Challenges of next‐generation sequencing in conservation management: Insights from long‐term monitoring of corridor effects on the genetic diversity of mouse lemurs in a fragmented landscape

Abstract

Long‐term genetic monitoring of populations is essential for efforts aimed at preserving genetic diversity of endangered species. Here, we employ a framework of long‐term genetic monitoring to evaluate the effects of fragmentation and the effectiveness of the establishment of corridors in restoring population connectivity and genetic diversity of mouse lemurs Microcebus ganzhorni . To this end, we supplement estimates of neutral genetic diversity with the assessment of adaptive genetic variability of the major histocompatibility complex (MHC). In addition, we address the challenges of long‐term genetic monitoring of functional diversity by comparing the genotyping performance and estimates of MHC variability generated by single‐stranded conformation polymorphism (SSCP)/Sanger sequencing with those obtained by high‐throughput sequencing (next‐generation sequencing [NGS], Illumina), an issue that is particularly relevant when previous work serves as a baseline for planning management strategies that aim to ensure the viability of a population. We report that SSCP greatly underestimates individual diversity and that discrepancies in estimates of MHC diversity attributable to the comparisons of traditional and NGS genotyping techniques can influence the conclusions drawn from conservation management scenarios. Evidence of migration among fragments in Mandena suggests that mouse lemurs are robust to the process of fragmentation and that the effect of corridors is masked by ongoing gene flow. Nonetheless, results based on a larger number of shared private alleles at neutral loci between fragment pairs found after the establishment of corridors in Mandena suggest that gene flow is augmented as a result of enhanced connectivity. Our data point out that despite low effective population size, M. ganzhorni maintains high individual heterozygosity at neutral loci and at MHC II DRB gene and that selection plays a predominant role in maintaining MHC diversity. These findings highlight the importance of long‐term genetic monitoring in order to disentangle between the processes of drift and selection maintaining adaptive genetic diversity in small populations.

MHC Genotyping by SSCP and Amplicon-Based NGS Approach in Chamois

“…The variety of artefacts that occur during the PCR step can far exceed the number of variants corresponding to true alleles, and the total number of sequence reads derived from artefacts can be higher than those from true alleles [33]. PCR may also be responsible for the allelic dropout caused by differences in amplification efficiency between alleles, especially in the SSCP/Sanger methodology [34]. Allelic dropouts and false alleles can cause important genotyping errors and thus significantly alter downstream analyses based on heterozygosity, allele diversity, and genotype composition, which has a direct implication in population genetics.…”

Trends in forest fragment research in Madagascar: Documented responses by lemurs and other taxa

“…Five species living in fragmented landscapes displayed evidence of genetic bottlenecks within their studied range (white‐collared lemur, Eulemur cinereiceps : Brenneman et al, 2012; L. edwardsi : Craul et al, 2009; V. variegata : Holmes et al, 2013; Perrier's sifaka, P. perrieri , and Tattersall's sifaka, P. tattersalli : Salmona et al, 2017). Evidence for contemporary gene flow between fragments was also reported for Microcebus ganzhorni , indicating the species’ ability to access the matrix (Montero et al, 2019). Relatively high levels of genetic diversity or lack of signals of genetic differentiation between sites were also reported for three other species, with authors suggesting that individuals are still able to migrate between sites ( P. perrieri : Salmona et al, 2014; P. tattersalli : e.g., Quéméré, Louis, Ribéron, Chikhi, & Crouau‐Roy, 2010) or that fragmentation has happened relatively recently (northern rufous mouse lemur, Microcebus tavaratra : Aleixo‐Pais et al, 2019) and genetic responses have not yet had time to manifest (Quéméré, Crouau‐Roy, Rabarivola, Louis, & Chikhi, 2010).…”

“…For LA, other tests of fragmentation employed included the impact of landscape changes over time (percent forest loss: Marrocoli, Hamann, & Allison, 2013; pre‐ and with forest corridor: Montero et al, 2019), percent canopy cover (e.g., Raharivololona & Ganzhorn, 2009), and qualitative assessments of the extent of fragmentation (e.g., Brenneman et al, 2012; Campera et al, 2014), among others (Supporting Information Appendix S3). Two LA examined fragmentation per se: Salmona et al (2017) by a population genetics analysis determining whether habitat loss, fragmentation, or a combination best explained the demographic history of two sifaka species ( P. tattersalli and Propithecus perrieri ) and Steffens and Lehman (2018) by the impact of the simulated removal of fragments or alteration of interfragment distance on lemur species in Ankarafantsika National Park, northwestern Madagascar.…”

Social and paternal female choice for male MHC genes in golden snub‐nosed monkeys (Rhinopithecus roxellana)

“…Genotyping of two DRB loci were conducted using amplicon-based next-generation sequencing, which is more effective and has fewer missing sequences than traditional sequencing methods for multiloci amplifications (Montero et al, 2018;Santos et al, 2017;Sommer et al, 2013). Genotyping of DQA1 and DQB1 was achieved by cloning and sequencing of 10 positive clones per individual.…”