Arthropod diversity in the alpine tundra using metabarcoding: Spatial and temporal differences in alpha- and beta-diversity.

All ecosystems face ecological challenges in this century. Therefore, it is becoming increasingly important to understand the ecology and degree of local adaptation of functionally important Arctic-alpine biomes by looking at the most diverse taxon of metazoans: the Arthropoda. This is the first study to utilize metabarcoding in the Alpine tundra, providing insights into the effects of micro-environmental parameters on alpha- and beta-diversity of arthropods in such unique environments. To characterize arthropod diversity, pitfall traps were set at three middle-alpine sampling sites in the Scandinavian mountain range in Norway during the snow-free season in 2015. A metabarcoding approach was then used to determine the small-scale biodiversity patterns of arthropods in the Alpine tundra. All DNA was extracted directly from the preservative EtOH from 27 pitfall traps. In order to identify the controlling environmental conditions, all sampling locations were equipped with automatic data loggers for permanent measurement of the microenvironmental conditions. The variables measured were: air temperature [°C] at 15 cm height, soil temperature [°C] at 15 cm depth, and soil moisture [vol.%] at 15 cm depth. A total of 233 Arthropoda OTUs were identified. The number of unique OTUs found per sampling location (ridge, south-facing slope, and depression) was generally higher than the OTUs shared between the sampling locations, demonstrating that niche features greatly impact arthropod community structure. Our findings emphasize the fine-scale heterogeneity of arctic-alpine ecosystems and provide evidence for trait-based and niche-driven adaptation. The spatial and temporal differences in arthropod diversity were best explained by soil moisture and soil temperature at the respective locations. Furthermore, our results show that arthropod diversity is underestimated in alpine-tundra ecosystems using classical approaches and highlight the importance of integrating long-term functional environmental data and modern taxonomic techniques into biodiversity research to expand our ecological understanding of fine- and meso-scale biogeographical patterns.

Comparison of destructive and nondestructive DNA extraction methods for the metabarcoding of arthropod bulk samples.

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.

Metabarcoding dietary analysis in the insectivorous bat Nyctalusleisleri and implications for conservation.

In this study, we aim to uncover diet preferences for the insectivorous bat Nyctalusleisleri (Leisler's bat, the lesser noctule) and to provide recommendations for conservation of the species, based on the analysis of prey source habitats. Using a novel guano trap, we sampled bat faeces at selected roosts in a forest in Germany and tested two mitochondrial markers (COI and 16S) and three primer pairs for the metabarcoding of bat faecal pellets. We found a total of 17 arthropod prey orders comprising 358 species in N.leisleri guano. The most diverse orders were Lepidoptera (126 species), Diptera (86 species) and Coleoptera (48 species), followed by Hemiptera (28 species), Trichoptera (16 species), Neuroptera (15 species) and Ephemeroptera (10 species), with Lepidoptera species dominating in spring and Diptera in summer. Based on the ecological requirements of the most abundant arthropod species found in the bat guano, we propose some recommendations for the conservation of N.leisleri that are relevant for other insectivorous bat species.

Metabarcoding Malaise traps and soil eDNA reveals seasonal and local arthropod diversity shifts.

Forest habitats host enormous diversity, but little is known about the seasonal turnover of arthropod species between the above- and below ground forest layers. In this study, we used metabarcoding approaches to uncover arthropod diversity in different forest types and seasons. Our study shows that metabarcoding soil eDNA and Malaise trap bulk samples can provide valuable insights into the phenology and life cycles of arthropods. We found major differences in arthropod species diversity between soil samples and Malaise traps, with only 11.8% species overlap. Higher diversity levels were found in Malaise traps in summer whereas soil samples showed a diversity peak in winter, highlighting the seasonal habitat preferences and life strategies of arthropods. We conclude that collecting time series of bulk arthropod samples and eDNA in the same locations provides a more complete picture of local arthropod diversity and turnover rates and may provide valuable information on climate induced phenological shifts for long-term monitoring.

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence-capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence-capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence-capture recovered 80%-90% of the control sample species. DNA sequence-capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low-cost, whereas DNA-sequence capture for biodiversity assessment is still in its infancy, is more time-consuming but provides more taxonomic assignments.

Sperm size is negatively related to relative testis size in West African riverine cichlid fishes.

Fishes show a great diversity of mating systems and fertilization mechanisms. This diversity creates an enormous potential for sperm competition. Typically, monogamous species face a low risk of sperm competition and invest less into sperm, and thus show smaller relative testis mass compared to polygamous species with high sperm competition. In cichlids, sperm competition risk is very variable. In lacustrine East African cichlids, large sperm are interpreted as an adaptation to sperm competition, as in those species sperm length correlates with sperm swimming speed. The aim of the present study was to examine variation in sperm and testis traits of substrate breeding cichlids from West African river systems and its relationship to sperm competition. Therefore, sperm traits (total sperm size, flagellum-, midpiece-, and head size) and sperm number were related to the gonadosomatic index (GSI), an indicator of sperm competition, in eight species of two large informal tribes, the chromidotilapiines and the haplotilapiines. We found significant differences between species in all examined sperm traits, sperm number, and GSI with pronounced differences between chromidotilapiines and haplotilapiines. We used a generalized least-squares approach to control for non-independence of data. GSI was positively correlated with sperm number but negatively correlated with total sperm size (also negatively with the flagellum and head size but not significantly with midpiece size). Sperm number and sperm size were negatively correlated suggesting a trade-off between sperm size and quality. Our results suggest that large sperm can evolve in species with relatively low sperm expenditure and probably in absence of high sperm competition between males.

No experimental evidence for sneaking in a west african cichlid fish with extremely long sperm.

Alternative reproductive tactics are widespread in fishes, increasing the potential for sperm competition. Sperm competition has enormous impact on both variation in sperm numbers and sperm size. In cichlids, the sperm competition risk is very divergent and longer sperm are usually interpreted as adaptation to sperm competition. Here we examined whether sneaking tactics exist in Pelvicachromis taeniatus, a socially monogamous cichlid with biparental brood care from West Africa. The small testis indicates low gonadal investment which is typical for genetically monogamous species. In contrast, sperm length with up to 85 µ m is extraordinarily long. We examined the reproductive behaviour of ten groups with a male-biased sex ratio under semi-natural conditions via continuous video recording. We recorded spawning site preferences and correlates of reproductive success and conducted paternity tests using microsatellites. Safe breeding sites that could be successfully defended were preferred. All offspring could be assigned to their parents and no multiple paternities were detected. Body size of spawning pairs predicted their spawning probability and offspring hatching rate suggesting benefits from mating with large individuals. Our study suggests low risk of sperm competition under the given conditions in P. taeniatus and thus first evidence for genetic monogamy in a substrate breeding cichlid.

Microsatellite support for active inbreeding in a cichlid fish.

In wild animal populations, the degree of inbreeding differs between species and within species between populations. Because mating with kin often results in inbreeding depression, observed inbreeding is usually regarded to be caused by limited outbreeding opportunities due to demographic factors like small population size or population substructuring. However, theory predicts inclusive benefits from mating with kin, and thus part of the observed variation in inbreeding might be due to active inbreeding preferences. Although some recent studies indeed report kin mating preferences, the evidence is still highly ambiguous. Here, we investigate inbreeding in a natural population of the West African cichlid fish Pelvicachromis taeniatus which showed clear kin mating preferences in standardized laboratory experiments but no inbreeding depression. The presented microsatellite analysis reveals that the natural population has, in comparison to two reference populations, a reduced allelic diversity (A = 3) resulting in a low heterozygosity (H(o) = 0.167) pointing to a highly inbred population. Furthermore, we found a significant heterozygote deficit not only at population (F(is) = 0.116) but also at subpopulation level (F(is) = 0.081) suggesting that inbreeding is not only a by-product of population substructuring but possibly a consequence of behavioral kin preferences.