# Unique among high passes: Phylogenetic inferences from DNA barcoding of the butterfly fauna of Ladakh Trans-Himalaya, India

## TL;DR

This study uses DNA barcoding to analyze the genetic diversity of butterflies in the cold-desert region of Ladakh, India, revealing insights into their phylogeny and conservation.

## Contribution

The study creates a DNA barcode reference library for 23 butterfly species in Ladakh, including novel barcodes for six species.

## Key findings

- Interspecific divergence in two mountain butterfly genera was below the 3% threshold, challenging standard DNA barcoding assumptions.
- Most species showed minimal divergence when global barcodes were added, but a few showed a two-fold increase.
- Bayesian clustering mostly aligned with morphological classifications, with exceptions in taxonomically complex genera.

## Abstract

The butterfly assemblage of Ladakh Trans-Himalaya demands a thorough
analysis of their population genetic structure owing to their typical
biogeographic affinity and their adaptability to extreme cold-desert climates.
No such effort has been taken till date, and in this backdrop, we created a
barcode reference library of 60 specimens representing 23 species. Barcodes were
generated from freshly collected leg samples using the Sanger sequencing method,
followed by phylogenetic clade analyses and divergence calculation. Our data
represents 22% of Ladakh’s Rhopaloceran fauna with the novel barcode
submission for six species, including one Schedule II species, Paralasa
mani. Contrary to the 3% threshold rule, the interspecific
divergence between two species pairs of typical mountain genus Hyponephele and
Karanasa was found to be 2.3% and 2.2%, respectively. The addition of
conspecific global barcodes revealed that most species showed little increase in
divergence value, while a two-fold increase was noted in a few species. Bayesian
clade clustering outcomes largely aligned with current morphological
classifications, forming monophyletic clades of conspecific barcodes, with only
minor exceptions observed for the taxonomically complicated genus
Polyommatus and misidentified records of
Aulocera in the database. We also observed variations
within the same phylogenetic clades forming nested lineages, which may be
attributed to the taxonomic intricacies present at the subspecies level
globally, mostly among Eurasian species. Overall, our effort not only
substantiated the effectiveness of DNA Barcoding for the identification and
conservation of this climatically vulnerable assemblage but also highlighted the
significance of deciphering the unique genetic composition among this
geographically isolated population of Ladakh butterflies.