Corresponding author: Valentina Todisco ( email@example.com )
Academic editor: Michael Ohl
© 2017 Valentina Todisco, Vazrick Nazari, Paul D.N. Hebert.
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: Todisco V, Nazari V, Hebert PDN (2017) Preliminary molecular phylogeny and biogeography of the monobasic subfamily Calinaginae (Lepidoptera, Nymphalidae). Zoosystematics and Evolution 93(2): 255-264. https://doi.org/10.3897/zse.93.10744
Calinaga, Calinaginae, Nymphalidae, mtDNA, butterfly, Indochina, Oligocene
Calinaga (Moore, 1857) is an enigmatic butterfly genus mainly distributed in the Indochina region, but also extending to Taiwan and the Himalayas (Fig.
The systematics of the genus has long been a topic for taxonomic discussion.
Located in the continental portion of Southeast Asia, lying to the southeast of the Himalayas and south of China, the Indochina bioregion has long been recognized as an area with globally important levels of biodiversity (
For the first time in this study, we conduct an analysis of molecular data (mitochondrial COI-5’ and ribosomal protein S5 nuclear gene (RpS5)) and we examine morphological characters (genitalia and wing pattern) for 51 specimens of Calinaga from widely distributed sites in the Indochina region (Fig.
Given the scarcity of information on the evolutionary history of Calinaga, the geographic and taxonomic coverage in our datasets will help to advance understanding of the diversification of this charismatic insect group in the Indochina bioregion and of other genera with similar distribution patterns.
(A) Approximate geographic distributions (
Taxon= Calinaga species identified in this study. Sample numbers (N), collection locality, population code, mtDNA haplotype code and GenBank Accession numbers.
|taxon||N||Collection locality||Population code||Haplotype code||GenBank Accession|
|COI KX233–||RpS5 KX283–|
|aborica||2||Burma: N Sagaing, Tailing Hka river||CABO-BURSA||H9||590/612||390/398|
|lhatso||1||China: Shaanxi, Tsnling Mts, Foping nature reserve, 1600 m||CLHA-CHSTF||H4||570||379|
|davidis||2||China: Qinghai, Ningyou Jiuzhi country||CDAV-CHNJQ||H15, H23||576/581||382/384|
|davidis nubilosa||2||China: Qinghai, Ningyou Jiuzhi country||CBUD-CHQNJ||H13||577/593||383/391|
|davidis||1||China: Sichuan, Yingxiuwan, 1000 m||CDAV-CHSYI||H22||596||394|
|davidis nubilosa||1||China: W Sichuan, Dayi district, 23Km north of Huashuiwan, 1500m||CBUD-CHSDH||H22||603||–|
|davidis nubilosa||1||China: W Sichuan, Shimian–Mianning road, 2300 m||CBUD-CHSSM||H20||599||–|
|davidis nubilosa||1||China: W Sichuan, 30km W of Tianquan, 1250 m||CBUD-CHSTI||H15||580||–|
|davidis nubilosa||3||China: W Sichuan, Ya`an–ergaz Shan, 1400 m||CBUD-CHSYS||H12, H13||601/614/609||–|
|davidis||2||China: S Sichuan, SW of Dechang Miyi, 2100 m||CBUD-CHSDM||H18, H21||572/615||–|
|davidis buphonas||3||China: Yunnan, Wei–Xi 10km SE, 2000–2400 m||CDAV-CHYWE||H11||613/584/616||387/399|
|davidis buphonas||1||China: Yunnan, Surroundings of Dali city, 2400 m||CBUD-CHYDA||H11||594||392|
|davidis||2||China: Yunnan, Ningjing Shan Tse–Kou Mekong River, 2100 m||CBUD-CHYNM||H11||619/605||401/395|
|davidis genestieri||1||China: Yunnan, Ningjing Shan Wei–Shi, 2200 m||CBUD-CHYNS||H11||583||386|
|davidis genestieri||1||China: Yunnan, Wei–Xi, 2200 m||CBUD-CHYWX||H10||618||–|
|davidis||1||China: Yunnan, 40km N of Xhinxrong, 1850 m||CBUD-CHYXH||H22||586||–|
|sudassana||2||China: W Yunnan, Salween valley, 6 Km of Fugong, 1300 m||CBUD-CHYSA||H5||611/587||397|
|davidis||2||China: W Yunnan, Salween valley, 16Km N of Gongshan, 1675 m||CBUD-CHYSG||H17||617/620||400/402|
|davidis cercyon||2||China: N Yunnan, Sanba: Blotopel (Zhongdian), 2400 m||CBUP-CHYSB||H19||598/591||–|
|davidis buphonas||2||China: N Yunnan, 75Km NW of Lijang, 2000 m||CBUD-CHYLI||H11||578/571||–|
|lhatso||1||China: N Yunnan, 55km N of Zhongdian, 2450 m||CLHA-CHYZH||H1||592||–|
|sudassana||2||India: Nagaland Naga hills||CBUD-INDNNH||H3, H4||608/597||–|
|sudassana||1||Thailand: Wiang Papao, Chiang Rai||CSUD-TAIRAI||H2||574||–|
|sudassana||2||N Thailand: Samoeng||CSUD-TAISAM||H2||604/610||–|
|sudassana||3||Vietnam: Dong Van, Meo Wac district, Ha Glang, 1700 m||CSUD-VNDVM||H7||589/573/575||389/380/381|
|sudassana||6||Vietnam: Hoang Lien national Park Sapa, Cat Cat village and Muong Hoa river, 1350 m||CSUD-VNHLK||H7, H8||
|davidis||1||Stratford Butterfly Farm, UK||NW64-3||H18||AY090208||EU141406|
|lactoris||1||China: Tianma National Nature Reserve in Jinzhai County, Anhui||–||H14||HQ658143||–|
We examined 51 Calinaga individuals from 29 localities across India, South China (Yunnan, Shaanxi, Qinghai, Sichuan), Laos, Vietnam, Myanmar and Thailand (Fig.
Morphological identifications in this study were made based on illustrations of type material in
DNA was extracted from one leg of each individual on Biomek FX liquid handling robot using a semi-automated DNA extraction protocol (
The entire nuclear RpS5 was initially amplified using the primers RpS5f/r (
Sequences were obtained by using an ABI 3730xl sequencer following the manufacturer’s recommendations and they were edited and assembled using CodonCode Aligner 6.0.2. All sequences of Calinaga were submitted to GenBank (Accession Numbers in Table
Two sequences from C. buddha (COI: EF683684, AY090208; RpS5: EU141406) and one from C. davidis (COI: HQ658143), as well as COI and RpS5 sequences for eight outgroups for species belonging to closely related lineages of Nymphalidae (Charaxinae and Satyrinae) from
We used NETWORK 5 (
We used the BEAST package 1.8.3 (
Recent phylogenetic studies calibrated with fossil data (
Bayesian phylogeny of Calinaga estimated in BEAST using concatenated data. Purple squares are calibration points (root: 75 ± 3; Satyrinae + Charaxinae 70 ± 3.5, Charaxes + Euxanthe 22 ± 1). Monophyly was enforced on nodes marked with orange squares. The inset map shows the biogeographic regions used in DIVA analysis: A) Southwestern China ecozone, B) Himalaya-Tibetan plateau region, C) Northern Sino-Himalaya, D) Southern Sino-Himalaya, E) Indochina. Colored dots correspond to haplogroups on the tree.
Haplotype and nucleotide diversity were calculated using DnaSP 5.0 (
Dispersal-vicariance optimization of ancestral areas implemented in DIVA 1.1 (
The morphological study on the wing pattern of our specimens, based on illustrations in the most relevant literature, suggested the presence of four species: C. aborica, C. davidis, C. lhatso and C. sudassana (Table
Male genitalia in Calinaga were found to be very uniform and thus genitalic characters were of little use in discrimination of species. Although
For COI (658 bp), 54 sequences (51 sequences analyzed in this study plus 3 sequences retrieved from GenBank) showed 23 different haplotypes (Fig.
Bayesian phylogeny analysis was used to reconstruct phylogenetic relationships (Fig.
The Bayesian tree for concatenated genes resulted in a well-supported Calinaga clade (Fig.
The MJ network analysis (Fig.
According to Fs and R2 statistics, calculated for Haplogroup A “davidis” (N= 20) and Haplogroup B “sudassana” (N= 30) mtDNA sequence sets (Table
The most recent common ancestor of Calinaga has been estimated to have split from Satyrinae + Charaxinae sometime between 67-84 Mya (
N, number of mtDNA sequences; H, number of unique mtDNA haplotypes; h, mtDNA haplotype diversity (±SD); π, mtDNA nucleotide diversity (±SD). Tests of demographic equilibrium and mismatch analysis in mtDNA phylogeographic groups: FS, Fu’s FS statistic; R2, Rozas and Ramon-Onsins’ R2 statistic; τ, sudden demographic expansion parameter, with 5% and 95% confidence limits; t, true time since population expansion, from τ = 2 μ T (where μ is the substitution rate per gene). Significantly small values of FS and R2 are in bold.
|Haplogroup||N||H||h||π (X 102)||Fs||R2||τ||τ (5%)||τ (95%)||t (ka)||t (5%)||t (95%)|
Results of the ancestral area reconstructions showed that the common ancestor of Calinaga probably occurred before ~43 Mya in area ADE (Fig.
Despite six species initially identified (Fig.
The question of where and when Calinaga originated and how its species are related have not been previously investigated. Although no fossils are known for the genus, previous phylogenetic studies on Nymphalidae that have used fossils for molecular calibration have dated the last common ancestor of Calinaga + (Satyrinae + Charaxinae) at 67-84 Mya (
Our results suggest that the common ancestor of Calinaga probably occurred before ~43 Mya (63-25 Mya) in the region now corresponding to the Southwestern China ecozone, Southern Sino-Himalaya and Indochina (ADE: Fig.
Our molecular dating suggests that C. lhatso and Haplogroup B“sudassana” split into distinct lineages in Early Miocene ~21 Mya (38-8 Mya), possibly isolating the last common ancestor of C. lhatso in the Southern Sino-Himalaya (A). Southern Sino-Himalaya and Indochina (AE) possibly represented the ancestral area for C. lhatso + Haplogroup B“sudassana”. Considering that before ~22 Mya the Indochinese peninsula did not exist, it is more likely that the ancestral area of this clade was mainly in Southern Sino-Himalaya (A). The common ancestor of Haplogroup B“sudassana” began diversifying around ~12 Mya (24-4 Mya) and it probably expanded its range from Southern Sino-Himalayan region (A) to Indochina peninsula (E). Conversely, the origin of C. aborica + Haplogroup A “davidis” must be traced back to Oligocene (~30 Mya), when the first uplift of the Himalayan–Tibetan Plateau occurred (29 – 65 Mya:
In addition, our analyses show that Haplogroup A “davidis” and Haplogroup B“sudassana” experienced demographic expansion in the Pleistocene, ~168 kya (70-363 kya) and ~260 ka (16-403 kya) respectively. Therefore, the genetic diversity and the present-day distributions of Calinaga should be attributed not only to the dramatic geological events in Oligocene/Miocene, but also to the impacts of the cycles of glacial advance/retreat that occurred in this region during the Pleistocene (
Finally, the taxon C. formosana, for which we examined several specimens but could not obtain any sequences (Supplementary Material S1), is morphologically allied to the Haplogroup A “davidis”. Since Taiwan formed ~4-5 Mya at a complex convergent boundary between the Philippine Sea Plate and the Eurasian Plate (“The Penglai Orogeny”:
The genus Calinaga probably originated in the South-East Tibet in Eocene following the immense geological and environmental impact caused by the collision between Indian and Asian subcontinents. The extrusion of Indochina from the continent during the Oligocene/Miocene further prompted dramatic orogenetic changes that promoted isolation and speciation events in the genus. More recently, in the Pleistocene, climatic changes further modified the distribution of species and probably facilitated vicariant speciation events.
Since we did not sample or sequence specimens from all of the available names under Calinaga, we cannot make any definitive statements about the number of valid species warranted to be recognized as such, although the existence of many superfluous names is evident. From the names of the genus and the species coined by early British lepidopterists including F. Moore, it is apparent that they drew inspiration from Hindu mythological characters. In Sanskrit, Nāga refers to mythical reptilian creatures found in Indian religions (Hinduism, Buddhism and Janism) who were often worshipped as deities. Among them, “Kaliya” (or Kalya, “Kalia-Naga”, Calinaga) was a particularly notorious and poisonous one living in Yamuna river in Vrindavan (Uttar Pradesh). After an encounter with Krishna, Kaliya surrendered and was sent to exile (Bhagavata Purana, 16:10). It seems that the modern taxonomy of Calinaga is in need of a Krishna to conquer these superfluous names and cleanse its taxonomy albeit after careful examination of the types and sequencing of additional material.
This study was supported by grants to PDNH from NSERC and from the Government of Canada through Genome Canada and the Ontario Genomics Institute in support of the International Barcode of Life Project. We are extremely grateful to Niklas Wahlberg for providing valuable insights in the early stages of this study, to Valerio Sbordoni for providing specimens, to Vadim Tshikolovets for providing identification materials, and to Prof. R.I. Vane-Wright, Rodolphe Rougerie and an anonymous reviewer for their helpful comments.