Discussion
In the present paper we investigated the DNA taxonomy of a megadiverse
assemblage of chafer beetles (Sericini) with particular focus on the
performance of commonly used species delimitation methods. The setup of
examining barcodes of a single locality was chosen to investigate
molecular species delimitation performance using data without geographic
bias. While we know that match ratios strongly vary in tropical taxa
(e.g., from 0.14 to 1.00; Ahrens et al., 2016), we theoretically
expected that match ratios would go against one due to the exclusion of
geography-induced variance. Instead, for different standard species
delimitation methods, we could also not report match ratios higher than
0.77. Interestingly, the 3% threshold clustering that is commonly used
in metabarcoding approaches did not perform worse than more
sophisticated approaches (like PTP, or TCS), however, an accuracy of
only less than 80% is not really what one could call a reliable
taxonomy assessment.
DNA-based species delimitation approaches may oversplit morphological
entities (Ahrens et al., 2016), while at the same time the opposite may
be also the case (Dalstein et al., 2019), even in the same taxon (as
demonstrated here for the tribe Sericini). This particularly proved to
be true in presence of incomplete lineage sorting and hybridisation and
if geographic bias is not excluded (match ratio < 0.5;
Dalstein et al., 2019). Extreme over-splitting has been reported for
both mtDNA and nDNA, when sex-biased dispersal occurs (Eberle et al.,
2019) and were the general dispersal is in consequence also very
limited.
Over-splitting in our data is caused by the relatively deep coalescence
in 21% of the species, which widely corresponds with the missing match
to the morphospecies, which is also reflected by the lack of a classical
barcoding gap (Fig. 4). The impact is high with only 31 out of the 56
morphospecies matching perfectly the boundaries of inferred MOTUs (Fig.
2). The nature of maternal inheritance of mtDNA and its very low
recombination rate is probably the major reason for these patterns of
deep coalescence. Historically acquired genetic differentiation, for
example in previously isolated populations, is maintained in secondarily
mixing populations. The more often such isolated populations occur in
time and space, for example due to climatic fluctuation during the
Pleistocene in geographically highly structured areas such as Southeast
Asia, the more often we encounter such ”paleogeographically induced”
infraspecific variation which leads to the same result as current
geographic variation. This effect consequently impedes species
delimitation methods in the same way, particularly in a single marker
system (e.g., cox1 ).
Similarly, in our data we could also report cases of incomplete lineage
sorting and/or hybridisation. In three cases, morphospecies were not
monophyletic (Microserica sp 11/ Microserica sp 13 vs.Microserica varians ; Neoserica sp 29 vs. Neoserica
martinui ; Maladera sp 27 vs. Maladera sp 9.), while
another three morphospecies shared identical haplotypes (Maladerasp 3a, sp 3b; sp 4). In all cases, we may exclude cross contamination
based on the position of the single samples on the DNA-extraction
microtiter-plates. These cases do occur in only rather closely related
species, which might show similar life traits (e.g., daytime activity inMicroserica ), chemical communication, or mating behaviour (which
is however, unknown for all species). In those instances, lumping of
morphospecies in DNA based species delimitation seems to be more likely;
however, also over-splitting was observed (e.g. Microserica ).
Despite strong divergence in male genital morphology, hybridization
between closely related species of Sericini have been reported (e.g., .
The rather divergent structure of the aedeagus of the different species
might function with females by mechanical isolation (lock-and-key
hypothesis) that prevents mating between different species . However,
although there have been some recent work on the morphology of female
genitalia, our knowledge on copulation functionality and mechanics is
still not sufficient to tell if morphological structures of males and
female genitalia actually function as a barrier, if only through tactile
recognition by cryptic female choice .
Again, the present study demonstrates the necessity of an integrative
taxonomy in the sense of Yeates et al. (2011) (see also . We showed that
the use of different clustering- and tree-based delimitation methods
(Carstens et al., 2013) with the same single maker reproduces the same
erroneous signal in slightly different variations. It is thus critical
to corroborate results with data from other sources (e.g., genital or
larval morphology, feeding traits, behaviour, etc.; e.g. Janzen et al.,
2009) to allow for independent testing of species boundaries.
Sericini chafers proved to be a valuable model system, because of robust
morphospecies assignments that were facilitated by the highly dissimilar
and morphologically complex male genitalia that perfectly serve as a
species diagnostic trait .
Overall, the initial hypothesis of impeccable DNA-based species
boundaries in syntopically co-occurring species assemblages clearly had
to be rejected. This was rather unexpected, especially since there was
no additional evidence from other sources that these over-splittings
could relate to cryptic diversity (Janzen et al., 2009, 2017; Janzen &
Hallwachs, 2011).
Given the highly simplified parameters of DNA based species delimitation
in this one-site species assemblage, it becomes clear how complex
species delimitation with DNA-based methods is. Performance with mean
error rates of more than 30% are under the expectations for proper use
for applied sciences and conservation management. Even more
sophisticated methods did not perform better than over-simplified
threshold clustering methods as used for example in metabarcoding. Once
more, we highlight the necessity of morphology for the verification ofde novo species delimitation results and the constant need of
integrative taxonomic approaches.