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The phylogenetic history of the old world butterfly subtribe Mycalesina extended: the Mycalesis (Lepidoptera: Nymphalidae) of Sri Lanka

https://doi.org/10.1016/j.aspen.2018.12.013Get rights and content

Highlights

  • Phylogenetic relationships of Sri Lankan Mycalesis are resolved.

  • Sri Lankan species diverged in the Miocene, when India and Sri Lanka were separated.

  • All five Sri Lankan species can be identified based on genetic characteristics.

  • The endangered Mycalesis rama was confirmed as an endemic species of Sri Lanka.

  • Occasional hybridization between some species may occur in Sri Lanka.

Abstract

Sri Lanka, together with the Western Ghats, is one of the world's biodiversity hotspots, yet little is known about the genetic diversity of the butterflies here. Within the framework of the recently reconstructed molecular phylogeny for the subtribe Mycalesina, we determined the phylogenetic relationships and biogeographic history of the five species of Mycalesis butterflies that are found in Sri Lanka. Sequences of Elongation Factor 1-α (EF1-α), Wingless (Wgl) and the barcode region of the Cytochrome c Oxidase sub unit 1 (COI) were used to confirm the identity of species, to resolve taxonomic queries and to infer the phylogenetic history of the group.

Time-calibrated analysis of genetic data suggests that the Mycalesis species that occur in Sri Lanka diverged in the Miocene (5–23 million years ago). The results of phylogenetic analyses confirmed the following; the endemicity of Mycalesis rama to Sri Lanka; the subspecies classification of M. perseus typhlus and, the close sister relationship of the Sri Lankan M. patnia patnia to M. p. junonia of India. However, whether or not M. subdita of Sri Lanka and India are genetically similar remains unknown. Lastly, genetic evidence emerged suggesting that M. mineus forms a cryptic species complex in the Oriental region, and that in Sri Lanka, there may be occasional hybridization between M. mineus and M. perseus. As a case study of island colonization and diversification by the Mycalesis species, this study further extends our understanding of the Old World butterfly subtribe of Mycalesina.

Introduction

Mycalesis is a large genus belonging to the subtribe Mycalesina Reuter, 1896 (subfamily Satyrinae, family Nymphalidae). This group of tropical butterflies are cosmopolitan and abundant, with a well-documented radiation across the Old World (Aduse-Poku et al., 2015). Within the Mycalesina, the genera Bicyclus and Heteropsis are the most species rich, followed by the genus Mycalesis, which has species distributed in both the Oriental and Australasian regions. Members of the Mycalesina, including the genus Mycalesis, have been used as models in numerous studies on genetic and phenotypic variation (e.g., Benedick et al., 2007; Sekar and Karanth, 2013; Ho et al., 2016).

For Mycalesis species, the island of Sri Lanka provides a range of suitable tropical climates and habitats. Sri Lanka has a total area of 65,525 km2 and is located in the Indian Ocean, between latitude 5o54’ and 9o52’ North and longitude 79o39’and 81o53’ East. The island has a wet tropical climate with nearly constant temperatures but highly variable rainfall (Panabokke, 1996) and is divided into three broad climatic zones based on precipitation: the dry zone, intermediate zone and wet zone (Panabokke, 1996; Dharmaratne, 2005) (Fig. 1). Sri Lanka, together with the Western Ghats of India, is a biodiversity “hotspot” with a high species density of flora, amphibians and reptiles (Mittermeier et al., 2011). Given the wide range of habitats, understanding how the Mycalesis species have colonized Sri Lanka and evolved here within this small island of <70,000 km2 may provide a key to understanding the diversification of the larger scale mycalesine radiation across the Old World tropics. For large taxonomic groups such as the Mycalesina, which contains >300 species (Aduse-Poku et al., 2015), various studies focusing on different sub-groups may be necessary to more fully understand the group as a whole. For example, multiple studies were required to help build a robust phylogeny of the Hesperiidae (Warren et al., 2009; Jiang et al., 2013; Yuan et al., 2015; Fan et al., 2016; Sahoo et al. 2016; Toussaint et al., 2018).

Of the five Mycalesis species that have been recorded in Sri Lanka (Fig. 2), the National Red List documents M. rama (Moore, 1892) and M. subdita (Moore, 1892) as endemic to Sri Lanka (van der Poorten, 2012). However, the latter has been reported in both Sri Lanka and India (Moore, 1890–1892; Bingham, 1905; Evans, 1920, Evans, 1932; Talbot, 1947; Mathew and Soumya, 2013; Anonymous, 2016). The subspecies, M. patnia patnia Moore, 1857, is considered endemic to Sri Lanka (MOERE, 2014). The remaining two species of Mycalesis recorded in Sri Lanka: M. perseus (Fabricius, 1775) and M. mineus (Linnaeus, 1758), are distributed throughout the Oriental region. M. perseus in Sri Lanka has been classified as M. p. typhlus (Fruhstorfer, 1908) and M. mineus as M. m. polydecta (Cramer, 1777) based on morphological characters (Evans, 1932; van der Poorten and van der Poorten, 2016). Little is known about the genetic variation of these Sri Lankan species or their relationships to other taxa found elsewhere in the world. Although molecular phylogenies have been reconstructed recently for the subtribe Mycalesina (Aduse-Poku et al., 2015; Kodandaramaiah et al., 2010), none of them included Sri Lankan exemplars. Thus, no information exists on the diversification of the Mycalesis here or the radiation of the species onto the island. Here, we address this gap in Mycalesina phylogenetics.

Members of the genus Mycalesis in Sri Lanka are distributed across different climatic zones and elevations, and are known to feed on a wide variety of grasses (Poaceae). M. perseus has the widest distribution and is common up to 700 m above sea level. Its larval diet has been recorded as including the plants Oryza sativa, Leersia hexandra and Axonopus compressus (Poaceae) (van der Poorten and van der Poorten, 2012). M. p. patnia is also widely distributed throughout the island at all elevations and is common in grassy vegetation in forests and wooded home gardens (van der Poorten and van der Poorten, 2016) favouring habitats with Cyrtococcum trigonum and Isachne globosa (Poaceae) (van der Poorten and van der Poorten, 2012). M. mineus is found in grasslands of the wet zone and intermediate zone (Uva, Sabaragamuwa and Central province) from 200 to 750 m above sea level. M. subdita is common in the intermediate zone and dry zone below 750 m. Though the habitats of M. mineus and M. subdita seldom overlap, larvae of both species are reported to feed on Panicum maximum and Axonopus compressus, which are naturalized widespread plants throughout the island (van der Poorten and van der Poorten, 2012, van der Poorten and van der Poorten, 2016). The endemic species, M. rama, is considered ‘Endangered’ according to IUCN Red List criteria. This species is confined to patches of bamboo (Ochlandra stridula) forest of the low country wet zone (van der Poorten and van der Poorten, 2016) and its larvae are believed to feed on bamboo.

Except for M. p. patnia, the four remaining species are morphologically similar (van der Poorten and van der Poorten, 2016), which makes identification difficult. Identifying females is particularly challenging, as species identification is based mainly on male characters. Seasonal polyphenism adds considerable variation which further complicates identification.

There are several other taxonomic issues within genus Mycalesis that need to be resolved. One issue is the status of M. perseus in Sri Lanka. It is currently unclear whether the Sri Lankan subspecies, M. perseus typhlus (Evans, 1932) and Indian subspecies, M. p. tabitha warrant their current subspecies statuses (Kunte, 2012) or whether the Sri Lankan subspecies, M. p. typhlus, should be raised to species rank as M. typhlus (MOERE, 2014).

The objectives of this study were therefore to: 1) characterize the genetic variation of Mycalesis in Sri Lanka in order to describe the phylogenetic history of the group; 2) add to current knowledge on the phylogenetic history of the subtribe Mycalesina; and finally, 3) establish whether genetic characteristics are useful for the identification of Mycalesis species. The latter is particularly important considering the morphological similarity and seasonal variations in morphology, which make using morphological characters for identification problematic at times.

To answer these questions, three DNA regions were selected for this study, the barcode region of the mitochondrial gene Cytochrome c Oxidase sub unit 1 (COI), and two nuclear DNA (nDNA) regions, Elongation Factor 1- α (EF1-α) and Wingless (Wgl). The COI barcode region has been used successfully to delineate species of butterflies (Laiho and Ståhls, 2013; Ashfaq et al., 2013), to detect cryptic species (Dincă et al., 2011, Dincă et al., 2015; Seraphim et al., 2014; Lukhtanov et al., 2015) as well as morphologically similar species (Hebert, Penton, et al., 2004a; Gillespie et al., 2013), and to discern population genetic structure and phylogenetic relationships among species (Dai et al., 2012; Ashfaq et al., 2013; Seraphim et al., 2014; Silva-brandão et al., 2015; Karthika et al., 2017). The COI, EF1-α and Wgl regions have also been successfully used to delineate lepidopteran clades and identify phylogenetic relationships (Wahlberg et al., 2005; Kodandaramaiah et al., 2010; Price et al., 2011; Aduse-Poku et al., 2015).

Section snippets

Sample collection and identification

Adult specimens were collected in Sri Lanka from 2012 to 2014 from all three climatic zones (wet zone, dry zone and intermediate zone) within three elevational bands (lowland, midland and highland) (Fig. 1). Field nets and traps baited with overripe fruit were used for capture of the specimens. GPS coordinates and elevation at the location of each captured specimen were recorded with a Magellan eXplorist 510 navigation system. Field collections of M. rama were restricted due to the endangered

Estimating times of divergence

A Bayesian MCMC approach in BEAST 2 v2.1.3 (Bouckaert et al., 2014) was used to estimate divergence times. The relaxed log normal clock model, calibrated Yule tree model prior with gamma distributed birth-death rate, and 50 million MCMC generations were specified as parameters. Secondary age calibration points from Aduse-Poku et al. (2015) were selected: the stem age of the outgroup was constrained to be 39.8 ± 0.5 million years ago (Mya) with a normal distribution prior between minimum and

Results

A total of 221 specimens of Sri Lankan Mycalesis (♂ = 161, ♀ = 60), were collected (Fig. 1, Table A.1). Sexes were identified using morphological characters and specimens selected for DNA analysis included both males and females.

The sequencing success rate of the COI barcode region was 98% (139 sequences out of 142 attempted sequences), with >600 bp of the 658 bp region amplified and sequenced in all samples. The sequences were added to BOLD under the project folder MYCSL (//www.boldsystems.org

Extended phylogeny of subtribe Mycalesina

A total of 70 sequences from 16 Mycalesis species and three sequences each representing the seven in-group genera, Bicyclus, Hallelesis, Heteropsis, Telinga, Mydosama, Lohora and Nirvanopsis, were included to represent the Mycalesina. The combined molecular data included 2113 bp of DNA sequence data.

The tree constructed from this analysis (Fig. 4) retained the monophyly of genus Mycalesis (95% posterior probability, 54% ML bootstrap support). The results are congruent with the results shown by

Discussion

The present classification of butterflies in Sri Lanka is based on morphological data and subspecies classifications are based on geographic variation (Evans, 1932; Talbot, 1947; van der Poorten and van der Poorten, 2016). In the current study, three molecular markers were used to clearly differentiate the individual Sri Lankan species of Mycalesis as distinct taxonomic units, confirming species status as well as resolving several taxonomic queries. The results also indicated the effectiveness

Taxonomic inference

Sri Lankan and non-Sri Lankan specimens of M. perseus showed little divergence in either mtDNA or nDNA regions (mean intra-specific variation <2%) which confirmed the specimens as conspecific. Though there are no clear morphological or genetic guidelines to delimit subspecies, the Sri Lankan specimens have been previously identified as subspecies M. p. typhlus based on morphological data (van der Poorten and van der Poorten, 2016). It has therefore been argued that subspecies status should be

Historical biogeography of the Sri Lankan Mycalesis

Evans (1920) proposed that M. perseus and M. mineus were the oldest lineages of the genus (classified as the Calysisme group). Though M. perseus has not radiated to give rise to new species, Evans (1920) hypothesized that ancestral M. mineus gave rise to M. subdita (in Sri Lanka), M. perseoides, M. igilia, M. mercea, M. visala and others that have successfully colonized the Oriental region. This may explain the genetic similarity of M. mineus to the other species of Mycalesis in the Oriental

Conclusion

In conclusion, this study presents a more complete phylogeny of genus Mycalesis. While we have contributed to a better understanding of the taxonomic status, identification, phylogenetic relationships and the biogeographic history of the Sri Lankan and regional species of Mycalesis, we have also identified critical areas that would require further study in the future.

Acknowledgements

The National Research Council, Sri Lanka (Grant 11-010) and The Open University of Sri Lanka are gratefully acknowledged for financial support. We thank the Department of Wildlife and Conservation (ref: WL/3/2/1/9 and WL/3/2/7/13) and the Forest Department (ref: RandE/RES/2010/OUSL) for granting permission to collect specimens and enter protected forest reserves. We are grateful to K.M. Supun Deshaprema and J.A. Roshan Madushanka for helping with field work, and to Niklas Wahlberg, Natalie dos

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