how little is known about the entomofauna of Fiji and how the use of DNA barcoding has helped to uncover Fiji’s hidden secrets of biodiversity.

 

Written by

Cale Matthews

Cale Matthews

School of Biological Sciences, Flinders University, Adelaide, Australia

James Dorey

James Dorey

School of Biological Sciences, Flinders University, Adelaide, Australia

Scott Groom

Scott Groom

School of Agriculture, University of Adelaide, Australia

Olivia Davies

School of Biological Sciences, Flinders University, Adelaide, Australia

Elisha Freedman

Elisha Freedman

School of Biological Sciences, Flinders University, Adelaide, Australia

Justin Holder

School of Biological Sciences, Flinders University, Adelaide, Australia

Ben Parslow

School of Biological Sciences, Flinders University, Adelaide, Australia

Michael Schwarz

School of Biological Sciences, Flinders University, Adelaide, Australia

Mark Stevens

Mark Stevens

School of Biological Sciences, Flinders University, Adelaide, Australia

April 7, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5482

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A DNA Barcoding Review of the Entomofauna of Egypt

A DNA Barcoding Review of the Entomofauna of Egypt

A DNA Barcoding Review of the Entomofauna of Egypt

From insect diversity to pests to forensics, DNA barcoding plays a vital role in Egyptian biodiversity conservation and legislative protection efforts.
Egyptian hornet wasp (Vespa orientalis) predating on Dermaptera (Labidura sp.).
PHOTO CREDIT: Mohamed Gamal

Egypt has more than 23,587 identified plant and animal species in addition to thousands of algae, bacteria, and viruses1, and this unique biodiversity contributes to Egypt’s economy and supports the welfare of its citizens. Agricultural production accounts for more than 10 per cent of Egypt’s GDP while tourism revenues from marine activities on the Red Sea represent more than 30 billion LE annually. Protecting threatened species such as dolphins, sharks, and dugong contribute by more than 61 million LE per year and the marine fish production is estimated to be worth 5 billion LE2. Therefore, Egypt has paid particular attention to the conservation and legislative protection of its natural heritage.

Joining its International Barcode of Life (iBOL) partners, Egypt has been using DNA barcoding to better understand and plan for protection of biodiversity. So far, Egypt has published 20,980 DNA barcode sequence records, 25 per cent (5,368) of which have species names that represent 695 species.

In this review, we present an overview of the DNA barcoding carried out on the Egyptian entomofauna and introduce current advances of this promising technique. This review focuses on three main areas that highlight studies investigating insect diversity and distribution, insects in forensic applications as well as pest and parasite dynamics.

Insect diversity and distribution: DNA barcoding has been used to investigate the genetic diversity of Egyptian wasp populations with a wide geographical range3. Three species, Vespa orientalis, Polistes bucharensis, and Polistes mongolicus were accurately identified by their DNA barcodes with the COI phylogenetic signal revealing interesting insights across Jordan, Giza, Cyprus, and Greece. Despite the wide geographical range, only minor genetic diversity was observed among populations of the three wasp species, indicating unrestricted gene flow. 

DNA barcoding has also been used in a larger-scale insect diversity investigation in the understudied Saharo-Arabian zoogeographic region, revealing significant heterogeneity between Egypt, Pakistan, and Saudi Arabia4. The year-long deployment of Malaise traps in these countries collected 53,092 specimens, including 18,391 from Egypt. The DNA barcode sequences revealed the occurrence of 3,682 BINs belonging to 254 families. These results reflect the high species richness of the area, encouraging further research into biodiversity monitoring for the region.

Insects in forensic applications: The Egyptian Forensic Medicine Authority, the leading authority on forensic medicine in Egypt, handles a relatively large number of cases annually and relies on laboratories for assistance with molecular techniques to ensure fast and reliable identification of species of forensic interest (e.g. necrophagous insects). To date, few studies in Egypt have evaluated the use of DNA barcoding in the identification and establishment of reference libraries for insect species of important post-mortem interval indication.

PHOTO CREDIT: Samy Zalat

Egyptian records of blow flies (Calliphoridae). Maggots (larva) are scavengers and adults are plant visitors.

PHOTO CREDIT: Ramadan Mounir

Aly & Wen5 studied the applicability of a 296-bp cytochrome c oxidase I (COI) sequence as a reliable mitochondrial genetic marker for the identification of forensically important flies following previous research showing the efficacy of a short COI marker in this group6. The study analyzed 16 species of blowflies (Calliphoridae), flesh flies (Sarcophagidae), and house flies (Muscidae) originating from Egypt and China and concluded that a shorter COI fragment is simple, cheap, and reproducible but lacks agreement with traditional morphological classification. In a follow-up investigation, Aly7 examined the reliability of long (1173-bp) vs. short (272-bp) COI markers for 18 species of the same 3 dipteran families from Egypt and China. The results indicated that the longer COI marker performed better than the shorter marker for dipterous identification due to better monophyletic separation and concordance with taxonomic classifications.

A more in-depth survey of the genetic diversity of forensically important blowflies (Calliphoridae) revealed numerous haplotypes among 158 specimens collected from four locations in Egypt (Giza, Dayrout, Minya, and North Sinai)8. Three particularly important species (Chrysomya albiceps, Chrysomya , Chrysomya marginalis) were well-differentiated using COI supporting its use for subfamily-, genera-, and species-level identification of blowflies.

Most importantly for forensics use, COI is highly effective at identifying different developmental stages of forensically important flies, including larvae, pupae, and even empty, otherwise difficult to identify morphologically. Five different species of Diptera and their immature stages from Alexandria, Egypt including Chrysomya albiceps, Chrysomya megacephala, Calliphora vicina, Lucilia sericata, and Ophyra capensis, were correctly identified using mitochondrial DNA markers9.

Pest and parasite dynamics: DNA barcoding has also played an important role in the identification of pests and parasites. Seventeen species of mealybug pests (Hemiptera: Pseudococcidae) have been identified by DNA barcoding specimens collected from populations infesting various crops and ornamental plants in Egypt and France10. The genetic variation found between populations of the same species using a combination of three markers (28S-D2, COI, and ITS2) and morphological examination indicated cryptic taxa that might respond differently to management strategies.

High diversity and rapid diversification were found in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)11. P. humanus includes two morphologically indistinguishable subspecies: the head louse, P. humanus and the body louse, P. humanus. By analyzing sequence diversity of two mitochondrial genes (COI, cytb) in 837 specimens of Pediculus humanus from Egypt, Pakistan, and South Africa, high diversity and the occurrence of five mitochondrial lineages was revealed with implications for the spread of disease.

Conclusion: DNA barcoding of crop pests and pollinators, in addition to disease-carrying insect-vectors, will continue to be the top priority for the Egyptian government. Egypt actively enacts laws, carries out research, increases public awareness, engages local communities in the management of protected areas, and implements projects funded by Egypt and other international donors to protect biodiversity. These motivations place Egypt in a valuable position among other countries joining iBOL in support of BIOSCAN, a project that will build a global monitoring system for the planet.

References:

1. Egypt’s Fifth Biodiversity National Report (2014). Ministry of Environmental Affairs, Cairo, Egypt.

2. Coastal and marine biodiversity in Egypt (2018). United Nations Convention on Biological Diversity Conference (CBD COP14), Sharm El Sheikh. Ministry of Environment.

3. Abdel-Samie E, ElKafrawy I, Osama M, Ageez A (2018) Molecular phylogeny and identification of the Egyptian wasps (Hymenoptera: Vespidae) based on COI mitochondrial gene sequences. Egyptian Journal of Biological Pest Control. 28: 36. https://doi.org/10.1186/s41938-018-0038-z

4. Ashfaq M, Sabir JSM, El-Ansary HO, Perez K, Levesque-Beaudin V, Khan AM, Rasool A, Gallant C, Addesi Jo, Hebert PDN (2018) Insect diversity in the Saharo-Arabian region: revealing a little-studied fauna by DNA barcoding. PLoS ONE 13(7). https://doi.org/10.1371/journal.pone.0199965

5. Aly SM, Wen J (2013) Molecular identification of forensically relevant Diptera inferred from short mitochondrial genetic marker. Libyan Journal of Medicine 8:10. https://doi.org/10.3402/ljm.v8i0.20954

6. Zehner R, Amendt J, Schutt S, Sauer J, Krettek R, Povolny D. (2004) Genetic identification of forensically important flesh flies (Diptera: Sarcophagidae). International Journal of Legal Medicine 118(4): 245–247. https://doi.org/10.1007/s00414-004-0445-4

7. Aly SM (2014) Reliability of long vs short COI markers in identification of forensically important flies. Croatian Medical Journal. 55(1): 19–26. https://doi.org/10.3325/cmj.2014.55.19

8. Salem A, Adham F, Picard C (2015) Survey of the genetic diversity of forensically important Chrysomya (Diptera: Calliphoridae). Journal of Medical Entomology 52(3):320–328. https://doi.org/10.1093/jme/tjv013

9. Abdel Ghaffar HA, Moftah MZ, Favereaux A, Swidan M, Shalaby O, El Ramah S, Gamal R (2018) Mitochondrial DNA-based identification of developmental stages and empty puparia of forensically important flies (Diptera) in Egypt. Journal of Forensic Science & Medicine 4(3): 129–134. http://www.jfsmonline.com/text.asp?2018/4/3/129/242508

10. Abd-Rabou S, Shalaby H, Germain J, Ris N (2012) Identification of mealybut pest species (Hemiptera: Pseudococcidae) in Egypt and France, using a DNA barcoding approach. Bulletin of Entomological Research 102(5):515–523. https://doi.org/10.1017/S0007485312000041

11. Ashfaq M, Prosser S, Nasir S, Masood M, Ratnasingham S, Hebert PDN (2015) High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera). Scientific Reports, 14188. https://doi.org/10.1038/srep14188

 

 

 

 

 

 

 

 

 

 

 

Written by

Samy Zalat

Samy Zalat

Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.

Mona Mahmoud

Mona Mahmoud

Nature & Science Foundation, Cairo, Egypt.

April 7, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5515

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