Abstract

   Increasing human disturbance and climate change have a major impact on
   habitat integrity and size, with far‐reaching consequences for wild
   fauna and flora. Specifically, population decline and habitat
   fragmentation result in small, isolated populations. To what extend
   different endangered species can cope with small population size is
   still largely unknown. Studies on the genomic landscape of these
   species can shed light on past demographic dynamics and current genetic
   load, thereby also providing guidance for conservation programs. The
   pygmy hog (Porcula salvania) is the smallest and rarest wild pig in the
   world, with current estimation of only a few hundred living in the
   wild. Here, we analyzed whole‐genome sequencing data of six pygmy hogs,
   three from the wild and three from a captive population, along with 30
   pigs representing six other Suidae. First, we show that the pygmy hog
   had a very small population size with low genetic diversity over the
   course of the past ~1 million years. One indication of historical small
   effective population size is the absence of mitochondrial variation in
   the six sequenced individuals. Second, we evaluated the impact of
   historical demography. Runs of homozygosity (ROH) analysis suggests
   that the pygmy hog population has gone through past but not recent
   inbreeding. Also, the long‐term, extremely small population size may
   have led to the accumulation of harmful mutations suggesting that the
   accumulation of deleterious mutations is exceeding purifying selection
   in this species. Thus, care has to be taken in the conservation program
   to avoid or minimize the potential for further inbreeding depression,
   and guard against environmental changes in the future.

   Keywords: conservation genomics, deleterious variants, inbreeding,
   population genomics

1. INTRODUCTION

   During the last glacial maximum, the ranges of most temperate species
   shifted and shrunk as temperatures decreased (Davis & Shaw, [34]2001).
   During the Holocene, human populations expanded rapidly, and negatively
   affected biotic recoveries and natural range expansions through both
   hunting and land clearing (Ellis, [35]2015). Thus, the combined effects
   of climatic changes and human activities have reduced population sizes
   of many species throughout the world to a critically small size over
   the past 10,000 years (Miraldo et al., [36]2016; Pimm &
   Raven, [37]2017).

   Small, fragmented, and isolated populations lead to reduced genetic
   variation and increased inbreeding and genetic drift (Lynch
   et al., [38]1995, [39]2016). Inbreeding can have a negative effect on
   population viability through inbreeding depression, which is a
   consequence of an increase of harmful mutations in the homozygous state
   in inbred individuals (Kardos et al., [40]2017; Pekkala
   et al., [41]2012, [42]2014). In some populations, purging of harmful
   mutations can result in lower load. Purifying selection facilitated by
   inbreeding as it increases the homozygosity of partially recessive
   deleterious variants (Hedrick & Garcia‐Dorado, [43]2016). However, in
   extremely small populations, genetic drift tends to prevail over
   natural selection, limiting the potential for purifying selection
   against deleterious variation, and even allowing deleterious variants
   to increase in frequency (W. C. Funk et al., [44]2016; Lynch
   et al., [45]2016). Importantly, low levels of genetic variation are
   expected to reduce the opportunities for selection and to limit
   adaptive potential in populations that experience rapid environmental
   changes, for example, new diseases and climate fluctuation (Hamilton &
   Miller, [46]2016; Piertney & Oliver, [47]2006).

   Studies on demographic history and erosion of genomic variation of
   endangered populations can show the impact of losing genomic diversity
   and accumulation of genetic load. For instance, in the endangered
   Cheetah (Acinonyx jubatus) population, long‐term decline and subsequent
   bottlenecks have resulted in excessive deleterious mutations, reducing
   reproductive success (Dobrynin et al., [48]2015; Merola, [49]1994).
   However, not all populations with low genetic diversity suffer from
   inbreeding depression. Similar patterns of long‐term decline are
   apparent in the genomes of island foxes, which resulted in extensive
   runs of homozygosity and increased genetic load. Yet, the lack of
   apparent phenotypical defects suggests that deleterious variants were
   purged from the island fox population in parallel with further
   adaptation to the local environment (Robinson et al., [50]2016,
   [51]2018). It is, therefore, important to understand demographic
   history as well as temporal changes in mutational load in small,
   fragmented populations in order to predict the impact of inbreeding and
   increase the chances of long‐term population persistence.

   The pygmy hog (Porcula salvania) is the smallest and the rarest wild
   suid in the world, and so far known as the sole living representative
   of the genus Porcula. The pygmy hog has been classified as a critically
   endangered species by the International Union of Conservation of Nature
   (IUCN) since 2008. The pygmy hog is confined to the tall grass savanna
   of the Himalayan foothills. Since the early 20th century, human
   settlement and agriculture led to accelerated fragmentation and loss of
   pygmy hog habitat (Peet et al., [52]1999). The pygmy hog was believed
   to be extinct in most of its natural range in the Terai and Duars
   region (Oliver & Deb Roy, [53]1993) until they were rediscovered in
   1971. Currently, only one viable wild population remains, in Manas
   National Park, northern Assam, India. Considering its critical status
   and the unique habitat it lives in, a recovery program for this
   species, the Pygmy Hog Conservation Programme (PHCP), was initiated in
   1995 (PHCP, [54]2008). Starting with six wild‐caught hogs, the breeding
   program exceeded early expectations. The captive population is now
   around 80 (Huffman, [55]2016). Although the PHCP has benefited from
   several decades of planned breeding and pedigree management, so far
   there has been no information on the genetic diversity in the
   individuals that were used to establish the breeding program. This
   information is essential to inform the breeding program to prevent
   inbreeding issues.

   It is still largely unknown whether the small population has
   experienced purifying selection of harmful mutations and whether
   current inbreeding leads to inbreeding depression in this population.
   To infer their demographic history, and eventual inbreeding concerns,
   we studied whole‐genome data of six pygmy hogs: three from the wild and
   three from the breeding program. By comparing the pygmy hog information
   with 30 pigs belonging to six other old‐world pig species
   (Table [56]S1), we interpreted our findings in the context of these
   other pig species, whose demographic history has been well studied. For
   example, we included the critically endangered Javan warty pig (Sus
   verrucosus), which is highly inbred due to recent zoo management
   (Semiadi & Meijaard, [57]2006). We also include much more widespread
   species, such as the European wild boar (Sus scrofa), which have
   experienced profound population bottlenecks due to glaciations and,
   historically, hunting and habitat loss (Groenen et al., [58]2012).

   In this study, we aim at using a comparative genomics approach to infer
   past population dynamics and assess the consequences of severe
   population decline. Our results provide a detailed genomic estimation
   of the pygmy hog's population history, genomic diversity, inbreeding
   status, and genetic load. These results provide a strong foundation in
   evaluating the conservation status of the pygmy hog and highlighting
   the importance of genomic monitoring in population management of pygmy
   hogs and other endangered species, both in situ and ex situ.

2. MATERIALS AND METHODS

2.1. Whole‐genome resequencing, variant calling, and filtering

   The pygmy hog samples used for this research are derived from three
   wild and three captive individuals. On these samples, whole‐genome
   Illumina PE 100 bp resequencing was performed at SciGenom Laboratories
   in Chennai, India. A selection of other Suidae species was included
   (Table [59]S1). All these samples were also sequenced with the Illumina
   sequence technology. The whole‐genome sequencing data were trimmed
   using sickle (Version 1.33, [60]https://github.com/najoshi/sickle) with
   default parameters. The trimmed reads were aligned to the Sscrofa 11.1
   reference genome. Since there are multiple closely related species to
   the reference species, we used the unique alignment option of MOSAIK
   aligner (Version 2.2.30) (Lee et al., [61]2014) to increase mapping
   accuracy (Pightling et al., [62]2014). Local re‐alignment was performed
   using GATK (Version 3.7) RealignmentTargetCreator and IndelRealigner
   and variants were called using GATK UnifiedGenotyper (McKenna
   et al., [63]2010), with the –stand_call_conf option set to 50, the
   –stand_emit_conf option set to 20, and the ‐dcov option set to 200.
   Variants with a read depth between 0.5 and 2.0 times of the average
   sample genome coverage were selected and stored in variant calling
   format (Table [64]S1).

2.2. Mitochondrial genome assembly and analysis

   As no pygmy hog mitochondrial sequence was available, we reconstructed
   one, using the short‐read data from the high‐coverage individual
   (Table [65]S1). We assembled the mitochondrial genome through iterative
   mapping using MITObim v1.8 (Hahn et al., [66]2013) on 100 million
   trimmed and merged reads, subsampled using seqtk (version 1.3 r106),
   [67]https://github.com/lh3/seqtk. Mitochondrial reconstruction was
   performed in three independent runs using three different starting bait
   reference sequences. The references included the domestic pig