Introduction
More than 5,800 animal species to date are endangered, as the Earth
experiences a mass extinction event (Ceballos et al., 2015).
Conservationists have multiple factors to consider in order to arrest
population declines. Extrinsic of population declines include habitat
loss and degradation, over-exploitation, emerging infectious diseases,
invasive species, and climate change (Butchart et al. 2010). Intrinsic
drivers of extinction, such as genetic factors, also play a key role for
population viability, especially when species are reduced to small,
isolated populations that can be negatively affected by genetic load
(Hedrick, 2001). In this scenario, finding a suitable mate is
challenging and reproduction with related individuals can occur, leading
to inbreeding depression. Inbreeding has largely been documented in
small populations in the wild (Hedrick, 2001; reviewed in Spurgin &
Gage, 2019), impacting individual and population fitness through the
fixation of detrimental alleles. An increase of detrimental alleles in
endangered species increases their susceptibility to extrinsic
ecological drivers of extinction (Frankham, 2005). One ex situconservation tool used to mitigate the decrease of genetic diversity in
endangered species are Captive Breeding Programs (CBPs). Captive
populations of endangered species have the difficult goal of ensuring
the survival of stable, self-sustaining populations for later
reintroduction into the native habitat (Mallinson, 1995). A key
challenge of CBPs is to maintain genetic diversity and avoid inbreeding
depression with a small number of founders (Bouman, 1977; Ralls et al.,
1979).
The majority of captive breeding schemes rely on studbooks that document
pedigree information within the CBPs. While studbooks can be useful to
minimize inbreeding effects (Pelletier et al., 2009; Witzenberger &
Hochkirch, 2011), information from pedigrees can be flawed in some
captive populations (Bowling et al., 2003; Marshall et al., 1999; Signer
et al., 1994). Molecular genetic analyses can provide more insights into
the relationships within captive populations and their genetic
structure. Recently, genetic studies of endangered species have
increased, using highly variable loci non-coding for fitness traits such
as microsatellite markers (Witzenberger & Hochkirch, 2011).
Microsatellites are known to be highly informative for small populations
that have reached bottleneck events and are considered a tool to measure
neutral genetic variation, and generally represent the extent and
pattern of molecular variation within a population (Selkoe & Toonen,
2006). However, both empirical and simulated data indicate that
patterns of variation and divergence in adaptive traits are not always
associated with concomitant variation in neutral markers (Hedrick, 2001;
Larson, 2012; Reed & Frankham, 2001), and some conservation biologists
advocate for genetic diversity analysis for adaptive variation in CBPs
(Hughes, 1991; Sommer, 2005). One targeted adaptive region is the Major
Histocompatibility Complex (MHC) because its genes play a crucial role
in the adaptive immune system. Historical events such as bottlenecks
and founder effects, but also constraints of the mating system, such as
limited sexual selection in CBPs (Schulte-Hostedde and Mastromonaco
2015), can be reflected in low numbers of MHC alleles (Schad et al.,
2004; Hapke et al., 2004). However, in some free-ranging populations,
genetic variation at the MHC might persist due to balancing selection,
through heterozygote and/or rare allele fitness advantage, despite low
levels of variability shown by neutral markers (Jarvi et al., 2004; Rico
et al., 2016). These studies support the importance of balancing
selection as a mechanism to maintain variation in small populations, and
the difficulty of using neutral markers as surrogates for variation in
fitness-related loci. Furthermore, MHC genes, particularly of class I,
contribute to participate in pheromone recognition, playing a role in
sexual selection as well (Penn, 2002).
MHC genes have a crucial role in adaptive immunity in jawed vertebrates,
by encoding proteins that bind peptide antigens and present them at the
cell surface to lymphocytes for their activation (Ujvari & Belov,
2011). T and B lymphocytes are known to interact directly with gut
microbial communities to prevent invasion, pathogenesis, and detrimental
immune response towards commensals (Ost & Round, 2018). T cells react
to foreign molecules via cell co-receptors: glycoproteins CD8 and CD4
encoded by MHC class II and class I genes, respectively (Penn & Potts
1999). MHC-I molecules are present on all host nucleated cells and
antigen presenting cells, and are known to act at the intracellular
level, while MHC-II molecules are strictly found on antigen presenting
cells and target extracellular microbes (Ost & Round, 2018). MHC
presentation of antigens to T cells is thus the basis for initiating
antigen specificity and, immune responses are considered specific to the
organisms they target.
MHC genes are considered one of the most diverse loci in jawed
vertebrates and good candidates for genetic diversity analysis in
endangered species (Hughes, 1991). High genetic diversity in these loci
could allow targeting more combinations of gut microbes, reflected in
variable immunity or tolerance among individuals through rare allele and
heterozygous advantage in balancing selection. Bolnick et al. (2014)
examined the role of MHC-II motifs (amino acid sequences) in gut
microbial community variation in sticklebacks (Gasterosteus
aculeatus ) and found that common MHC motifs were linked to increases in
microbial abundance and diversity, and rare motifs had the opposite
impacts. Similarly, the microbiota was less phylogenetically diverse in
individuals with high MHC-II diversity in the plumage of blue petrels
(Halobaena caerulea , Leclaire et al., 2018), the gut of
laboratory mouse strains (BALB/c, Khan et al., 2019), and the fur
microbiota of fur seals (Arctocephalus gazella , Grosser et al.,
2019). However, no study to date has investigated the MHC-gut microbiota
relationships in endangered species under CBPs.
While the host environment has a strong impact on its gut microbial
community, the genetics and biology of the host should also be taken
into account to fully understand the complex dynamics that occur in this
system (Koskella et al., 2017; Spor et al., 2011). This is especially
true when considering the gut microbiota of endangered species, where a
small number of founders in CBPs are likely to experience overall low
genetic variation and diversity. Transition from natural to captive
settings for breeding can become more challenging in these conditions
when considering host-associated microbes (Trevelline et al., 2019; West
et al., 2019). Within this context, Foster et al. (2017) proposed a
theoretical framework known as the leash model, which posits that the
host is under strong selection to evolve mechanisms to keep the
microbiota under control, or “on a leash”. The presence of a
genetically diverse microbiota leads to the dominance of the fastest
growing microbes instead of the microbes that are most beneficial to the
host (Foster et al., 2017). The targeting of microbial taxa to either
promote or limit their proliferation could thus be beneficial to the
host, through its adaptive immune response. We therefore hypothesize
that less host control, expressed by more genetically diverse gut
microbes, should happen in individuals with reduced genetic diversity in
both neutral and adaptive markers. To test this hypothesis, we
investigated the genetic diversity and gut microbial community
assemblages in the critically endangered European mink (Mustela
lutreola ).
The European mink (E-mink) is a semi-aquatic carnivore from theMustelidae family. Once widespread throughout Europe, it was
evaluated as “critically endangered” in 2011 (Maran et al., 2016).
There have been drastic declines in population and range, historically
due to overexploitation and still today driven by habitat loss,
degradation and fragmentation, road collisions, and the impacts of the
alien American mink (Mustela vison ). E-mink populations are now
restricted to enclaves in western France and northern Spain (referred as
the western population), the delta of the Danube in Romania, and Ukraine
and Russia (referred as the eastern population, Maran et al., 2016), the
latter being the focal origin of a captive breeding effort in Estonia
with successful reintroduced populations on Hiiumaa Island.
Two major studies have documented the genetic diversity of the
free-ranging E-mink populations (Michaux et al., 2005; Cabria et al.,
2015). The western population was characterized by a single
mitochondrial DNA haplotype against 17 haplotypes in the overall eastern
population. Western E-mink had also a much lower microsatellite genetic
diversity and allelic richness compared to the eastern population. The
authors concluded that the western free-ranging population reached a
recent bottleneck, and potentially inbreeding depression due to
geographic isolation. However, no proof of fitness reduction in this
population through inbreeding has been reported as of yet (Carbonell et
al., 2015). The antigen-binding site, encoded by exon 2 of the DRB MHC
class II gene, was also investigated in the eastern captive population
by Becker et al. (2009). They detected nine alleles within the 20
individuals investigated, estimating low to moderate variability when
comparing to other endangered species in similar situations to the
E-mink. However, no comparison is yet available for the captive western
population.
Both populations are currently in a CBP. The majority of the eastern
breeding stock is held at the Tallinn Zoo and extending to other zoos in
Europe such as Zoodyssée in France and is only composed of captive-born
individuals for over thirty of generations (Maran, pers. comm., 2021).
This stock is managed under an EAZA Ex situ Program (EEP). On the other
hand, the western breeding stock is held in facilities in Spain. Due to
the recent implementation of a CBP in Spain, most of the breeding stock
originates from the free-ranging western population captures in Spain
within the last seven years (i.e. seven generations), and wild-born
individuals from Spain are still being introduced as founders to this
date. Those populations are considered as two distinct stocks and are
bred separately, although few cross breeds are currently being conducted
in Spanish facilities and at the Tallinn Zoo.
Both captive E-mink populations therefore offer a range of variation in
neutral and adaptive genetic diversity. Due to extreme population
variation over time and the emergence of small and isolated populations,
the E-mink provides a unique framework to study the relationship between
host genetics and gut microbial communities. Following the ecosystem on
a leash model, the aims of this study were to (i) characterize the
genetic diversity in the two captive E-mink populations with neutral and
adaptive genetic markers as well as their gut microbial communities,
(ii) examine the relationship between gut microbial diversity and
genetic diversity, and (iii) investigate if gut microbial community
structure and composition is linked to specific MHC motifs.