For all audiences:
Incorporating immature stages into standardised inventories of
mega-diverse groups has a major impact on our understanding of
biodiversity patterns
Marc Domènech1, Owen S.
Wangensteen2, Alba Enguídanos1,
Jagoba Malumbres-Olarte3,4 & Miquel A.
Arnedo1
1 Department of Evolutionary Biology, Ecology and
Environmental Sciences & Biodiversity Research Institute (IRBio),
Universitat de Barcelona, Av. Diagonal 643, E-08028, Barcelona, Spain.
2 Norwegian College of Fishery Science, University of
Tromsø, The Arctic University of Norway, Norway.
3 cE3c – Centre for Ecology, Evolution and
Environmental Changes / Azorean Biodiversity Group and Universidade dos
Açores – Faculty of Agrarian and Environmental Sciences, Rua Capitão
João d’Ávila, São Pedro, 9700-042 Angra do Heroísmo, Terceira, Açores,
Portugal.
4 LIBRe – Laboratory for Integrative Biodiversity
Research, Finnish Museum of Natural History, University of Helsinki,
P.O.Box 17 (Pohjoinen Rautatiekatu 13), 00014 Helsinki, Finland
Correspondence : Marc Domènech, Department of Evolutionary
Biology, Ecology and Environmental Sciences & IRBio, Universitat de
Barcelona, Barcelona, Spain. Email: mdomenan@gmail.com
ABSTRACT
Because of their challenging taxonomy, arthropods are traditionally
underrepresented in biological inventories and monitoring programs.
However, arthropods are the largest component of biodiversity, and no
assessment can be considered informative without including them.
Arthropod immature stages are often discarded during sorting, despite
frequently representing more than half of the collected individuals. To
date, little effort has been devoted to characterising the impact of
discarding non-adult specimens on our diversity estimates.
Here, we use a metabarcoding approach to analyse spiders from white oak
communities in the Iberian Peninsula collected with standardised
protocols, to assess (1) the contribution of juvenile stages to local
diversity estimates, and (2) their effect on the diversity patterns
inferred across communities. We further investigate the ability of
metabarcoding to inform on abundance. We obtained 363 and 331 species as
adults and juveniles, respectively. Species represented only by
juveniles represented an increase of 35% with respect to those
identified from adults in the whole sampling. Differences in composition
between communities were greatly reduced when immature stages were taken
into account, especially across latitudes. Moreover, our results
revealed that metabarcoding data are to a certain extent quantitative,
but some sort of taxonomic conversion factor may be necessary to provide
accurate informative estimates.
Although our findings do not question the relevance of the information
provided by adult-based inventories, they also reveal that juveniles
provide a novel and relevant layer of knowledge that, especially in
areas with marked seasonality, may influence our interpretations,
providing more accurate information from standardised biological
inventories.
Keywords : Araneae, Diversity, DNA barcoding, Iberian Peninsula,
Metabarcoding, Spiders
Running title: Incorporating juveniles in diversity estimates
INTRODUCTION
Global human activity is altering the species richness and abundance of
biological communities (Stuart Chapin III et al 2000; Socolar,
Valderrama-Sandoval, & Wilcove, 2019; Barlow et al., 2016). These
disturbances are accelerating the invasion rate of exotic species
(Hulme, 2009) and driving numerous species to extinction, sometimes even
before they are described, in what has been referred to as thesixth mass extinction (Dirzo et al, 2014; Barnosky et al, 2011).
Bioinventories and early detection methods for monitoring ecosystem
changes are essential to identify and tackle unanticipated threats to
biodiversity (Telfer et al 2015; Barnosky et al 2013). However, sampling
and identifying highly abundant and diverse groups such as arthropods is
a daunting task. Several methods have been devised to overcome this
limitation. For instance, rapid biodiversity assessment protocols are
fast yet efficient sampling strategies specifically designed to retrieve
the greatest amount of information from a particular area, minimising
the number and length of sampling periods (Oliver and Beattie 1996).
Arthropods represent the largest and most abundant component of animal
biodiversity. Therefore, no biodiversity monitoring program can be
considered credible unless it takes them into account (Taylor and Doran
2001). Moreover, because of their high reproductive rates and short
generation times, arthropods have the potential to inform on
biodiversity changes at finer spatial and shorter temporal scales than
vertebrates (Kremen et al 1993, Yen & Butcher 1997). However, the poor
taxonomic knowledge of many arthropods limits their use as
bioindicators, and they are frequently underrepresented in biodiversity
assessments and conservation programs (Cardoso, Borges, Triantis,
Ferrández, & Martín, 2012). On the other hand, recent studies suggest
that arthropod populations, which play a fundamental role in ecosystem
functioning, are declining at an alarming rate (Leather 2018).
Because of their rapid and divergent evolution, male copulatory
apparatus and, to a lesser degree, female external reproductive systems
are the main structures for species identification across most arthropod
groups (Eberhard, 1985). Spiders are no exception. Morphological
taxonomic identification is almost exclusively based on genitalic
characters, i. e., the structure of the copulatory bulb in males and the
vulva and epigyne (external modifications of the genital area) in
females. These features are only visible in the last moult, which makes
immatures difficult or impossible to identify at species level (Dobyns,
1997; Coddington, Young, & Coyle, 1996). Thus, in most inventories and
diversity studies immature stages are discarded during sorting. However,
immature specimens may account for between 40% and 70% of the
collected specimens in biodiversity surveys (Soukainen et al, 2020,
Malumbres-Olarte et al, 2020a, Malumbres-Olarte, Cardoso, & Crespo,
2019; Cardoso, Silva, Oliveira, & Serrano, 2004; Russell-Smith & Stork
1995, Silva 1996), or even up to 94% in extreme cases (Kuntner &
Baxter, 1997). Disregarding immatures may significantly influence the
inference of the temporal and spatial patterns of biodiversity, so their
incorporation is desirable to obtain reliable estimates of diversity in
short-term sampling protocols (Toti, Coyle, & Miller, 2000; Sorensen,
Coddington, & Scharff, 2002).
It is known that the life cycles of different groups of spiders differ
in the number of generations per year and in the time of the year when
they are present as adults or juveniles (Aitchison, 1984; Nadal,
Achitte-Schmutzler, Zanone, Gonzalez, & Avalos, 2018), even within the
same species. For example, some wolf spiders are known to have annual
life cycles maturing as adults in March-April and reproducing in
May-June, although they can also have two clutches in the same year
depending on the weather conditions (Rádai, Kiss, & Samu, 2017). Since
rapid biodiversity assessment protocols are usually conducted once, they
provide a “photograph” of the species present in a certain area at one
particular time, so considering only adult individuals would completely
dismiss all the species that are present as immature stages in that
particular time of the year. To our knowledge, only one study (Norris,
1999) has partially addressed the effect in diversity estimates of
incorporating juvenile spider stages. This study, which included only a
few species that could reliably be morphologically identified at
immature stages, already pointed out that numerous species were only
found as juveniles and that relative abundances changed drastically when
these immature stages were taken into account.
The use of DNA-based approaches for species identification, e.g., DNA
barcoding (Hebert, Ratnasingham, & DeWaard, 2003), ease the
identification of immature stages. DNA barcode sequences of immature
individuals can be assigned to species through comparison to reference
databases containing barcodes of adult-based morphologically identified
species (Richard et al, 2010; Meiklejohn, Wallman, & Dowton, 2012).
However, this technique still poses some drawbacks. For example, it
requires manually extracting and amplifying each specimen individually,
which for large samplings with hundreds or thousands of juveniles can be
very time-consuming. Moreover, the economic cost of extracting,
amplifying and sequencing such a large amount of samples would also be
considerable.
DNA metabarcoding is a more recently developed molecular technique
consisting in the automated identification of multiple species from a
single bulk sample containing entire or partial organisms or from
environmental samples (water, soil, etc.) containing remains of DNA
(e.g., Bohmann et al., 2014; Yu et al., 2012; Morinière et al., 2016).
This approach represents a clear advantage with respect to DNA
barcoding, as it allows the simultaneous processing of a large number of
specimens at once, greatly reducing the workload and processing time. In
addition, it is more cost efficient for large numbers of specimens, as
the number of sequences obtained from a single metabarcoding run is in
the order of millions (Sales et al., 2020; Watts et al., 2019). The
downside of using this approach is that individual specimens cannot be
traced back or are sometimes even lost in the process of preparing the
bulk sample, making it impossible to revise the voucher specimens if
interesting sequences were found.
Another potential drawback of the use of metabarcoding for biodiversity
assessment is its presumed inability to provide abundance information.
To what extent the number of sequence reads of a certain taxon correctly
represents its abundance or biomass in the sample has been a matter of
much debate. Several studies have specifically addressed this issue
(Elbrecht and Leese, 2015; Piñol, Mir, Gomez-Polo, & Agustí, 2015; Lamb
et al., 2019; Deagle et al., 2019), but the answer remains inconclusive.
While some studies consider the quantitative power of metabarcoding
limited (Elbrecht and Leese, 2015; Piñol et al., 2015), others found
metabarcoding to give an accurate estimate of a taxon’s abundance under
certain conditions (Ratcliffe et al., 2020) or by applying correction
factors that may vary among taxa (Kennedy et al., 2020; Thomas, Deagle,
Eveson, Harsch, & Trites, 2016), or have even used it to quantitatively
analyse dietary data (Soinien et al., 2015). One explanation for these
different conclusions may be that the range of concentrations analysed
varies considerably across studies, as suggested by Deagle et al.
(2019). While a positive relationship between number of reads and
biomass is commonly found, only a certain part of the variation in the
number of reads seems to be explained by differences in the biomass in
the sample, while the rest of the variation seems to be due to factors
such as primer specificity
(Elbrecht and Leese, 2015), different extraction success between
different tissues or species (Schiebelhut, Abboud, Gómez-Daglio, Swift,
& Dawson, 2016) or the efficiency of the blocking primers of predator
DNA in the case of diet studies (Piñol et al., 2015).