Research Article |
Corresponding author: Vladimir A. Lukhtanov ( lukhtanov@mail.ru ) Academic editor: Valentina G. Kuznetsova
© 2019 Vladimir A. Lukhtanov, Yaroslavna Iashenkova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Lukhtanov VA, Iashenkova Y (2019) Linking karyotypes with DNA barcodes: proposal for a new standard in chromosomal analysis with an example based on the study of Neotropical Nymphalidae (Lepidoptera). Comparative Cytogenetics 13(4): 435-449. https://doi.org/10.3897/CompCytogen.v13i4.48368
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Chromosomal data are important for taxonomists, cytogeneticists and evolutionary biologists; however, the value of these data decreases sharply if they are obtained for individuals with inaccurate species identification or unclear species identity. To avoid this problem, here we suggest linking each karyotyped sample with its DNA barcode, photograph and precise geographic data, providing an opportunity for unambiguous identification of described taxa and for delimitation of undescribed species. Using this approach, we present new data on chromosome number diversity in neotropical butterflies of the subfamily Biblidinae (genus Vila Kirby, 1871) and the tribe Ithomiini (genera Oleria Hübner, 1816, Ithomia Hübner, 1816, Godyris Boisduval, 1870, Hypothyris Hübner, 1821, Napeogenes Bates, 1862, Pseudoscada Godman et Salvin, 1879 and Hyposcada Godman et Salvin, 1879). Combining new and previously published data we show that the species complex Oleria onega (Hewitson, [1852]) includes three discrete chromosomal clusters (with haploid chromosome numbers n = 15, n = 22 and n = 30) and at least four DNA barcode clusters. Then we discuss how the incomplete connection between these chromosomal and molecular data (karyotypes and DNA barcodes were obtained for different sets of individuals) complicates the taxonomic interpretation of the discovered clusters.
karyotype, DNA barcoding, COI, meiosis, metaphase, Lepidoptera, Nymphalidae, Biblidinae, Danainae, Ithomiini, Peru
Chromosomal data are an important source of information for taxonomic, evolutionary and comparative phylogenetic studies (
To avoid this problem, here we suggest linking each karyotyped sample with its DNA barcode. It was empirically demonstrated that the mitochondrial DNA barcode, a relatively short fragment of the mitochondrial COI gene (658 base pairs) (i.e., a negligible part of the genome in terms of size), could differentiate up to 95% of species in many taxa (
Obtaining barcodes is currently a simple technical task, which can be carried out in almost any laboratory or on a commercial basis. Our personal experience, based on a molecular analysis of the fauna of Central Asia, Eastern Europe and Western Asia (
Additionally, combination of DNA barcodes and karyotypes represents a powerful tool for detection, delimitation and description of unrecognized species (
The approach based on combination of chromosomal and DNA barcode data has been already used in different studies on butterflies (
In this paper, we demonstrate the algorithm, features and capabilities of the proposed approach with the butterflies of the Neotropical fauna.
The samples were collected in Peru in 2013 by V.A.Lukhtanov. The information on localities where the specimens were collected is presented in the Table
List of the samples of the genera Oleria Hübner, 1816, Ithomia Hübner, 1816, Vila Kirby, 1871, Pseudoscada Godman et Salvin, 1879, Godyris Boisduval, 1870, Hypothyris Hübner, 1821, Napeogenes Bates, 1862 and Hyposcada Godman et Salvin, 1879 collected by V.A.Lukhtanov and used in the study.
Id | BOLD Id | Genus | Species | N | Exact site | Latitude / Longitude | Altitude | Collection date |
A107 | NOB001-17 | Oleria | didymaea ramona | n = 22 | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A108 | NOB002-17 | Ithomia | salapia | n = 34 | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A111 | NOB004-17 | Vila | emilia | n = 30 | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A112 | NOB005-17 | Vila | emilia | – | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A113 | NOB006-17 | Vila | emilia | n = 30 | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A115 | NOB007-17 | Vila | emilia | n = 30 | 60 km SSW Ikitos, Puente Itaya | 04°11'47"S, 73°28'39"W | 114 m | 30 August 2013 |
A121 | NOB008-17 | Oleria | gunilla serdolis | n = 11 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A122 | NOB009-17 | Oleria | gunilla serdolis | n = 11 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A123 | NOB010-17 | Oleria | gunilla serdolis) | n = 11 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A125 | NOB011-17 | Oleria | onega | n = 15 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A127 | NOB012-17 | Oleria | gunilla serdolis | n = 11 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A124 | NOB013-17 | Oleria | onega | n = 15 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A129 | n/a | Pseudoscada | timna | n = 15 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A130 | NOB014-17 | Ithomia | salapia | n = 34 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A131 | NOB015-17 | Godyris | zavaleta | n = 33,35 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A132 | NOB016-17 | Ithomia | salapia | n = 35 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A133 | NOB017-17 | Ithomia | salapia | n = 36 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A135 | NOB018-17 | Ithomia | salapia | n = 36 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A136 | NOB019-17 | Hypothyris | euclea | n = 14 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A137 | NOB020-17 | Napeogenes | sylphis | n = 14 | Tingo Maria | 09°21'02"S, 76°03'21"W | 835 m | 4 September 2013 |
A140 | NOB021-17 | Hyposcada | kena | n = 14 | Cayumba | 09°29'25"S, 75°56'46"W | 1020 m | 5 September 2013 |
A141 | NOB022-17 | Oleria | onega | n = 15 | Cayumba | 09°29'25"S, 75°56'46"W | 1020 m | 5 September 2013 |
A142 | NOB023-17 | Oleria | onega | n = 15 | Cayumba | 09°29'25"S, 75°56'46"W | 1020 m | 5 September 2013 |
A143 | NOB024-17 | Oleria | onega | n = 15 | Cayumba | 09°29'25"S, 75°56'46"W | 1020 m | 5 September 2013 |
A144 | NOB025-17 | Oleria | onega | n = 15 | Cayumba | 09°29'25"S, 75°56'46"W | 1020 m | 5 September 2013 |
A145 | NOB026-17 | Godyris | dircenna | n = 36 | Cayumba | 09°29'43"S, 75°58'01"W | 786 m | 6 September 2013 |
Gonads were removed from the abdomen and placed into freshly prepared fixative (3:1; 96% ethanol and glacial acetic acid) directly after capturing the butterfly in the field. Testes were stored in the fixative for 3–36 months at +4 °C. Then the gonads were stained in 2% acetic orcein for 30–60 days at +18–20 °C. Karyotypes (Figs
Male metaphase I (MI) and II (MII) plates of Ithomiini and Biblidinae 1 A111, Vila emilia, MI, n = 30 2 A107, Oleria didymaea ramona, MI, n = 22 3 A108, Ithomia salapia, MI, n = 34 4 A132, Ithomia salapia, MII, n = 35 5 A133, Ithomia salapia, MI, n = 36 6 A135, Ithomia salapia, MII, n = 36 7 8 A122 Oleria gunilla serdolis, MI, n = 11 9 A124, Oleria onega, MI, n = 15 10 A125, Oleria onega, MII, n = 15 11 A141, Oleria onega, MI, n = 15 12 A142, Oleria onega, MI, n = 15 13 A143, Oleria onega, MII, n = 15 14 A144, Oleria onega, MI, n = 15 15 A131, Godyris zavaleta, MI, n = 33 16 A136, Hypothyris euclea, MI, n = 14 17 A137, Napeogenes sylphis, MI, n = 14 18 A140, Hyposcada kena, MII, n = 14 19 A129, Pseudoscada timna, MI, n = 30. Scale bar: 10 μ in all figures.
Standard COI barcodes (658-bp 5’ segment of mitochondrial cytochrome oxidase subunit I) were studied. Legs were sampled from the karyotyped specimens, and sequence data from the DNA barcode region of COI were obtained at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) using primers and protocols described in
The DNA-barcode-based species identification was carried out by using the BOLDSYSTEMS Identification Engine (http://www.boldsystems.org/index.php/IDS_OpenIdEngine).
The Bayesian majority rule consensus tree of the analyzed samples (Figs
Fragment of the Bayesian majority rule consensus tree of the analyzed samples of Ithomiini inferred from COI sequences. I, II and III are the recovered clusters of the Oleria onega species complex (see Fig.
Vila emilia (Cramer, 1779)
Fig.
The meiotic karyotype was found to include 30 bivalents of similar size.
Oleria didymaea ramona (Haensch, 1909)
Fig.
The meiotic karyotype was found to include 22 bivalents of similar size.
Ithomia salapia Hewitson, [1853]
Figs
The meiotic karyotype was found to include 34 bivalents in a single studied specimen from Puente Itaya (Peru, 60 km SSW Ikitos). One bivalent was slightly larger than the rest ones. The meiotic karyotype was found to include 35–36 bivalents of similar size in the specimens from Tingo Maria.
Oleria gunilla serdolis (Haensch, 1909)
Figs
The meiotic karyotype was found to include 11 bivalents. Two bivalents were larger than the other nine ones.
Oleria onega (Hewitson, [1852])
Figs
The meiotic karyotype was found to include 15 bivalents. The bivalents had different sizes and shapes.
Godyris zavaleta (Hewitson, [1855])
Fig.
The meiotic karyotype was found to include cells with 33 and 35 chromosomal elements, presumably bivalents. 34 bivalents were counted in a single studied specimen from Tingo Maria.
Hypothyris euclea (Godart, 1819)
Fig.
The meiotic karyotype was found to include 14 bivalents of similar size.
Napeogenes sylphis (Guérin-Méneville, [1844])
Fig.
The meiotic karyotype was found to include 14 bivalents of similar size.
Hyposcada kena (Hewitson, 1872)
Fig.
The meiotic karyotype was found to include 14 bivalents. The bivalents had different sizes and shapes.
Pseudoscada timna (Hewitson, [1855])
Fig.
The meiotic karyotype was found to include 30 bivalents of similar size. The bivalents formed a gradient size row.
All studied species were found to be significantly differentiated with respect to the DNA barcode region and formed distinct clusters on the BI tree (Fig.
The Neotropics is one of the most species-rich regions of the world, and the nymphalids are the most speciose butterfly family (
Chromosomal studies represent only a small part of the Neotropical nymphalid diversity (
In this study we suggest a plan for further analysis of the Neotropical Nymphalidae based on a parallel analysis of chromosomal and molecular markers.
Using this approach, we confirm the previously published data on the karyotypes of Godyris dircenna (n = 36), Hypothyris euclea (n = 14), Napeogenes sylphis (n = 14) and Oleria gunilla (n = 11) (
Haploid chromosome number n=30 is found by us in Pseudoscada timna, whereas n = 31 was reported for this taxon by
We provide the first data on karyotypes of Vila emilia and demonstrate a high interspecific chromosome number variation in this genus (previously n = 15 was reported for an unidentified Vila species from western Brazil;
We show chromosome number n = 14 for Hyposcada kena confirming high level of interspecific variation in the genus Hyposcada (from n = 12 to n = 19) (
Different chromosome numbers were previously reported for Godyris zavaleta by
Even more interesting data were obtained regarding the species Oleria didymaea (Hewitson, 1876) and O. onega. We found n = 22 in the taxon identified by us as Oleria didymaea ramona (Haensch, 1909), whereas n= 15 was reported for taxon identified as Oleria alexina didymaea (
Based on chromosome numbers, we hypothesize that Oleria onega is a complex of at least three species with different chromosome numbers: n = 15 (our data), n = 22 and n = 30 (
The incomplete connection between the chromosomal and molecular data (karyotypes and DNA barcodes were obtained for different sets of individuals) complicates the taxonomic interpretation of the discovered clusters. Nevertheless, we predict that in future linking karyotypes with DNA barcodes will result in a significant rearrangement of taxonomy of the genus Oleria.
We thank Fedor Konstantinov (St. Petersburg State University) for help in butterfly collecting and research. The study was supported by the projects RFBR 18-04-00263-a, RFBR 17-04-00828-a and state research project AAAA-A19-119020790106-0.