DNA Barcoding and Genomics in the Megadiverse Amazon Altitude Fields

DNA Barcoding and Genomics in the Megadiverse Amazon Altitude Fields

DNA BARCODING AND GENOMICS IN THE MEGADIVERSE AMAZON ALTITUDE FIELDS

Scientists are contributing to the most profound molecular representation of biodiversity in any Brazilian environment.
Entrance of a ferruginous cave

The entrance of a ferruginous cave at the Bocaina mountain in the Carajás region, Pará State, Brazil. PHOTO CREDIT: João Marcos Rosa.

Industrial activities in the Brazilian Amazon are highly regulated by governmental agencies. However, the lack of knowledge about megadiverse areas is a problem for the establishment of best conservation practices; this is the case for mining operations in the eastern Amazonian Carajás, a region comprised of a mosaic of national parks, indigenous peoples’ conservation areas, and nature reserves. Of particular interest are the ferruginous altitude fields known as the Canga. Our floristic survey described the presence of 1,094 species from just over 200 previously known1. The lack of biodiversity data is even more significant for the ferruginous caves where only around 10 invertebrate species are identified to the species level. Unfortunately, this is typical for the Amazon basin.

To provide reliable scientific data that contributes to the implementation of best conservation practices, Instituto Tecnológico Vale is developing DNA barcode reference libraries for the flora, cave invertebrates, and bats of the region, and providing deeper genomic references for species that are endangered or difficult to identify. To achieve this goal, we established the necessary infrastructure to conduct DNA sequencing using Sanger, Illumina, and PacBio technologies, coupled with high-performance computing, artificial intelligence algorithms, and highly trained personnel.

 

Botanists collecting samples

Botanists collecting samples in a temporary lake in ferruginous altitude fields in the Carajás region, Pará State, Brazil.
PHOTO CREDIT: João Marcos Rosa

To date, over 8,575 barcodes for 3,548 specimens of plants and invertebrates have been produced, while a large number of species remain to be identified by morphological attributes. Morphological specimen determination is conducted by in-house specialists, as well as by an extensive network of specialists in universities and museums across Brazil and abroad.

For this purpose, nuclear and chloroplast or mitochondrial markers as well as low coverage to whole genome sequencing or restriction site-associated DNA sequencing (RADSeq) are being employed to unravel the vast genetic diversity of the biota of Carajás2-4. For several endemic plants, such as species of Asteraceae, Melastomataceae5, Convolvulaceae6, and Isoetaceae2, diversity analyses, based on next-generation sequencing, aim to characterize the genetic variability among and within populations, as well as the identification of markers under selective pressure. These methods also contribute to the understanding of population structure and the process of gene flow between populations affected by natural factors and industrial operations. Models of environmental distribution, including parameters sensitive to climate change, were determined for several taxonomic groups, including plants and bats7.

Specimens of the pinheirinho-da-canga (Paepalanthus fasciculoides) highly adapted to inhabit the canga (ancient ferruginous rock outcrops) at the altitude fields in the Carajás region, Pará State, Brazil.
PHOTO CREDIT: João Marcos Rosa

We are also establishing eDNA methods, as well as metagenomics and metaproteomics data for environmental monitoring of ferruginous fields phytophysiognomies, areas under rehabilitation processes, and caves8. Together these data constitute the most profound molecular representation of any environment in Brazil. We have contributed a total of 3,072 specimens to the Barcode of Life Data System (BOLD) comprising 398 genera (291 new) in addition to the 408 different genera collected through the national effort for angiosperms in Brazil. We have also provided 571 cave fauna specimens. It is important to highlight that all of these data generated are being provided to the public and its use will be critical to the conservation of such a unique collection of species.

References:

1. Brazil Flora Group (2018) Growing knowledge: an overview of Seed Plant diversity in Brazil. Rodriguésia 66(4): 1085–1113. http://dx.doi.org/10.1590/2175-7860201566411

2. Nunes GL, Oliveira RRM, Guimarães JTF, Giulietti AM, Caldeira C, Vasconcelos S, et al., (2018) Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae. PLoS ONE 13(8): e0201417. https://doi.org/10.1371/journal.pone.0201417

3. Ramalho AJ, Zappi DC, Nunes GL, Watanabe MTC, Vasconcelos S, Dias MC, Jaffé R, Prous X, Giannini TC, Oliveira G and Giulietti AM (2018) Blind testing: DNA barcoding sheds light upon the identity of plant fragments as a subsidy for cave conservation. Frontiers in Plant Science 9:1052. https://doi.org/10.3389/fpls.2018.01052

4. Oliveira RRMO, Vasconcelos S, Pires ES, Pietrobon T, Prous X and Oliveira G (2019) Complete mitochondrial genomes of three troglophile cave spiders (Mesabolivar, pholcidae), Mitochondrial DNA Part B 4(1): 251–252. https://doi.org/10.1080/23802359.2018.1547139

5. Carvalho CdS, Lanes ECM, Silva AR, Caldeira CF, Carvalho-Filho N, Gastauer M, Imperatriz-Fonseca VL, Nascimento W, Oliveira G, Siqueira JO, Viana PL, Jaffe R (2019) Habitat loss does not always entail negative genetic consequences. bioRxiv 528430. https://doi.org/10.1101/528430

6. Lanes ÉC, Pope NS, Alves R, Carvalho Filho NM, Giannini TC, Giulietti AM, Imperatriz-Fonseca VL, Monteiro W, Oliveira G, Silva AR, Siqueira JO, Souza-Filho PW, Vasconcelos S and Jaffé R (2018) Landscape genomic conservation assessment of a narrow-endemic and a widespread morning glory from Amazonian Savannas. Frontiers in Plant Science 9:532. https://doi.org/10.3389/fpls.2018.00532

7. Costa WF, Ribeiro M, Saraiva AM, Imperatriz-Fonseca VL, Giannini TC (2018) Bat diversity in Carajás National Forest (Eastern Amazon) and potential impacts on ecosystem services under climate change. Biological Conservation 218: 200–210. https://doi.org/10.1016/j.biocon.2017.12.034

8. Gastauer M, Vero MPO, de Souza KP, Pires ES, Alves R, Caldeira CF, Ramos SJ, Oliveira G (2019) A metagenomic survey of soil microbial communities along a rehabilitation chronosequence after iron ore mining. Scientific Data 6:190008. https://doi.org/10.1038/sdata.2019.8

Written by

Guilherme Oliveira

Guilherme Oliveira

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Gisele Nunes Lopes

Gisele Nunes Lopes

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Rafael Valadares

Rafael Valadares

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Ronnie Alves

Ronnie Alves

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Santelmo Vasconcelos

Santelmo Vasconcelos

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

April 7, 2019
doi: 10.21083/ibol.v9i1.5498

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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
doi:10.21083/ibol.v9i1.5515

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