Conservation Genetics

The European flat oyster (Ostrea edulis), like numerous other oyster species, has been exploited for millennia and cultivated and translocated for centuries. Following a severe population decline, and in the context of ongoing conservation and restoration programs, genetic considerations must now be addressed to avoid mistakes. The objective of our study was to complement population genetic studies conducted at various scales along European coasts. Our sampling primarily targeted the French Atlantic, English Channel, and Mediterranean coasts, aiming to provide a fine-scale genetic characterization of populations in these regions. By integrating SNP array and low-coverage sequencing datasets, we obtained a comprehensive overview of the population genetic structure of Ostrea edulis across western Europe. Most previously identified clusters in Western Europe were confirmed. In France, populations assigned to these clusters exhibited notable within-patch homogeneity. However, two key findings emerged: (1) an extensive overlap zone between the Atlantic and western Mediterranean clusters, spanning at least from southern Portugal to southern France, and (2) the detection of a novel, clearly distinct cryptic cluster east of the English Channel, whose geographic range remains to be better delineated. These insights are critical for informing management decisions, particularly as restoration and conservation plans are currently being implemented across the species range.

EU has reclassified the sika deer (Cervus nippon) as an undesirable invasive species based on reports that hybridization with the indigenous red deer (C. elaphus) may produce fertile offspring. Since sika-derived DNA previosuly introduced into the red deer population (introgression) cannot be removed, the crucial question is whether new (F1) hybridisation occur. To address this, we analysed the chromosomes in 56 sika and 22 red deer. All red deer had a chromosome number 2n=68. In contrast, the chromosome number in sika ranged from 64 to 67, due to the variable presence of two sika-specific Robertsonian translocations (ROB1,ROB2). In the free-ranging sika population in Jutland, >90% of the sika deer were homozygote for at least one of these ROBs, excluding that they could be F1-hybrids. Moreover, ROB2 was in Hardy-Weinberg equilibrium, further supporting the absence of gene flow between the two species. In contrast, ROB1 was in Hardy-Weinberg disequilibrium, suggesting negative fitness of heterozygotes, including potential F1-hybrids. In Jaegersborg Deer Park, the eight examined sika deer had the same genotype (absence of ROB1, homozygosity of ROB2), supporting that it is a founder population which may have been isolated for [~]100 years. Again, none of these can be F1-hybrids due to the homozygosity of ROB2. We conclude that F1-hybridisation between sika and red deer either does not occur or occur very rarely in Denmark. The study establish the Danish sika-populations as unique models for adressing important biological questions: What underlies the absence of hybridisation? Why are ROBs frequent in sika deer but not in the closely related red deer? How fast do new species/subspecies develop in isolated founder populations? Which factors determine, that some ROBs have little heterozygous effects, whereas others are selected against, with implications for the role of ROBs as genetic barriers promoting speciation, and for fertility problems in some human ROB carriers.

Urbanization has been previously shown to affect evolution of plants and animals by restricting gene flow, increasing genetic drift, and causing divergent selection between rural and urban populations, however, the effect of urbanization on terrestrial gastropod evolution has only been minimally researched. Most research on terrestrial gastropods primarily focusing on invasive pest species, and little attention is given to native species like Ariolimax columbianus (Pacific banana slug). There is no current published research on the phylogeography of A. columbianus. In this study, surface epithelial cells from 66 Ariolimax columbianus individuals were collected from 11 locations around Corvallis, Oregon, and one site in northern California. These samples were amplified and sequenced at two loci: cytochrome oxidase 1 (CO1) and the internal transcribed spacer 2 (ITS-2). This data was used to generate maximum-likelihood phylogenetic trees to better understand the population genetics of these local native gastropods. The CO1 phylogenetic analysis showed two major haplotypes around the city of Corvallis - northwestern and southern, while the ITS-2 phylogenetic analysis showed three general haplotypes around the city - northern, western, and southern. The results in this study show the presence of genetic breaks between many of these populations, possibly due to a combination of factors including anthropogenic fragmentation, natural barriers, and the low active dispersal ability of A. columbianus. However, the extent that each of those factors has on disrupting gene flow and explaining patterns of genetic variation in this species requires further research. The present study acts as a foundation for the development of future research on banana slug phylogenetics and population genetic structure.

A core aim of ex situ conservation is to represent wild genetic diversity in managed living collections. For the climate-threatened tropical montane cloud forest (TMCF) flora of northeast Australia, an ex situ metacollection of plants and seeds has been established by the Tropical Mountain Plant Science (TroMPS) project. In this study we used reduced-representation sequencing (DArTseq) of wild, herbarium, and ex situ material alongside provenance information for ten species, to pursue two central aims: to characterise landscape-scale genetic structure across species' ranges, and to evaluate how well the assembled metacollections represent that wild diversity. Analyses revealed consistent patterns of genetic differentiation among mountain top populations across multiple species, reflecting the isolating influence of lowland gaps between upland habitats, with the degree of differentiation varying among species. These results provide the first genetic baseline for Australian TMCF flora and reinforce the importance of treating individual mountain top populations as distinct units for conservation management. Additionally, the project provided valuable insights into the logistical challenges of coordinated multi-institutional collecting, informing strategies for metacollection design more broadly. Evaluation of the metacollection revealed both strengths and gaps in representation across species, providing an evidence base to refine the current holdings and guide future targeted collecting to strengthen their long-term conservation value.

Rosa gallica L., the French rose, is a perennial, tetraploid, heterozygous species that naturally propagates by seed and sucker. It occurs in the wild, primarily in Europe, and also exists as cultivated varieties. R. gallica cultivars were extensively bred and cultivated in France at the beginning of the 19th century. Although several hypotheses have been proposed regarding the species expansion based on historical records, none have been assessed using molecular data. Indeed, its genetic diversity has so far been investigated only at local or regional scales, hindering the identification of the evolutionary factors shaping its present-day distribution. Using 29 sequenced microsatellites, we genotyped a comprehensive sample of 1618 individuals, including wild R. gallica from 219 sites across the species range, rose cultivars, and specimens from other Rosa species. We then detected clonal lineages and characterized the range-wide genetic diversity and structure, aiming to disentangle the roles of natural and human factors in shaping the distribution of R. gallica, with particular focus on France. French diversity appears particularly structured compared to the rest of the range, suggesting multiple origins within France. Populations in South Alps, Central Eastern Europe, and Eastern France appear to have recolonized naturally from a single southern glacial refugium. In contrast, populations in the western part of France likely resulted from more recent natural or human-mediated dispersal. Finally, clonal lineages containing both wild and cultivated individuals were predominantly found in France, highlighting the role of human-mediated dispersal in 28 of the 98 French sites studied. These findings show that the present-day natural range of R. gallica was shaped primarily by post-glacial recolonization, but also reveal a contribution of human activities to its recent dispersal, particularly in France, where cultivated varieties were intensively bred and exchanged.

A central goal when assessing patterns of population structure for conservation is to identify populations with unique genetic compositions. The use of genomic sequencing to identify distinct populations has become an increasingly popular method of delineating conservation units. Reduced costs associated with sequencing make it possible to generate larger, more informative datasets to assess genetic diversity within and among populations. In species that exhibit nest-site philopatry, genetic population structure can emerge on much finer scales, particularly in maternally inherited mitochondrial genomes. We demonstrate the feasibility and insight gained by using whole mitochondrial genome sequencing for evaluating population genetic structure and comparing to previous single marker studies in a vulnerable turtle. We used whole mitochondrial genome sequences from diamond-backed terrapin (Malaclemys terrapin) to evaluate whether nest-site philopatry generates fine-scale genetic structure among M. terrapin nesting beaches in western Mobile Bay (Alabama, USA). We then compared haplotype diversity between the Alabama population and M. terrapin populations from the Atlantic and Gulf coasts and evaluated the utility of using whole mitochondrial genomes rather than a subset of loci to characterize unique haplotypic diversity. We found no genetic structure associated with nest-site philopatry within Alabama, but none of the haplotypes in this region were shared with other Gulf Coast sites. This genetic structure is consistent with strong female natal philopatry within western Mobile Bay relative to the Gulf of Mexico and suggests that the Mobile Bay population is genetically unique relative to other M. terrapin populations and merits a unique conservation and management plan.

Understanding how contemporary population declines affect the genomic diversity and structure of threatened species is important for effective conservation. The Eurasian curlew (Numenius arquata) is experiencing severe population declines across Europe, with Ireland among the most extreme, showing declines exceeding 90% over 40 years. Genomic data are increasingly incorporated into policy and used to assess conservation status by estimating genetic diversity, differentiation, inbreeding, effective population size, and adaptive divergence. Such data for curlew is scarce, and the population structure among northern and north-western European breeding populations remains unclear. To address this, we generated whole-genome resequencing data for 56 curlews across Ireland, Britain and Sweden. Irish and British populations showed minimal interpopulation differentiation, but both were substantially differentiated from Sweden. This was apparent from principal component analysis, and admixture and FST analyses. Measures of genetic diversity (nucleotide diversity, heterozygosity, Watterson's{theta} ) were similar across populations. A slightly elevated Tajima's D in Ireland, along with elevated FROH in Ireland and Britain relative to Sweden, may be the early genomic signs of recent population declines. We identified locally selected candidate genes. These had putative roles in metabolic processes, the immune response, and were potentially associated with distinct migratory behaviours and environmental conditions. We find a potential lag in genomic effects of decline being detectable following population contraction. We also show highly migratory species can exhibit differentiation in ecologically relevant traits, potentially driven by high site fidelity. These findings warrant consideration in translocation planning and broader conservation strategies.

Color polymorphism can facilitate local adaptation, maintain intraspecific diversity, or reflect early stages of speciation. Clownfishes (Amphiprion spp.) typically display a simple black, orange, and white pattern, but the saddleback clownfish (Amphiprion polymnus) shows striking variation in melanism and the number of vertical bars, which are thought to play a role in species recognition. In 2024, a revision on iNaturalist split A. polymnus into multiple species based solely on color pattern and geographic range. This raises the question of whether these morphs represent true species or intraspecific polymorphism, which we tested using genomic and image-based data. We sampled 97 individuals from seven populations across the species range and quantified color patterns from standardized photographs. Phenotypic and genomic analyses reveal a complex pattern of divergence. Image analysis identified three distinct phenotypic clusters, with A. polymnus, A. annamensis, and A. laticlavius each showing consistent differences in saddle shape and vertical bar extent. ADMIXTURE resolved three distinct genetic groups corresponding to the morphs. Pairwise FST (0.54-0.71) and dxy indicate extremely high differentiation between A. polymnus and A. annamensis, consistent with species-level divergence, whereas A. laticlavius shows much lower differentiation from A. polymnus (FST 0.09-0.18) and higher differentiation from A. annamensis (FST 0.64-0.66). Overall, phenotypic and genomic data show structured variation, but the status of A. laticlavius remains ambiguous. Our study reveals clear and structured divergence across the full range, yet the taxonomic interpretation of this variation remains inherently challenging. The key question remains: do these patterns reflect a single polymorphic species or a complex of closely related species?

Due to the sparse and uneven availability of genomic resources, it remains challenging to appraise genomic attributes for species of conservation concern. While long-read sequencing enables assessment of genetic diversity at unprecedented scale, accessing high-quality tissues remains a challenge for non-model species. Here, we explore an alternative sampling strategy for tissues where "gold-standard" cryopreservation is infeasible. Focusing on the Australian scincid lizard Cryptoblepharus pulcher, we compare DNA obtained from various ethanol-preserved tissue types and DNA extraction kits, and ask whether high-molecular weight DNA can still be retrieved. PacBio HiFi sequencing of the most promising sample yielded a highly contiguous reference-level assembly, validating this approach in vertebrates, specifically lizards. After scaffolding the assembly to chromosome-level, we then used a comparative approach to shed light on the evolution and demise of C. egeriae, a closely related, now Extinct-in-the-Wild species. Surprisingly, despite being a wide-spread continental analogue with a similar ecology, C. pulcher has lower genetic diversity and long-term historical population size than C. egeriae, an island endemic. However, C. pulcher also shows fewer runs-of-homozygosity, supporting prior reports that C. egeriae experienced recent inbreeding. Together, these findings demonstrate that a practical and cost-effective preservation strategy can still yield high-quality genomic resources in vertebrates, as well as valuable insights that are relevant in an age of biodiversity decline.

The sperm whale is an emblematic species of the Azores archipelago. After a century of intensive whaling, the species has become a focal point for ecotourism since the late 1980s, enabling collaborative research during tourism activities. This study assessed the feasibility of using a citizen science based non-invasive sampling approach to evaluate the genetic diversity of local sperm whales. Seven local whale-watching companies contributed to collect 70 fecal samples and 34 sloughed skin fragments between 2019 and 2024. A 588-bp fragment of mitochondrial DNA Control Region (MCR) was successfully sequenced from 56 fecal samples and all skin fragments, yielding 90 MCR sequences assigned to 79 individual sperm whales. MCR sequences from both sources of the same individuals were in perfect agreement, validating the use of fecal samples for mitochondrial haplotype analysis - the first study to do so in sperm whales. Four MCR haplotypes, including a newly determined one, evidenced different maternal lineages within the Macaronesia population. Haplotypes showed a clear geographic pattern around Sao Miguel Island: one was restricted almost exclusively to the south coast, while the other three occurred predominantly on the north. This contrasted distribution is likely correlated to different matrilineal groups.

Genomic health estimates help inform conservation and management decisions, with genetic load and runs-of-homozygosity (ROH) being two key metrics. White-tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) are found throughout North America, with some populations declining or of conversation concern. Using genome-wide data from samples across their range, we provide the first estimate of genetic load in mule deer, and revisit ROH estimates using model-based approaches. These updated estimates of ROH notable showed elevated inbreeding in the Key deer, consistent with current conservation designations. We also detected a relatively high number loss-of function mutations in mule deer that we attributed to historical bottlenecks. Notably, we observed an increased overall genetic load in O. hemionus from the Pacific Northwest.

BackgroundAedes albopictus is among the worlds most invasive mosquito species and an important vector of dengue, Zika, and chikungunya viruses. Its global spread has been strongly associated with human-mediated transport and international trade, particularly through commodities such as used tires and ornamental plants. However, integrative studies combining population genetics, microbial symbiosis, and trade connectivity remain limited in Latin America, constraining understanding of invasion dynamics and dispersal processes. MethodsAedes albopictus populations from five Colombian departments sampled between 2019 and 2023 were analyzed using eight microsatellite loci and a [~]1.3-kb mitochondrial COI fragment. Wolbachia infection and lineage composition (wAlbA/wAlbB) were evaluated by PCR, and arbovirus detection (DENV, CHIKV, ZIKV) was performed using multiplex RT-PCR. Nuclear and mitochondrial differentiation (FST, {Phi}ST), mito-nuclear discordance, and trade-related connectivity were evaluated in relation to geographic, national transport, and international trade variables derived from customs databases. ResultsMicrosatellite analyses revealed admixed but structured populations consistent with multiple introductions and contemporary gene flow. Colombian populations showed nuclear genetic affinities with Asian, European, and North American populations, supporting a complex invasion history involving multiple geographically distributed lineages. In contrast, mitochondrial COI data showed evidence of regional genetic structure and heterogeneous mito-nuclear discordance among several population pairs. Notably, nuclear and mitochondrial markers captured contrasting dimensions of the invasion process: nuclear differentiation was positively associated with international trade intensity, particularly shipment frequency and used tire importation, whereas mitochondrial differentiation retained stronger regional structure and showed no detectable association with trade-related variables. Wolbachia prevalence ranged from 34% to 100% across departments and showed exploratory patterns consistent with localized mitochondrial differentiation. Natural detection of DENV and CHIKV RNA in larvae provided evidence of local arbovirus circulation. ConclusionsIntegrating population genetics, trade connectivity, and Wolbachia screening supports a scenario in which the Colombian invasion of Ae. albopictus has been shaped by multiple introductions, contemporary human-mediated connectivity, and partially discordant mito-nuclear histories. These findings highlight how different genomic compartments retain complementary signatures of invasion dynamics, with contemporary trade-associated connectivity primarily reflected in nuclear structure and regional lineage persistence retained in mitochondrial variation. Author SummaryThe Asian tiger mosquito, Aedes albopictus, is one of the worlds most invasive mosquito vectors and continues to expand across Latin America through human transportation and trade networks. However, the processes shaping its spread in the region remain poorly understood. We combined population genetics, international trade data, Wolbachia screening, and arbovirus surveillance to investigate the invasion dynamics of Ae. albopictus in Colombia. Our results revealed evidence of multiple introductions and ongoing genetic admixture, with international trade connectivity emerging as an important predictor of contemporary nuclear genetic structure. In contrast, mitochondrial DNA retained stronger regional patterns, generating heterogeneous mito-nuclear discordance among populations. These findings suggest that different genomic compartments retain distinct signatures of the invasion process, with trade-associated connectivity reflected primarily in nuclear variation and stronger regional structure preserved in mitochondrial lineages. More broadly, our study highlights the complex invasion dynamics of Ae. albopictus in Latin America illustrates how integrating genetics and human connectivity data can improve understanding of invasive vector spread.

Hybridization is widespread among marine fishes and can contribute to adaptation, diversification, and the generation of novel phenotypes. In clownfishes, only two wild hybrids Amphiprion leucokranos and A. thiellei have been described, yet those naturally occurring clownfish hybrids remain rarely documented. Both hybrids involve crosses with A. sandaracinos. During field surveys in the Philippines, we identified a previously undocumented clownfish individual with an unusual phenotype resembling both A. sandaracinos and A. perideraion. To characterize its origin, we combined genomic, mitochondrial, and phenotypic comparisons to previously described clownfish hybrid systems. Genome-wide PCA and admixture analyses supported mixed ancestry between A. sandaracinos and A. perideraion. Reconstruction of the mitochondrial genome placed the individual within the A. sandaracinos mitochondrial lineage. Together, these results support a hybrid origin and suggest predominant A. sandaracinos ancestry, consistent with a backcrossed descendant rather than a first-generation hybrid. Comparisons with the previously characterized hybrid A. leucokranos further revealed similarities in genomic composition and phenotype across independently derived clownfish hybrid systems. Our findings identify a previously undocumented natural clownfish hybrid and suggest that integrating genomic and field-based approaches may reveal additional cryptic hybrid systems and improve understanding of hybridization in clownfishes.

Among Madagascar primates, the sportive lemurs (family Lepilemuridae) have seen their species diversity increase from eight in 2005 to 26 in 2009 mostly by applying the phylogenetic species concept to DNA barcode data. Despite the genus being speciose, only one case of sympatry is known from northern Madagascar, where two sportive lemur species described based on low mtDNA divergence, Lepilemur ankaranensis and Lepilemur milanoii, were found to co-occur at the center of their joint distribution range. Here, to clarify the taxonomy of these two species and examine their sympatry, we apply an integrative taxonomic framework to genomic and morphological data from 84 individuals of L. ankaranensis and L. milanoii, encompassing their entire distribution range and the forest of Analafiana, beyond their southernmost limit. Using clustering, multivariate, and isolation by distance analyses, we find no evidence of a sympatric zone and show that despite clear genetic differentiation between regions, the genomic and morphological diversity of the L. ankaranensis, L. milanoii-Analafiana group is clinal and explained by geographic distance. These results clarify that L. milanoii is a junior synonym of L. ankaranensis and that the Analafiana forest population belongs to L. ankaranensis, extending its distribution. It further implies that the 'sympatric' zone, the Andrafiamena forest, hosts conspecific individuals with slightly differentiated mtDNA backgrounds, rather than slightly differentiated sympatric species. Lastly, we re-evaluate the IUCN conservation metrics of L. ankaranensis, which continue to qualify as Endangered (EN) under the B1ab(i-v) criteria.

The Hesperiine genus Atrytonopsis Godman, 1900, occurs broadly across the American Southwest. Atrytonopsis margarita (Skinner, 1913) and Atrytonopsis python (W. H. Edwards, 1882) have look-alike appearances, concurrent flights, and geographic distributions which converge in New Mexico. Their similar wing markings and intertwined taxonomic history has made it challenging to fully understand the identity and occurrence of each. Burns (2015) revealed differences in genitalia, clarifying that they are distinct species. Genomic DNA analysis of more than 100 specimens now illuminates their genetic uniqueness, phylogenetic relationship, field identification challenges and details of their geographic distributions.

Ectomycorrhizal (EM) fungi are vital to forest ecosystems, supporting tree growth and survival. However, their inclusion in conservation policy and action remains limited and little is known about the status of their genetic diversity, which is essential for their long-term survival and adaptation. The Global Biodiversity Framework adopted a genetic indicator based on the effective population size, Ne, to monitor genetic diversity in all species. To date, it is still uncertain how Ne, a key parameter, can be reliably assessed in species with complex life history traits. Ectomycorrhizal fungi are a highly diverse group of taxa displaying haplodiplontic life cycles with partially clonal reproduction. Here, we review the literature to understand how these life history traits might affect Ne and its estimation in six species of EM fungi. We estimated Ne in 19 populations using eight genetic and genomic datasets from selected studies. We compared Ne estimates using Linkage Disequilibrium (LD) and Sibship Frequency (SF) methods. We tested how Ne estimates change due to partial clonality and genetic structure gradients and whether the number of genetic markers influence the precision of the estimates. We show a systematic bias in Ne estimations when large clones are present and when populations are not correctly delimited. We found both methods are not robust to these factors, which makes them unreliable for conservation assessment purposes in EM fungi. This study provides new perspectives for further research into the links between life history traits and the effective population size of ectomycorrhizal fungi.

The global study of freshwater clams in the genus Corbicula is frequently confounded by invasive androgenetic lineages that experience mitochondrial DNA capture and clonal propagation. In contrast, the endemic Corbicula of Sulawesi's ancient lakes reproduce sexually, offering a uniquely reliable system for mitochondrial population genetics. This study provides the first population-genetic framework for two endemic species, Corbicula possoensis (Lake Poso) and C. linduensis (Lake Lindu), using the cytochrome c oxidase subunit I (COI) marker. We analysed 90 newly generated COI sequences from C. possoensis (six stations) and C. linduensis (three stations), integrated with reference sequences from GenBank, to assess genetic diversity, population structure, and phylogeographic patterns. Hierarchical AMOVA revealed deep divergence between the two lakes ({Phi}_CT = 0.607), consistent with prolonged independent isolation rather than a single shared vicariance event, as the two species do not form a sister pair in the phylogeny. Within Lake Poso, C. possoensis exhibited exceptionally high genetic diversity (24 haplotypes; h = 0.876; {pi} = 0.016) and pronounced micro-geographic structuring into three phylogeographic zones (North: Tentena and Siuri; East: Tando Nceppo and Busogo Beach; Southwest: Bancea and Pendolo), each characterised by distinct haplogroups. Remarkably, the maximum divergence between zones (K2P = 2.33%) approached the interspecific distance between C. possoensis and C. linduensis (K2P = 2.42%), indicating that within-lake mitochondrial divergence has reached near-interspecific levels. Conversely, C. linduensis displayed near-panmixia and extreme genetic depauperation (3 haplotypes; h = 0.246; {pi} = 0.0004), indicating long-term demographic stasis within a restricted habitat. The deep phylogeographic zonation in C. possoensis suggests that its discrete populations should be treated as separate Management Units (MUs) in conservation planning to preserve locally adapted gene complexes, whereas the severely depauperate gene pool of C. linduensis renders it critically vulnerable to environmental disturbance and invasive species, warranting urgent IUCN Red List assessment. To validate these mitochondrial boundaries and inform future conservation strategies, multi-marker and genome-wide reassessments are strongly recommended.

The Atlantic surfclam, Spisula solidissima solidissima supports multimillion dollar harvests in the western North Atlantic, including Georges Bank and Mid-Atlantic Bight populations along the continental shelf. Surfclam populations in this part of the Exclusive Economic Zone (3-200 nm from shore) are managed as a single unit, but demographic connectivity has never been tested directly. This study analyzed >8,500 SNPs in 548 Atlantic surfclams sampled across the USA harvested range to infer population structure, genomic diversity, and gene flow patterns. Genomic analyses included eight sampled and 10 reference Spisula solidissima similis samples, a morphologically cryptic nominal subspecies previously known only from nearshore habitats. Novel morphologically cryptic population structure was identified in S.s. solidissima. One operational taxonomic unit (OTU-A) was found only in the inshore region of southern New England, south of Cape Cod. The other population unit, OTU-B, was found offshore and in Cape Cod Bay. The federal fishing grounds only had OTU-B clams. Populations within each OTU were connected by gene flow, justifying current fishery management practices. Nearshore state waters had mixed OTU stocks. Hybridization was analyzed between the two OTUs and between the two subspecies based on nuclear SNPs and asymmetrical mitochondrial DNA introgression. Finally, we demonstrated the utility of a novel SNP panel for diagnosing all three taxa and their hybrids.

Connectivity is critical to sustaining endangered carnivores in speciose yet fragmented landscapes such as in the global south. Corridors to mitigate fragmentation are designed based on charismatic species or habitat-based approaches, but their multispecies effectiveness for maintaining functional connectivity remains poorly tested. We combined landscape genetic analyses across five sympatric carnivores - Panthera tigris, Panthera pardus, Prionailurus viverrinus, Felis chaus, and Melursus ursinus, to evaluate how landscape features shape functional connectivity in a globally important felid landscape. We then assessed the efficiency of existing tiger corridors and single-species surrogates for maintaining multispecies functional connectivity. Species exhibited contrasting responses to landscape variables, producing distinct resistance surfaces and connectivity corridors. Spatial similarity of connectivity between species pairs was highly variable (r = 0.14-0.93), but no single species effectively captured connectivity patterns of the broader carnivore community (maximum mean overlap of <0.7 across species). Moreover, genetically optimized corridors were at least 70% more efficient in capturing connectivity compared to existing tiger corridors, demonstrating mismatches between structural and functional connectivity. Our results highlight limitations of surrogate-based corridor planning and demonstrate that integrating multispecies functional connectivity can substantially improve conservation planning in human-dominated landscapes.

High-throughput amplicon sequencing has transformed microsatellite (STR) genotyping by overcoming many of the limitations of fragment-length analysis, enabling more accurate, cost-effective, and standardized genotyping. Yet, protocols specifically designed for high-throughput sequencing (HTS)-based STR genotyping from low-template and degraded DNA remain scarce, despite the prevalence of these challenging sample types in ecological and conservation contexts. We present a methodology for the de novo development of robust STR multiplex panels together with a laboratory protocol for efficient and reliable STR genotyping by sequencing with low quantity and quality DNA samples. The protocol comprises (i) an automated bioinformatic pipeline to design large sets of short tetranucleotide markers optimized for multiplex amplicon sequencing of degraded and low-template DNA; (ii) guidelines for efficient in vitro optimization of multiplex amplification using directly low quantity/quality template DNA; and (iii) a library preparation procedure that improves detection of low-level allele signal while enabling quality assessment of STR amplicon sequencing under limiting DNA conditions. We demonstrate the approach by developing and validating STR panels for non-invasive genotyping of three large carnivore species: a 44-plex for the grey wolf (Canis lupus), a 41-plex for the Eurasian lynx (Lynx lynx), and a 30-plex for the brown bear (Ursus arctos). Multiplex performance was high, with [&ge;]91% of samples successfully genotyped at [&ge;]50% of loci (allele size range 28-110 bp across panels) and correctly assigned to known individuals, negligible levels of noise in the controls, and high discriminatory power (PIDsibs [&le;]2.4 x 1e-12), also owing to sequence variation among same-length alleles at 15-50% of loci. The approach is broadly applicable to animal and plant species, a wide range of sample types, and large-scale analysis such as genetic monitoring. Our study reinforces the value of STR amplicon sequencing for ecological and conservation applications while highlighting the importance of marker design and laboratory workflows tailored to HTS-based genotyping for accurate and efficient implementation.