Abstract

   The Himalayan Arc is recognized as a global biodiversity hotspot. Among
   its numerous cryptic and undiscovered organisms, this composite
   high-mountain ecosystem harbors many taxa with adaptations to life in
   high elevations. However, evolutionary patterns and genomic features
   have been relatively rarely studied in Himalayan vertebrates. Here, we
   provide the first well-annotated transcriptome of a Greater Himalayan
   reptile species, the Ladakh Ground skink Asymblepharus ladacensis
   (Squamata: Scincidae). Based on tissues from the brain, an embryonic
   disc, and pooled organ material, using pair-end Illumina NextSeq 500
   RNAseq, we assembled ~77,000 transcripts, which were annotated using
   seven functional databases. We tested ~1600 genes, known to be under
   positive selection in anurans and reptiles adapted to high elevations,
   and potentially detected positive selection for 114 of these genes in
   Asymblepharus. Even though the strength of these results is limited due
   to the single-animal approach, our transcriptome resource may be
   valuable data for further studies on squamate reptile evolution in the
   Himalayas as a hotspot of biodiversity.

   Keywords: adaptation, evolution, genomic, high elevation, Himalayas,
   Scincidae

1. Introduction

   The Himalayan arc represents one of the world’s most important faunal
   and floral hotspots with high species diversity and endemism [[30]1],
   which result from the Tertiary orogeny of this mountain chain, its
   complex topography as well as its great climatic heterogeneity and
   isolation. The genesis of the Tibetan highlands and the Himalayas since
   the Paleogene, with the Greater Himalayas starting to rise presumably
   the earliest in the post-Eocene (for a review, see the supplementary in
   Hofmann et al. [[31]2]), triggered the evolution of unique biodiversity
   under gradual high-altitude adaptation, as already shown for anurans
   [[32]3,[33]4,[34]5,[35]6,[36]7]. Besides amphibians, there are also
   several reptiles that can cope with life at high altitude in those
   regions, e.g., Thermophis [[37]8], Phrynocephalus [[38]9], and some
   Laudakia species [[39]10]. Potential constraints to upslope migration
   of reptiles (and amphibians) to high-elevation environments are the
   substantial UV-radiation, the thermal extremes, and especially the
   oxidative stress, referred to as high-altitude hypoxia, which interacts
   with temperature in a context-dependent manner to influence thermal
   performance and limits in terrestrial ectotherms [[40]11,[41]12].
   Recent advances in high-throughput sequencing technologies have led to
   a growing number of genomic studies that address the molecular basis of
   high-altitude adaptation, some of them focused also on reptiles
   [[42]13,[43]14,[44]15]. However, such data have been scarce in
   non-model species of the Greater Himalayas (but see [[45]16]). This
   results from the general understudied biodiversity of this
   high-mountain range, presuming a relatively large number of cryptic and
   undiscovered species [[46]17], even among vertebrates. Molecular data
   from Himalayan organisms can contribute to understanding of the
   taxonomic and functional diversity spectra across this species-rich,
   fragile ecosystem. These data resources are even more important because
   Himalayan biodiversity is threatened at the very core; rapid warming
   due to climate change, especially at higher elevations, as well as
   higher rates of forest degradation and deforestation, infrastructural
   development, trade routes, and hydropower dams are driving species loss
   at a very alarming speed [[47]18,[48]19]. To allow future studies in
   evolutionary biology at a genomic level and to generally provide
   sufficient and relevant data for Himalayan reptiles, in the present
   study, we have generated a new genomics data set based on RNAseq for a
   scincid species from the Greater Himalayas. Using these data, we
   specifically aimed to identify genes known to play roles in adaptation
   of terrestrial ectothermic vertebrates to high elevations. Since
   exposure to oxidative stress can particularly affect the physiology
   during early development [[49]20] and in oxygen-sensitive organs
   [[50]21,[51]22], such as the nerve system, we focused on embryonic and
   brain tissue samples.

   Our target species is a scincid lizard in the genus Asymblepharus, the
   Ladakh Ground Skink, A. ladacensis (GÜNTHER, 1864), which is endemic to
   the western part of the Himalayas. The genus further contains the
   following species ([52]Figure 1): A. alaicus (ELPATJEVSKY, 1901), A.
   eremchenkoi PANFILOV, 1999, A. himalayanus (GÜNTHER, 1864), A.
   mahabharatus EREMCHENKO, SHAH & PANFILOV, 1998, A. nepalensis
   EREMCHENKO & HELFENBERGER, 1998, and A. tragbulensis (ALCOCK, 1898).
   Another two species, Asymblepharus medogensis JIANG, WU, GUO, LI & CHE,
   2020 and A. nyingchiensis JIANG, WU, WANG, DING & CHE, 2020, have been
   described very recently from Mêdog, Nyingchi in SE Tibet, China.
   According to a large-scale phylogenetic study of squamates [[53]23],
   the sampled specimen of A. sikimmensis (BLYTH, 1854) is nested within
   Scincella and was therefore suggested to be transferred to this genus.
   However, it remains unclear whether this single specimen had been
   misidentified as A. sikimmensis since originally it was labeled in the
   museum collection as Scincella potanini (voucher catalogue number
   CAS:HERP:194923, see
   [54]http://portal.vertnet.org/o/cas/herp?id=urn-catalog-cas-herp-194923
   (accessed on 29 July 2021) [[55]24].

Figure 1.

   [56]Figure 1
   [57]Open in a new tab

   Map of Asymblepharus species based on GBIF ([58]www.gbif.org; accessed
   on 20 July 2021) records of preserved specimens and georeferenced
   localities in the taxonomic reptile database
   ([59]https://reptile-database.reptarium.cz/; accessed on 20 July 2021).
   The location of our RNA sample of the female A. ladacensis (photo) is
   indicated by a green circle with a dot in the middle and an arrow. *
   Note, according to a large-scale phylogeny of squamates, A. sikimmensis
   is nested within Scincella; however, it remains unclear whether this
   single “A. sikimmensis” specimen, on which the sequence data are based,
   had been taxonomically correctly identified. Therefore, we also show
   the GBIF records of specimens collected as A. sikimmensis. Records of
   A. eremchenkoi in the databases could not be georeferenced due to
   insufficient information on the collection site. Photo credit: Sylvia
   Hofmann.

   In general, Asymblepharus is a genus with a still poorly known endemic
   distribution, origin, and evolutionary history. No studies of its
   population genetic structure and genetic diversity exist to date for
   any Asymblepharus taxon, and the current taxonomic relationships of its
   lineages are in flux [[60]23,[61]24,[62]25]. The three Himalayan
   species A. himalayanus, A. ladacensis, and A. tragbulensis have
   frequently been assigned to the genus Himalblepharus Eremchenko, 1987.
   According to literature data ([[63]26] and references therein) and