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Singing from the grave: DNA from a 180 year old type specimen confirms the identity of Chrysoperla carnea (Stephens).

Price BW, Henry CS, Hall AC, Mochizuki A, Duelli P, Brooks SJ - PLoS ONE (2015)

Bottom Line: Sequencing diagnostic molecular markers from type material enables accurate species designation, especially where modern taxonomic hypotheses confirm morphologically cryptic species complexes.One such example is Chrysoperla carnea (Stephens), which belongs to a complex of about 20 cryptic species, most of which can only be reliably distinguished by their pre-mating courtship songs or by DNA analysis.Archival DNA extraction and sequencing from the 180 year old lectotype specimen, combined with Bayesian and Likelihood based phylogenetic analyses of modern specimens from the entire complex, were used to establish unambiguously the true identity of Chrysoperla carnea.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Department, Natural History Museum, London, England.

ABSTRACT
Historically serving as repositories for morphologically-based taxonomic research, natural history collections are now increasingly being targeted in studies utilizing DNA data. The development of advanced molecular techniques has facilitated extraction of useable DNA from old specimens, including type material. Sequencing diagnostic molecular markers from type material enables accurate species designation, especially where modern taxonomic hypotheses confirm morphologically cryptic species complexes. One such example is Chrysoperla carnea (Stephens), which belongs to a complex of about 20 cryptic species, most of which can only be reliably distinguished by their pre-mating courtship songs or by DNA analysis. The subtle morphological variation in the group has led to disagreement over the previous designation of the lectotype for C. carnea, an issue that has been further compounded because Chrysoperla carnea is a highly valued biological control agent in arable crops. Archival DNA extraction and sequencing from the 180 year old lectotype specimen, combined with Bayesian and Likelihood based phylogenetic analyses of modern specimens from the entire complex, were used to establish unambiguously the true identity of Chrysoperla carnea.

No MeSH data available.


Terminalia of Chrysoperla carnea lectotype.Tip of abdomen is shown following DNA extraction.
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pone.0121127.g002: Terminalia of Chrysoperla carnea lectotype.Tip of abdomen is shown following DNA extraction.

Mentions: Specimens were imaged before and after dissection (Figs. 1 and 2). A non-type specimen from the Stephen’s collection (BMNH(E) 1239047) was used to validate the method prior to attempting the extraction from the lectotype specimen (BMNH(E) 1239048). All lab equipment and areas were cleaned with DNA Away surface decontaminant (Thermo Scientific), and new consumables and reagents were used to prevent cross contamination of DNA between extraction of the test specimen and subsequent extraction of the lectotype. In each case a single leg was removed and genomic DNA was extracted using the Qiagen QIAamp DNA Micro kit as per manufacturer’s protocol “Isolation of genomic DNA from tissues”, modified by initially grinding the leg with a plastic micro-pestle in 20μl 1xTE buffer before the addition of lysis buffer (180 μl buffer ALT and 20μl proteinase K). The tissues were incubated at 56°C overnight (approx. 17 hours). In addition 1μg of carrier RNA was added to buffer AL at the appropriate stage. Cytochrome oxidase subunit (COI) sequences were amplified in eight separate reactions using the primer pairs designed by referring to COI sequences of the genus Chrysoperla deposited in the DDBJ/EMBL/GenBank database (S1 Table, S1 Fig.) in addition to C1-J-1718 and TL2-N-3014 [27]. Each reaction consisted of 1mM total dNTPs, 3mM MgCl2, 1.25u Bio-Taq DNA polymerase (Bioline), 0.1μM each primer and 1x reaction buffer (67mM Tris-HCl, 16mM (NH4)2SO4, 10mM KCl). Cycling conditions were: initial denaturation 94°C for 1 m followed by 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s, with a final elongation of 10 m at 72°C. To increase the potential DNA yield from the lectotype the abdomen was extracted separately from the leg using the Qiagen QIAamp DNA Micro kit as per manufacturer’s protocol with the following modifications to minimise damage to external morphology: the abdomen was removed and soaked whole in buffer ATL with proteinase K at 56°C overnight, rather than ground or vortexed. 1μg of carrier RNA was added to buffer AL. Following extraction the abdomen was washed in 500μl of TE buffer, then in a series of TE/ethanol dilutions (50% ethanol for 5 hours at 4°c then 70% ethanol for 16 hours at 4°c) before being stored in 100% ethanol and imaged with a Zeiss Axio Zoom v.16 stereo microscope (Fig. 2). All PCR products were cleaned using Millipore PCR filter purification plates as per manufacturer’s instructions, then sequenced bi-directionally using BigDye terminator reaction mix v3.1 in a 3730xl DNA analyser (Applied Biosystems) at the NHM sequencing facility.


Singing from the grave: DNA from a 180 year old type specimen confirms the identity of Chrysoperla carnea (Stephens).

Price BW, Henry CS, Hall AC, Mochizuki A, Duelli P, Brooks SJ - PLoS ONE (2015)

Terminalia of Chrysoperla carnea lectotype.Tip of abdomen is shown following DNA extraction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390323&req=5

pone.0121127.g002: Terminalia of Chrysoperla carnea lectotype.Tip of abdomen is shown following DNA extraction.
Mentions: Specimens were imaged before and after dissection (Figs. 1 and 2). A non-type specimen from the Stephen’s collection (BMNH(E) 1239047) was used to validate the method prior to attempting the extraction from the lectotype specimen (BMNH(E) 1239048). All lab equipment and areas were cleaned with DNA Away surface decontaminant (Thermo Scientific), and new consumables and reagents were used to prevent cross contamination of DNA between extraction of the test specimen and subsequent extraction of the lectotype. In each case a single leg was removed and genomic DNA was extracted using the Qiagen QIAamp DNA Micro kit as per manufacturer’s protocol “Isolation of genomic DNA from tissues”, modified by initially grinding the leg with a plastic micro-pestle in 20μl 1xTE buffer before the addition of lysis buffer (180 μl buffer ALT and 20μl proteinase K). The tissues were incubated at 56°C overnight (approx. 17 hours). In addition 1μg of carrier RNA was added to buffer AL at the appropriate stage. Cytochrome oxidase subunit (COI) sequences were amplified in eight separate reactions using the primer pairs designed by referring to COI sequences of the genus Chrysoperla deposited in the DDBJ/EMBL/GenBank database (S1 Table, S1 Fig.) in addition to C1-J-1718 and TL2-N-3014 [27]. Each reaction consisted of 1mM total dNTPs, 3mM MgCl2, 1.25u Bio-Taq DNA polymerase (Bioline), 0.1μM each primer and 1x reaction buffer (67mM Tris-HCl, 16mM (NH4)2SO4, 10mM KCl). Cycling conditions were: initial denaturation 94°C for 1 m followed by 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s, with a final elongation of 10 m at 72°C. To increase the potential DNA yield from the lectotype the abdomen was extracted separately from the leg using the Qiagen QIAamp DNA Micro kit as per manufacturer’s protocol with the following modifications to minimise damage to external morphology: the abdomen was removed and soaked whole in buffer ATL with proteinase K at 56°C overnight, rather than ground or vortexed. 1μg of carrier RNA was added to buffer AL. Following extraction the abdomen was washed in 500μl of TE buffer, then in a series of TE/ethanol dilutions (50% ethanol for 5 hours at 4°c then 70% ethanol for 16 hours at 4°c) before being stored in 100% ethanol and imaged with a Zeiss Axio Zoom v.16 stereo microscope (Fig. 2). All PCR products were cleaned using Millipore PCR filter purification plates as per manufacturer’s instructions, then sequenced bi-directionally using BigDye terminator reaction mix v3.1 in a 3730xl DNA analyser (Applied Biosystems) at the NHM sequencing facility.

Bottom Line: Sequencing diagnostic molecular markers from type material enables accurate species designation, especially where modern taxonomic hypotheses confirm morphologically cryptic species complexes.One such example is Chrysoperla carnea (Stephens), which belongs to a complex of about 20 cryptic species, most of which can only be reliably distinguished by their pre-mating courtship songs or by DNA analysis.Archival DNA extraction and sequencing from the 180 year old lectotype specimen, combined with Bayesian and Likelihood based phylogenetic analyses of modern specimens from the entire complex, were used to establish unambiguously the true identity of Chrysoperla carnea.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Department, Natural History Museum, London, England.

ABSTRACT
Historically serving as repositories for morphologically-based taxonomic research, natural history collections are now increasingly being targeted in studies utilizing DNA data. The development of advanced molecular techniques has facilitated extraction of useable DNA from old specimens, including type material. Sequencing diagnostic molecular markers from type material enables accurate species designation, especially where modern taxonomic hypotheses confirm morphologically cryptic species complexes. One such example is Chrysoperla carnea (Stephens), which belongs to a complex of about 20 cryptic species, most of which can only be reliably distinguished by their pre-mating courtship songs or by DNA analysis. The subtle morphological variation in the group has led to disagreement over the previous designation of the lectotype for C. carnea, an issue that has been further compounded because Chrysoperla carnea is a highly valued biological control agent in arable crops. Archival DNA extraction and sequencing from the 180 year old lectotype specimen, combined with Bayesian and Likelihood based phylogenetic analyses of modern specimens from the entire complex, were used to establish unambiguously the true identity of Chrysoperla carnea.

No MeSH data available.